CN218974139U

CN218974139U - Imaging system and super-resolution imaging system

Info

Publication number: CN218974139U
Application number: CN202220578729.7U
Authority: CN
Inventors: 史蒂夫·翔凌·陈; 郭明昊; 迈克尔·普雷维特; 周春红; 德里克·富勒
Original assignee: Element Bioscience Corp
Current assignee: Element Bioscience Corp
Priority date: 2021-03-16
Filing date: 2022-03-16
Publication date: 2023-05-05
Anticipated expiration: 2032-03-16
Also published as: CN217981206U

Abstract

The present application relates to imaging systems and super resolution imaging systems. An imaging system includes a sample container and an imager. Another imaging system flow cell, an objective lens, at least one image sensor, and an optical element. The super-resolution imaging system includes an excitation light source, a depletion light source, an objective lens, an optical path, and at least one image sensor.

Description

Imaging system and super-resolution imaging system

Technical Field

The present application relates to imaging systems and super resolution imaging systems.

Background

In a typical fluorescence-based genomic test assay, such as genotyping or nucleic acid sequencing (using a real-time, cyclic, or step-wise reaction scheme), dye molecules attached to nucleic acid molecules tethered to a substrate are excited using an excitation light source, fluorescent photon signals are generated at one or more spatially localized locations on the substrate, and then fluorescence is imaged onto an image sensor by an optical system. The images are then analyzed using analytical methods to find the location of the labeled molecules (or clonally amplified clusters of molecules) on the substrate and to quantify the fluorescent photon signal in terms of wavelength and spatial coordinates, which can then be correlated to the extent of a particular chemical reaction (e.g., hybridization event or base addition event) occurring at a specified location on the substrate. Image-based methods provide massive parallelism and multiplexing capabilities, which help reduce the cost and availability of such techniques. However, detection errors due to, for example, too close packing of the labeled molecules (or clonally amplified clusters of molecules) within a small area of the substrate surface or due to low contrast to noise ratio (CNR) in the image may lead to errors in attributing fluorescent signals to the correct molecules (or clonally amplified clusters of molecules).

Disclosure of Invention

The present disclosure discloses an imaging system comprising: a) Two different axially displaced surfaces; b) An objective lens; and c) at least one image sensor, wherein the imaging system has a Numerical Aperture (NA) of less than 0.6 and greater than 1.0mm ² And wherein the imaging system is capable of acquiring images of the two different axially displaced surfaces with substantially the same optical resolution without moving an optical compensator into the optical path between the objective lens and the at least one image sensor. In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the imaging system further comprises an autofocus mechanismThe autofocus mechanism includes an autofocus laser and an autofocus sensor, wherein the autofocus mechanism is configured to refocus the optical system between acquiring images of the two different axially displaced surfaces. In some embodiments, the objective lens has a magnification sufficient to image two or more fields of view on at least one of the two different axially displaced surfaces. In some embodiments, the two different axially displaced surfaces comprise two surfaces of a flow cell (flow cell). In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises a nucleic acid population of greater than 10,000 nucleic acid populations per square millimeter (mm) ² ) Is provided, the surface density of the labeled nucleic acid population disposed thereon. In some embodiments, the imaging system comprises 1, 2, 3, or 4 imaging channels comprising a tube lens having a depth of field sufficient to detect nucleic acid populations disposed on at least one of the two surfaces of the flow cell that have been labeled with 1, 2, 3, or 4 different detectable labels. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that a spatial sampling frequency of the imaging system is at least twice an optical resolution of the imaging system. In some embodiments, the active area of the at least one image sensor has a diagonal greater than or equal to about 15 millimeters (mm). In some embodiments, the imaging system further comprises at least one tube lens located between the objective lens and the at least one imaging sensor, and wherein the at least one tube lens is configured to correct an imaging performance index for imaging the two different axially displaced surfaces of the flow cell.

Disclosed herein is an imaging system configured to image a first interior surface and a second interior surface of a flow cell, the imaging system comprising: a) An objective lens; b) At least one image sensor; and c) at least one tube lens arranged on an optical path between the objective lens and the at least one image sensor; wherein the optical system has a Numerical Aperture (NA) of less than 0.6 and greater than 1.0mm ² Is a field of view (FOV); and wherein the at least one tube lens is configured to correct imaging performance such that images of the first inner surface of the flow cell and the second inner surface of the flow cell have substantially the same optical resolution.

In some embodiments, the flow cell has a wall thickness of at least 700 μm and the fluid-filled gap between the first and second inner surfaces is at least 50 μm. In some embodiments, images of the first inner surface and the second inner surface are acquired without moving an optical compensator into the optical path between the objective lens and the at least one image sensor. In some embodiments, the imaging system has a Numerical Aperture (NA) of less than 0.6. In some embodiments, the imaging system has a Numerical Aperture (NA) greater than 0.3. In some embodiments, the imaging system has a thickness of greater than 1.5mm ² Is a field of view (FOV). In some embodiments, the optical resolution of the images of the first and second inner surfaces is diffraction limited over the entire field of view (FOV). In some embodiments, the at least one tube lens includes an asymmetric convex-convex lens, a convex-flat lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens in that order. In some embodiments, the imaging system includes two or more tube lenses designed to provide optimal imaging performance for the first and second interior surfaces at two or more fluorescence wavelengths. In some embodiments, the imaging system further comprises a focusing mechanism configured to refocus the optical system between acquiring images of the first inner surface and the second inner surface. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the first inner surface or the second inner surface. In some embodiments, the first and second inner surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises >10,000 nucleic acid colonies/mm ² Is provided, the surface density of the labeled nucleic acid population disposed thereon. In some embodiments, when the nucleic acid community is labeled with cyanine dye 3 (Cy 3), the image of the first interior surface or the second interior surface taken using the imaging system exhibits a contrast-to-noise ratio (CN) of at least 5R), the imaging system included a dichroic mirror and bandpass filter set optimized for Cy3 emission, and images were acquired under non-signal saturation conditions while immersing the surface in 25mm ACES pH 7.4 buffer. In some embodiments, the imaging system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid populations disposed on at least one of the two different surfaces that have been labeled with 1, 2, 3, or 4 different detectable labels. In some embodiments, the imaging system is used to monitor the sequence of nucleotide bases on at least one of the first and second interior surfaces by affinity sequencing, by nucleotide base pairing sequencing, by nucleotide binding sequencing, or by nucleotide incorporation reaction, and detect the binding or incorporation of nucleotide bases. In some embodiments, the imaging system is used to perform nucleic acid sequencing. In some embodiments, the imaging system is used to determine the genotype of the sample, wherein determining the genotype of the sample comprises preparing nucleic acid molecules extracted from the sample for sequencing, and then sequencing the nucleic acid molecules. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the combination of the objective lens and the at least one tube lens is configured to optimize the modulation transfer function in the sample plane over a spatial frequency range of 700 cycles/mm to 1100 cycles/mm. In some embodiments, for a combination of an objective lens and at least one tube lens, the at least one tube lens is designed to correct a Modulation Transfer Function (MTF) in terms of one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast to noise ratio (CNR), or any combination thereof.

Also disclosed herein are methods of sequencing a nucleic acid molecule, the method comprising: a) Imaging the first surface and the axially displaced second surface using an optical system comprising an objective lens and at least one image sensor, wherein the optical system has a Numerical Aperture (NA) of less than 0.6 and greater than 1.0mm ² Is a field of view (FOV), and whereinAcquiring images of the first surface and the axially displaced second surface with substantially the same optical resolution without moving an optical compensator into the optical path between the objective lens and the at least one image sensor; b) Detecting a fluorescently labeled composition comprising a nucleic acid molecule or a complement thereof disposed on the first surface or the axially displaced second surface to determine the identity of a nucleotide in the nucleic acid molecule.

In some embodiments, the focusing mechanism is utilized to refocus the optical system between acquiring images of the first surface and the axially displaced second surface. In some embodiments, the method further comprises imaging two or more fields of view on at least one of the first surface or the axially displaced second surface. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating. In some embodiments, the hydrophilic coating further comprises a coating that is a coating of a polymer >10,000 nucleic acid colonies/mm ² Is provided, the surface density of the labeled nucleic acid population disposed thereon. In some embodiments, when the nucleic acid community is labeled with cyanine dye 3 (Cy 3), the surface images of the two surfaces obtained using the optical system exhibit a contrast to noise ratio (CNR) of at least 5, the optical system comprises a dichroic mirror and bandpass filter set optimized for Cy3 emission, and the images are obtained under non-signal saturation conditions while immersing the surfaces in 25mm ACES pH 7.4 buffer. In some embodiments, the optical system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid populations disposed on at least one of the first surface and the axially displaced second surface that have been labeled with 1, 2, 3, or 4 different detectable labels. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system comprises at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens One tube lens is configured to correct an imaging performance index for imaging a first inner surface of the flow cell and a second inner surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap between the first inner surface and the second inner surface of at least 50 μm. In some embodiments, the at least one tube lens includes an asymmetric convex-convex lens, a convex-flat lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens in that order. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of objective lens and tube lens is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance index includes a measurement of a Modulation Transfer Function (MTF) in terms of one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast to noise ratio (CNR), or any combination thereof. In some embodiments, the optical resolution of the images of the first surface and the axially displaced second surface is diffraction limited over the entire field of view (FOV). In some embodiments, sequencing of the nucleic acid molecule further comprises performing sequencing by affinity, sequencing by nucleotide base pairing, sequencing by nucleotide binding or sequencing by nucleotide incorporation reaction on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide base. In some embodiments, the method further comprises determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecule for sequencing and then sequencing the nucleic acid molecule.

Disclosed herein is an imaging system configured to image two different axially displaced surfaces, the imaging system comprising an objective lens and at least one image sensor, wherein the imaging system has a Numerical Aperture (NA) of less than 0.6 and greater than 1.0mm ² And wherein the optical compensator is moved into the optical path between the objective lens and the at least one image sensor without moving the optical compensator into the optical pathThe imaging system is capable of acquiring images of two different axially displaced surfaces having substantially the same optical resolution.

In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the imaging system further comprises a focusing mechanism for refocusing the optical system between acquiring images of two different axially displaced surfaces. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the two different axially displaced surfaces. In some embodiments, the two different axially displaced surfaces comprise two surfaces of a flow cell. In some embodiments, the two different surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises >10,000 nucleic acid colonies/mm ² Is provided, the surface density of the labeled nucleic acid population disposed thereon. In some embodiments, the imaging system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid populations disposed on at least one of the two different surfaces that have been labeled with 1, 2, 3, or 4 different detectable labels. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the imaging system includes at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct an imaging performance index for imaging the first inner surface of the flow cell and the second inner surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap between the first inner surface and the second inner surface of at least 50 μm. In some embodiments, the imaging system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the optical resolution of the images of two different axially displaced surfaces is diffraction limited over the entire field of view (FOV).

Disclosed herein are methods of nucleic acid molecule sequencing, the methods comprising: a) Imaging the first surface and the axially displaced second surface using an uncompensated optical system comprising an objective lens and at least one image sensor, wherein the optical system has a Numerical Aperture (NA) of less than 0.6 and a Numerical Aperture (NA) of greater than 1.0mm ² Is a field of view (FOV); b) Processing the images of the first surface and the axially displaced second surface to correct for optical aberrations such that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; and c) detecting the fluorescently labeled composition comprising a nucleic acid molecule or its complement disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

In some embodiments, the image of the first surface and the axially displaced second surface is acquired without moving an optical compensator into the optical path between the objective lens and the at least one image sensor. In some embodiments, images of the first surface and the axially displaced second surface are acquired by refocusing the optical system alone. In some embodiments, the method further comprises imaging two or more fields of view on at least one of the first surface or the axially displaced second surface. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating. In some embodiments, the hydrophilic coating further comprises a coating that is a coating of a polymer >10,000 nucleic acid colonies/mm ² Is provided, the surface density of the labeled nucleic acid population disposed thereon. In some embodiments, when the nucleic acid community is labeled with cyanine dye 3 (Cy 3), the surface images of the two surfaces obtained using the optical system comprising a dichroic mirror and bandpass filter set optimized for Cy3 emission exhibit a contrast to noise ratio (CNR) of at least 5, and the images are obtained under non-signal saturation conditions while immersing the surfaces in 25mm ACES pH 7.4 buffer. In some embodiments, the optical system includes 1, 2, 3, or 4 imaging channels configured toDetecting a nucleic acid population disposed on at least one of the first surface and the axially displaced second surface that has been labeled with 1, 2, 3, or 4 different detectable labels. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system includes at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct an imaging performance index for imaging the first inner surface of the flow cell and the second inner surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap between the first inner surface and the second inner surface of at least 50 μm. In some embodiments, the at least one tube lens includes an asymmetric convex-convex lens, a convex-flat lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens in that order. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the combination of objective lens and tube lens is configured to optimize the modulation transfer function over a medium to high spatial frequency range. In some embodiments, the imaging performance index includes a measurement of a Modulation Transfer Function (MTF) in terms of one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, image contrast to noise ratio (CNR), or any combination thereof. In some embodiments, the optical resolution of the images of the first surface and the axially displaced second surface is diffraction limited over the entire field of view (FOV). In some embodiments, sequencing of the nucleic acid molecule further comprises performing sequencing by affinity sequencing, by nucleotide binding sequencing, or by nucleotide incorporation reaction on at least one of the first surface and the axially displaced second surface, and detecting the bound or incorporated nucleotide base. In some embodiments, the method further comprises determining the genotype of the sample, wherein determining the genotype of the sample comprises preparing the nucleic acid molecule for sequencing The nucleic acid molecule is then sequenced.

Disclosed herein are systems for sequencing nucleic acid molecules, the systems comprising: a) An optical system comprising an objective lens and at least one image sensor, wherein the optical system has a Numerical Aperture (NA) of less than 0.6 and greater than 1.0mm ² And is configured to acquire images of the first surface and the axially displaced second surface; b) A processor programmed to: i) Processing the images of the first surface and the axially displaced second surface to correct for optical aberrations such that the images of the first surface and the axially displaced second surface have substantially the same optical resolution; and ii) detecting the fluorescently labeled composition comprising a nucleic acid molecule or a complementary sequence thereof disposed on the first surface or the axially displaced second surface to determine the identity of the nucleotides in the nucleic acid molecule.

In some embodiments, the image of the first surface and the axially displaced second surface is acquired without moving an optical compensator into the optical path between the objective lens and the at least one image sensor. In some embodiments, images of the first surface and the axially displaced second surface are acquired by refocusing the optical system alone. In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises >10,000 nucleic acid colonies/mm ² Is provided, the surface density of the labeled nucleic acid population disposed thereon. In some embodiments, the optical system comprises 1, 2, 3, or 4 imaging channels configured to detect nucleic acid populations disposed on at least one of the first surface or the axially displaced second surface that have been labeled with 1, 2, 3, or 4 different detectable labels. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodimentsIn an aspect, the system includes at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct an imaging performance index for imaging the first inner surface of the flow cell and the second inner surface of the flow cell. In some embodiments, the flow cell has a wall thickness of at least 700 μm and a gap between the first inner surface and the second inner surface of at least 50 μm. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths.

The fluorescence imaging system disclosed herein includes: a) At least one light source configured to provide excitation light within one or more specified wavelength ranges; b) An objective configured to collect fluorescence generated from a sample plane within a specified field of view of the sample plane after exposure to excitation light, wherein the objective has a numerical aperture of at least 0.3, wherein the working distance of the objective is at least 700 μm, and wherein the area of the field of view is at least 2mm ² The method comprises the steps of carrying out a first treatment on the surface of the And c) at least one image sensor, wherein the fluorescence collected by the objective is imaged onto the image sensor, and wherein the pixel size of the image sensor is selected such that the spatial sampling frequency of the fluorescence imaging system is at least twice the optical resolution of the fluorescence imaging system.

In some embodiments, the numerical aperture is at least 0.75. In some embodiments, the numerical aperture is at least 1.0. In some embodiments, the working distance is at least 850 μm. In some embodiments, the working distance is at least 1,000 μm. In some embodiments, the area of the field of view is at least 2.5mm ² . In some embodiments, the area of the field of view is at least 3mm ² . In some embodiments, the spatial sampling frequency is at least 2.5 times the optical resolution of the fluorescence imaging system. In some embodiments, the spatial sampling frequency is at least 3 times the optical resolution of the fluorescence imaging system. In some embodiments, the system further comprises an X-Y-Z displacement stage, such that the system is configured to acquire a series of two or more fluoroscopic images in an automated manner,wherein each image of the series is acquired for a different field of view. In some embodiments, the position of the sample plane is adjusted simultaneously in the X-direction, Y-direction, and Z-direction to match the position of the objective focal plane between acquired images for different fields of view. In some embodiments, the time required for simultaneous adjustment in the X, Y and Z directions is less than 0.4 seconds. In some embodiments, the system further comprises an autofocus mechanism configured to adjust the focal plane position prior to acquiring images of different fields of view if the error signal indicates that the difference between the focal plane and the position of the sample plane in the Z-direction is greater than a specified error threshold. In some embodiments, the specified error threshold is 100nm. In some embodiments, the specified error threshold is 50nm. In some embodiments, the system comprises three or more image sensors, and wherein the system is configured to image fluorescence of each of the three or more wavelength ranges onto a different image sensor. In some embodiments, the difference in position of the focal plane and the sample plane of each of the three or more image sensors is less than 100nm. In some embodiments, the difference in position of the focal plane and the sample plane of each of the three or more image sensors is less than 50nm. In some embodiments, the total time required to reposition the sample plane, adjust the focus, and acquire the image as necessary is less than 0.4 seconds per field of view. In some embodiments, the total time required to reposition the sample plane, adjust the focus, and acquire the image as necessary is less than 0.3 seconds per field of view.

Also disclosed herein is a fluorescence imaging system for duplex imaging of a flow cell, comprising: a) An objective lens configured to collect fluorescence generated within a specified field of view of a sample plane within the flow cell; b) At least one tube lens positioned between the objective lens and at least one image sensor, wherein the at least one tube lens is configured to correct an imaging performance index of a combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an inner surface of the flow cell. And wherein the wall thickness of the flow cell is at least 700 μm and the gap between the upper and lower inner surfaces is at least 50 μm; wherein the imaging performance index is substantially the same for imaging the upper or lower inner surface of the flow cell without moving an optical compensator in or out of the optical path between the flow cell and the at least one image sensor, without moving one or more optical elements of the tube lens along the optical path, and without moving one or more optical elements of the tube lens in or out of the optical path.

In some embodiments, the objective lens is a commercially available microscope objective lens. In some embodiments, the numerical aperture of commercially available microscope objectives is at least 0.3. In some embodiments, the working distance of the objective lens is at least 700 μm. In some embodiments, the objective lens is calibrated to compensate for a 0.17mm coverslip thickness (or flow cell wall thickness). In some embodiments, the fluorescence imaging system further comprises an electro-optic phase plate positioned adjacent the objective lens and between the objective lens and the tube lens, wherein the electro-optic phase plate provides correction for optical aberrations due to fluid filling a gap between the upper inner surface and the lower inner surface of the flow cell. In some embodiments, at least one tube lens is a compound lens comprising three or more optical components. In some embodiments, at least one tube lens is a compound lens comprising four optical components. In some embodiments, the four optical components include, in order, a first asymmetric convex-convex lens, a second convex-flat lens, a third asymmetric concave-concave lens, and a fourth asymmetric convex-concave lens. In some embodiments, at least one tube lens is configured to correct an imaging performance index of a combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an inner surface of a flow cell having a wall thickness of at least 1 mm. In some embodiments, at least one tube lens is configured to correct an imaging performance index of a combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an inner surface of a flow cell having a gap of at least 100 μm. In some embodiments, at least one tube lens is configured to correct an imaging performance index of a combination of the objective lens, the at least one tube lens, and the at least one image sensor when imaging an inner surface of a flow cell having a gap of at least 200 μm. In some embodiments, the system includes a single objective lens, two tube lenses, and two image sensors, and each of the two tube lenses is designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the system includes a single objective lens, three tube lenses, and three image sensors, and each of the three tube lenses is designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the system includes a single objective lens, four tube lenses, and four image sensors, and each of the four tube lenses is designed to provide optimal imaging performance at a different fluorescence wavelength. In some embodiments, the design of the objective lens or at least one tube lens is configured to optimize the modulation transfer function in the medium to high spatial frequency range. In some embodiments, the imaging performance index includes a measurement of Modulation Transfer Function (MTF) in terms of one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof. In some embodiments, the difference in imaging performance index for imaging the upper and lower interior surfaces of the flow cell is less than 10%. In some embodiments, the difference in imaging performance index for imaging the upper and lower interior surfaces of the flow cell is less than 5%. In some embodiments, the use of at least one tube lens provides at least equivalent or better improvement in imaging performance index for duplex imaging than conventional systems including an objective lens, a motion actuated compensator, and an image sensor. In some embodiments, the use of at least one tube lens provides at least a 10% improvement in imaging performance index for duplex imaging as compared to conventional systems including an objective lens, a motion actuated compensator, and an image sensor.

Disclosed herein is an illumination system for imaging-based solid phase genotyping and sequencing applications, the illumination system comprising: a) A light source; b) A liquid light guide configured to collect light emitted by a light source and deliver the light to a designated illumination field on a carrier surface containing tethered biological macromolecules.

In some embodiments, the illumination system further comprises a condenser lens. In some embodiments, the designated illumination area has at least 2mm ² Is a part of the area of the substrate. In some embodiments, for an imaging system for acquiring an image of a surface of a carrier, the light delivered to a specified illumination field has a uniform intensity throughout a specified field of view. In some embodiments, the specified field of view has at least 2mm ² Is a part of the area of the substrate. In some embodiments, the light delivered to the designated illumination field has a uniform intensity throughout the designated field of view when the Coefficient of Variation (CV) of the light intensity is less than 10%. In some embodiments, when the Coefficient of Variation (CV) of light intensity is less than 5%, the light delivered to the designated illumination field has a uniform intensity throughout the designated field of view. In some embodiments, the light delivered to the specified illumination field has a speckle contrast value of less than 0.1. In some embodiments, the light delivered to the specified illumination field has a speckle contrast value of less than 0.05.

Also disclosed herein is a system for nucleic acid molecule sequencing, comprising: a) An optical system, comprising: (i) a first surface and an axially displaced second surface; (ii) an objective lens; and (iii) at least one image sensor, wherein the optical system has a refractive index greater than 1.0mm ² And configured to acquire images of the first surface and the axially displaced second surface; and b) a processor operatively coupled to the optical system and comprising a software program that processes the images of the first surface and the axially displaced second surface to correct for optical aberrations such that the images of the first surface and the axially displaced second surface have the same optical resolution, and detects a fluorescently labeled composition comprising a nucleic acid molecule or a complementary sequence thereof disposed on the first surface or the axially displaced second surface to determine the identity of a nucleotide in the nucleic acid molecule.

In some embodiments, the optical compensator is not movedThe images of the first surface and the axially displaced second surface are acquired into an optical path between the objective lens and the at least one image sensor. In some implementations, the images of the first surface and axially displaced second surface are acquired by refocusing only the optical system. In some embodiments, the imaging system has a numerical aperture greater than 0.3. In some embodiments, the first surface and the axially displaced second surface comprise two surfaces of a flow cell. In some embodiments, the two surfaces of the flow cell are coated with a hydrophilic coating, and wherein the hydrophilic coating further comprises a nucleic acid population of greater than 10,000 nucleic acid populations per mm ² Is provided, the surface density of the labeled nucleic acid population disposed thereon.

In some embodiments, the optical system comprises 1, 2, 3, or 4 imaging channels comprising a tube lens having a depth of field sufficient to detect nucleic acid populations disposed on at least one of the first surface or the axially displaced second surface that have been labeled with 1, 2, 3, or 4 different detectable markers. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the optical system is at least twice the optical resolution of the optical system. In some embodiments, the optical system comprises at least one tube lens positioned between the objective lens and the at least one image sensor, and wherein the at least one tube lens is configured to correct an imaging performance index for imaging the first surface and the axially displaced second surface, wherein the first surface and the axially displaced second surface are two inner surfaces of a flow cell. In some embodiments, the imaging system further comprises at least one tube lens that is an infinity corrected tube lens. In some embodiments, the flow cell has a wall thickness of at least 700 μm and the gap between the first surface and the axially displaced second surface is at least 50 μm. In some embodiments, the optical system includes two or more tube lenses designed to provide optimal imaging performance at two or more fluorescence wavelengths. In some embodiments, the optical system has a numerical aperture NA of less than 0.6.

Also disclosed herein is an imaging system comprising: a) A sample container comprising a surface having a plurality of attachment elements, a single oligonucleotide molecule attached to each of said attachment elements, and an average distance between adjacent ones of said attachment elements being less than an abbe limit; and b) an imager positioned to image light switching occurring at the plurality of attachment elements by capturing the on and off events in a plurality of color channels while the on and off events occur for the attached oligonucleotide molecules when random light switching chemistry is applied to the attached oligonucleotide molecules simultaneously such that the attached oligonucleotide molecules fluoresce in up to four different colors.

In some embodiments, the sample container comprises a flow cell comprising the plurality of attachment elements at a plurality of sample locations. In some embodiments, each of the plurality of attachment elements on the sample container is within a field of view of the imager for imaging the light switch such that capture of the on and off events occurs simultaneously for the attached oligonucleotide molecules at the plurality of attachment elements. In some embodiments, the random light switching chemistry applied simultaneously to all of the attached oligonucleotide molecules comprises random optical reconstruction microscopy, DNA spot accumulation for nanotopography imaging, or direct random optical reconstruction microscopy random light exchange chemistry. In some embodiments, the concentration of the agent used for the random light switching is sufficient such that the probability of an on event for a given base of a given molecule occurring simultaneously with an on event for the same base of a molecule adjacent to the given molecule is less than 0.5%. In some embodiments, the rate at which the on and off events occur is such that the probability of an on event for a given base of a given molecule occurring simultaneously with an on event for the same base of a molecule adjacent to the given molecule is less than the error rate determined in a sequencing application in which the method is applied.

In some embodiments, the imaging system further comprises determining whether the illumination intensity or spot size of the on event detected in the color channel is greater than a predetermined threshold. In some embodiments, the average distance between the adjacent elements is less than 20nm. In some embodiments, the surface comprises at least one hydrophilic polymer coating. In some embodiments, the plurality of attachment elements comprises branched hydrophilic polymers having at least 8 branches. In some embodiments, the imager is positioned to exhibit a contrast to noise ratio CNR of at least 40 for the optically switched imaging of the surface. In some embodiments, the imager is positioned to exhibit a contrast to noise ratio CNR of at least 60 for the optically switched imaging of the surface.

Also disclosed herein is a super resolution imaging system comprising: (a) an excitation light source; (b) depleting the light source; (c) an objective lens; (d) An optical path comprising an optical assembly that produces an array of regions, wherein each region comprises an active region comprising light from the excitation light source, the active region surrounded by a depletion region comprising light from the depletion light source; (e) At least one image sensor that receives and integrates signals from said areas over time and generates integrated signals for individual points illuminated by a single annular area; and (f) a processor programmed to determine fluorescence of the individual spots from the integrated signal.

In some embodiments, the super resolution imaging system further comprises at least one tube lens disposed in the optical path between the objective lens and the at least one image sensor. In some embodiments, the at least one tube lens includes an asymmetric convex-convex lens, a convex-flat lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens in that order. In some embodiments, the individual spots correspond to fluorescent nucleic acid molecules on a solid support. In some embodiments, each region is a circular ring region. In some embodiments, the excitation light source comprises an excitation laser for each region in the array and the depletion light source comprises a depletion laser for each region in the array, and wherein, for each region in the array of regions, the optical path is for directing light from the respective excitation laser and depletion laser to the annular region produced. In some embodiments, the at least one image sensor comprises an image sensor for each respective region in the array of regions.

In some embodiments, the optical path includes a deflector that directs light from the excitation light source and directs light from the depletion light source in a time-dependent manner to generate the array of regions. In some implementations, the optical path includes a phase mask to split light from the excitation light source into a plurality of excitation light beams and to split light from the depletion light source into a plurality of depletion light beams to produce an array of the regions. In some implementations, the optical path includes a waveguide to create a standing wave within the waveguide with the light from the depletion light source. In some implementations, the at least one image sensor includes a single image sensor to detect light from the sample. In some embodiments, the single image sensor comprises a multi-channel photon sensor. In some embodiments, the multichannel photon sensor comprises a CCD image sensor. In some embodiments, the regions in the array of regions are distributed in a grid comprising a plurality of rows and a plurality of columns. In some embodiments, the super resolution imaging system further comprises a scanning system configured to move the sample to move the array of regions relative to the sample.

Also disclosed herein is a super resolution imaging system comprising: (a) an excitation light source; (b) depleting the light source; (c) an objective lens; (d) An optical path comprising an optical assembly that produces a plurality of pattern areas, wherein each pattern area comprises excitation light from the excitation light source and depletion light from the depletion light source; and (e) at least one image sensor configured to receive and integrate signals from the fluorophores illuminated by the pattern region, and to generate fluorescence of the fluorophores based on the integrated signals.

In some embodiments, the super resolution imaging system further comprises at least one tube lens disposed in the optical path between the objective lens and the at least one image sensor. In some embodiments, the fluorophore has a dark state with a lifetime of greater than or equal to 100ms. In some embodiments, the fluorophore comprises a dye having an off state that is stable for at least 10 seconds, and in some embodiments, the dye comprises a rhodamine, oxazine, or carbocyanine dye. In some embodiments, the pattern region includes a first activated light region surrounded by a second depleted light region.

Also disclosed herein is an imaging system comprising: a) A flow cell comprising a first inner surface and a second inner surface, wherein the first inner surface or the second inner surface comprises at least 5 x 10 ⁸ A nucleic acid population coupled thereto; b) An objective lens; c) At least one image sensor configured to acquire an image of a nucleic acid colony; and d) an optical element disposed in the optical path between the objective lens and the at least one image sensor. In some embodiments, the imaging system has a numerical aperture NA of greater than 0.2 and greater than 1.0mm ² Is defined in the figure. In some embodiments, the optical element is configured to correct imaging performance such that images of the first inner surface of the flow cell and the second inner surface of the flow cell have the same optical resolution.

In some embodiments, the flow cell has a wall thickness of at least 700 μm and a fluid filled gap between the first inner surface and the second inner surface is at least 50 μm. In some embodiments, the optical element is an optical compensator. In some embodiments, the imaging system further comprises a field programmable gate array FPGA. In some embodiments, the FOV is greater than 2.0mm ² . In some embodiments, the optical resolution of the images of the first and second interior surfaces is diffraction limited over the entire FOV.

In some embodiments, the optical element is a motion actuated compensator. In some embodiments, the imaging system further comprises a focusing mechanism configured to refocus the imaging system between acquiring images of the first inner surface and the second inner surface. In some embodiments, the imaging system is configured to image two or more fields of view on at least one of the first inner surface or the second inner surface. In some embodiments, when the nucleic acid population is labeled with cyanine dye 3Cy3, the image of the first interior surface or the second interior surface acquired using the imaging system comprising a dichroic mirror and band pass filter set optimized for Cy3 emission exhibits a contrast to noise ratio CNR of at least 5, and the image is acquired under non-signal saturation conditions while immersing the first interior surface or the second interior surface in 25mm ACES ph7.4 buffer. In some embodiments, the imaging system further comprises 2 imaging channels configured to detect nucleic acid populations disposed on at least one of the first and second interior surfaces that have been labeled with 2 different detectable labels. In some embodiments, the imaging system is used to monitor sequencing by nucleotide incorporation reactions on at least one of the first and second interior surfaces and detect bound or incorporated nucleotide bases. In some embodiments, the imaging system is used to monitor sequencing by nucleotide base pairing reactions on at least one of the first and second interior surfaces and detect bound or incorporated nucleotide bases. In some embodiments, the imaging system is used to monitor sequencing by nucleotide binding reactions on at least one of the first inner surface and the second inner surface and detect bound or incorporated nucleotide bases. In some embodiments, the imaging system is used to monitor sequencing by affinity reaction on at least one of the first inner surface and the second inner surface and detect bound or incorporated nucleotide bases.

In some embodiments, the imaging system further comprises means for extracting a plurality of nucleic acid molecules from the sample for sequencing. In some embodiments, the at least one image sensor comprises pixels having a pixel size selected such that the spatial sampling frequency of the imaging system is at least twice the optical resolution of the imaging system. In some embodiments, the combination of the objective lens and the optical element is configured to optimize a modulation transfer function in a spatial frequency range from 700 cycles/mm to 1100 cycles/mm in the sample plane. In some embodiments, a nucleic acid population is covalently coupled to the first interior surface or the second interior surface. In some embodiments, the first inner surface of the flow cell is disposed on the optical path between the objective lens and the second inner surface of the flow cell. In some embodiments, the imaging system further comprises a liquid between the first inner surface and the second inner surface. In some embodiments, the flow cell is a capillary flow cell. In some embodiments, the capillary flow cell is a multi-lumen capillary flow cell.

In some embodiments, the flow cell comprises two or more flow channels. In some embodiments, at least one optical element is located in a permanent position between the objective lens and the at least one image sensor. The imaging system further comprises a first linker coupled to a first set of the nucleic acid populations, wherein the first linker is covalently coupled to the first inner surface. In some embodiments, the imaging system further comprises a fluorescence filter. In some embodiments, the population of nucleic acids to which it is covalently coupled comprises a label that is detectable by the imaging system. In some embodiments, the imaging system further comprises a fluorescence excitation filter configured to selectively transmit fluorescence emitted by a fluorescent label coupled to the covalently coupled nucleic acid population. In some embodiments, at least one of the first inner surface and the second inner surface comprises at least 1 x 10 covalently coupled thereto ⁹ And (3) each of the nucleic acid communities. In some embodiments, at least one of the first inner surface and the second inner surface comprises at least 5 x 10 covalently coupled thereto ⁹ And (3) individual said nucleic acid colonies.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in the incorporated reference, the term herein controls.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

fig. 1A-1B are schematic diagrams illustrating non-limiting examples of imaged dual surface carrier structures.

Fig. 2A-2B are views showing non-limiting examples of a multi-channel fluorescence imaging module including a dichroic beamsplitter.

Fig. 3A-3B are schematic diagrams illustrating the optical paths within the multi-channel fluorescence imaging module of fig. 2A and 2B.

Fig. 4 is a graph showing the relationship between dichroic filter performance and incident beam angle.

Fig. 5 is a diagram showing the relationship between beam footprint size and incident beam angle on a dichroic filter.

Fig. 6A-6B schematically illustrate example configurations of dichroic filters and detection channels of a multi-channel fluorescence imaging module.

Fig. 7 is a schematic diagram illustrating improved dichroic filter performance corresponding to the imaging module configuration shown in fig. 6A and 6B.

Fig. 8 is a schematic diagram illustrating improved dichroic filter performance corresponding to the imaging module configuration shown in fig. 6A and 6B.

Fig. 9A-1, 9A-2, 9B-1 and 9B-2 are schematic diagrams illustrating reduced surface deformations resulting from the imaging module configuration of fig. 6A and 6B.

Fig. 10A-10B are schematic diagrams showing improved excitation filter performance (e.g., steeper transitions between passband and surrounding stopband) due to the use of s-polarization of the excitation beam.

11A-11B are diagrams illustrating Modulation Transfer Functions (MTFs) of the exemplary dual-surface imaging systems disclosed herein having a Numerical Aperture (NA) of 0.3.

Fig. 12A-12B are schematic diagrams illustrating MTFs of exemplary dual-surface imaging systems disclosed herein having NA of 0.4.

Fig. 13A-13B are schematic diagrams illustrating MTFs of exemplary dual-surface imaging systems disclosed herein having NA of 0.5.

Fig. 14A-14B are schematic diagrams illustrating MTFs of exemplary dual-surface imaging systems disclosed herein having NA of 0.6.

Fig. 15A-15B are schematic diagrams illustrating MTFs of exemplary dual-surface imaging systems disclosed herein having NA of 0.7.

Fig. 16A-16B are schematic diagrams illustrating MTFs of exemplary dual-surface imaging systems disclosed herein having NA of 0.8.

17A-17B are schematic diagrams showing calculated Style ratios (Strehl ratios) for imaging a second flow cell surface through a first flow cell surface.

Fig. 18 provides a schematic diagram of a dual wavelength excitation/four-channel emission fluorescence imaging system of the present disclosure.

Fig. 19 provides a ray trace for an objective design designed to image the surface of the opposite side of a 0.17mm thick coverslip.

Fig. 20 provides a plot of the modulation transfer function of the objective lens shown in fig. 19 as a function of spatial frequency when used to image the surface of the opposite side of a 0.17mm thick coverslip.

Fig. 21 provides a plot of the modulation transfer function of the objective lens shown in fig. 19 as a function of spatial frequency when used to image the surface of the opposite side of a 0.3mm thick coverslip.

Fig. 22 provides a plot of the modulation transfer function as a function of spatial frequency for the objective lens shown in fig. 19 when used to image a surface of a 0.1mm thick aqueous fluid layer spaced from the surface on the opposite side of a 0.3mm thick coverslip.

Fig. 23 provides a plot of the modulation transfer function of the objective lens shown in fig. 19 as a function of spatial frequency when used to image the surface of the opposite side of a 1.0mm thick coverslip.

Fig. 24 provides a plot of the modulation transfer function as a function of spatial frequency for the objective lens shown in fig. 19 when used to image a surface of a 0.1mm thick aqueous fluid layer spaced from the surface on the opposite side of a 1.0mm thick coverslip.

Fig. 25 provides a ray trace for a tube lens design that provides improved duplex imaging through a 1mm thick coverslip if used in conjunction with the objective lens shown in fig. 19.

Fig. 26 provides a plot of modulation transfer function as a function of spatial frequency for the combination of objective and tube lenses shown in fig. 25 when used to image the surface of the opposite side of a 1.0mm thick coverslip.

Fig. 27 provides a plot of modulation transfer function as a function of spatial frequency for the combination of objective and tube lenses shown in fig. 25 when used to image a surface of a 0.1mm thick aqueous fluid layer spaced from the surface on the opposite side of a 1.0mm thick coverslip.

Fig. 28 is a ray trace for the tube lens design (left) of the present disclosure.

Fig. 29 illustrates one non-limiting example of a single capillary flow cell with 2 fluid adaptors.

Fig. 30 is a diagram showing one non-limiting example of a flow cell cartridge.

Fig. 31 is a diagram illustrating one non-limiting example of a system.

Fig. 32 is a diagram illustrating one non-limiting example of a system.

Fig. 33 is a diagram illustrating one non-limiting example of a system.

Fig. 34 illustrates one non-limiting example of controlling the temperature of a capillary flow cell by using a metal plate placed in contact with the flow cell cassette.

FIG. 35 illustrates one non-limiting method for temperature control of a capillary flow cell including a non-contact thermal control mechanism.

36A-36C illustrate non-limiting examples of flow cell device fabrication. Fig. 36A shows the preparation of a one-piece glass flow cell. Fig. 36B shows the preparation of a two-piece glass flow cell. Fig. 36C shows the preparation of a three-piece glass flow cell.

FIGS. 37A 1-37A-4, FIGS. 37B-1-37B-3, and FIGS. 37C-1-37C-4 illustrate non-limiting examples of glass flow cell designs. FIGS. 37A 1-37A-4 show a one-piece glass flow cell design. FIGS. 37B-1 through 37B-3 illustrate two-piece glass flow cell designs. FIGS. 37C-1 through 37C-4 show a three-piece glass flow cell design.

Fig. 38 illustrates visualization of cluster amplification in a capillary cavity.

FIG. 39 provides a non-limiting example of a block diagram of a sequencing system disclosed herein.

FIG. 40 provides a non-limiting example of a flow chart of the sequencing methods disclosed herein.

Fig. 41 provides a non-limiting example of a schematic diagram of the structured lighting system disclosed herein.

FIG. 42 provides a non-limiting example of a flow chart for acquiring and processing a structured illumination image of a flow cell surface as disclosed herein.

43A-43B provide non-limiting schematic diagrams of multiplexed read heads as disclosed herein.

44A-44B provide non-limiting schematic diagrams of multiplexed read heads as disclosed herein.

Detailed Description

There is a need for fluorescence imaging methods and systems that provide increased optical resolution and improved image quality for genomic applications, resulting in a corresponding improvement in genomic test accuracy. Disclosed herein are optical system designs for high performance fluorescence imaging methods and systems that can provide any one or more of improved optical resolution (including high performance optical resolution), improved image quality, and higher throughput for fluorescence imaging-based genomic applications. The disclosed optical illumination and imaging system designs may provide any one or more of the following advantages: improved dichroic filter performance, increased dichroic filter frequency response uniformity, improved excitation beam filtering, larger field of view, increased spatial resolution, improved modulation transfer, contrast-to-noise ratio and image quality, higher spatial sampling frequency, faster transitions between image captures when repositioning the sample plane to capture a series of images (e.g., images of different fields of view), improved imaging system duty cycle, and higher throughput image acquisition and analysis.

In some cases, such as for dual-sided (flow cell) imaging applications, including the use of thick flow cell walls (e.g., wall (or cover slip) thickness >700 μm and fluid channels (e.g., height or thickness of the fluid channels is 50-200 μm), improvements in imaging performance can be achieved using novel objective designs that correct for optical aberrations introduced by imaging the thick cover slip and/or surfaces on the opposite side of the fluid channels from the objective.

In some cases, such as for dual-sided (flow cell) imaging applications, including the use of thick flow cell walls (e.g., wall (or cover glass) thickness >700 μm and fluid channels (e.g., height or thickness of the fluid channels is 50-200 μm), improvements in imaging performance can be achieved even when using commercially available off-the-shelf objectives by using novel tube lens designs (other than tube lenses in conventional microscopes that simply form images on an intermediate image plane) that in combination with the objective correct for optical aberrations caused by the thick flow cell walls and/or intermediate fluid layers.

In some cases, such as for multi-channel (e.g., two-or four-color) imaging applications, improvement in imaging performance may be achieved by: a plurality of tube lenses are used, one tube lens for each imaging channel, wherein each tube lens design has been optimized for the specified wavelength range used in the imaging channel.

In some cases, such as for dual-sided (flow cell) imaging applications, improvement in imaging performance may be achieved by: an electro-optic phase plate is used in combination with an objective lens to compensate for optical aberrations caused by the fluid layers separating the upper (proximal) and lower (distal) inner surfaces of the flow cell. In some cases, the design method may also compensate for vibrations introduced by, for example, a motion actuated compensator that moves into or out of the optical path depending on which surface of the flow cell is imaged.

Various multi-channel fluorescence imaging module designs are disclosed that may include illumination and imaging optical paths including folded optical paths (e.g., including one or more beam splitters or combiners, such as dichroic beam splitters or combiners) that direct an excitation beam to an objective lens and direct emitted light transmitted through the objective lens to a plurality of detection channels. Some particularly advantageous features of the fluorescence imaging modules described herein include specifying dichroic filter incident angles that result in steeper and/or more uniform transitions between passband and stopband wavelength regions of the dichroic filter. Such filters may be included within the folding optics and may include dichroic beam splitters or beam combiners. Additional advantageous features of the disclosed imaging optics designs may include the position and orientation of one or more excitation light sources and one or more detection light paths relative to the objective lens and dichroic filters that receive the excitation light beams. The excitation light beam may also be linearly polarized, and the linear polarization may be oriented such that the s-polarized light is incident on the dichroic reflective surface of the dichroic filter. Such features can potentially improve excitation beam filtering and/or reduce wavefront errors introduced into the emitted light beam due to surface distortions of the dichroic filter. The fluorescence imaging modules described herein may or may not include any of these features, and may or may not include any of these advantages.

Also described herein are devices and systems configured to analyze a number of different nucleic acid sequences by imaging, for example, an array of immobilized nucleic acid molecules or amplified nucleic acid clusters formed on a flow cell surface. The devices and systems described herein can also be used, for example, to perform sequencing on comparative genomes, track gene expression, perform microrna sequence analysis, epigenomic, aptamer and phage display library characterization, and perform other sequencing applications. The devices and systems disclosed herein include various combinations of optical, mechanical, fluid, thermal, electrical, and computing devices/aspects. Advantages conferred by the disclosed flow cell devices, cartridges and systems include, but are not limited to: (i) reduces the manufacturing complexity and cost of the devices and systems, (ii) significantly reduces the consumable costs (e.g., compared to existing nucleic acid sequencing systems), (iii) compatibility with typical flow cell surface functionalization methods, (iv) flexible flow control when combined with microfluidic components (e.g., syringe pumps and diaphragm valves, etc.), and (v) flexible system throughput.

Disclosed herein are capillary flow cell devices and capillary flow cell cartridges constructed from off-the-shelf, disposable, single-lumen (e.g., single fluid flow channels) or multi-lumen capillaries, which may also include a fluid adapter, cartridge mount, one or more integrated fluid flow control assemblies, or any combination thereof. Also disclosed herein are capillary flow cell based systems, which may include one or more capillary flow cell devices (or microfluidic chips), one or more capillary flow cell cartridges (or microfluidic cartridges), a fluid flow controller module, a temperature control module, an imaging module, or any combination thereof.

Design features of some of the disclosed capillary flow cell devices, cartridges, and systems include, but are not limited to, (i) unitary flow channel configurations, (ii) sealed, reliable, and repeated switching between reagent flows, which can be accomplished by: a simple loading/unloading mechanism, thereby reliably sealing the fluid interface between the system and the capillary, thereby facilitating capillary replacement and system reuse, and enabling precise control of reaction conditions, such as reagent concentration, pH, and temperature; (iii) A replaceable single fluid flow channel device or capillary flow cell cartridge includes a plurality of flow channels that are interchangeable to provide flexible system throughput, and (iv) compatibility with multiple detection methods (e.g., fluorescence imaging).

Although the disclosed capillary flow cell devices and systems, capillary flow cell cartridges, capillary flow cell-based systems, microfluidic devices and cartridges, and microfluidic chip-based systems are described primarily in the context of their use in nucleic acid sequencing applications, the various aspects of the disclosed devices and systems may be applied not only to nucleic acid sequencing, but also to any other type of chemical, biochemical, nucleic acid, cellular or tissue analysis application. It should be understood that the various aspects of the disclosed methods, apparatus and systems may be understood individually, collectively, or in combination with each other. Although primarily discussed herein in the context of fluorescence imaging (including, for example, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, etc.), those skilled in the art will appreciate that many of the disclosed optical design methods and features are applicable to other imaging modes, e.g., bright-field imaging, dark-field imaging, phase-contrast imaging, etc.

Definition: unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to cover "and/or" unless otherwise indicated.

As used herein, the term "about" means that the number adds to or points to 10% of the number. The term "about" is used in the context of a range to mean that the range minus 10% of its minimum value and plus 10% of its maximum value.

As used herein, the phrases "imaging module," "imaging unit," "imaging system," "optical imaging module," "optical imaging unit," and "optical imaging system" are used interchangeably and may include components or subsystems of a larger system including, for example, a fluidic module, a temperature control module, a displacement platform, automated fluid dispensing and/or microplate processing, a processor or computer, instrument control software, data analysis and display software, and the like.

As used herein, the term "detection channel" refers to an optical path (and/or optical components therein) within an optical system configured to pass an optical signal generated from a sample to a detector. In some cases, the detection channel may be configured to perform a spectroscopy measurement, such as using a detector (e.g., photomultiplier tube) to monitor the fluorescent signal or other optical signal. In some cases, a "detection channel" may be an "imaging channel," i.e., an optical path (and/or optical components therein) within an optical system configured to capture an image and communicate the image to an image sensor.

As used herein, a "detectable label" may refer to any of a variety of detectable labels or tags known to those of skill in the art. Examples include, but are not limited to, chromophores, fluorophores, quantum dots, up-converting phosphors, luminescent or chemiluminescent molecules, radioisotopes, magnetic nanoparticles, mass labels, and the like. In some cases, the preferred label may include a fluorophore.

As used herein, the term "excitation wavelength" refers to the wavelength of light used to excite a fluorescent indicator (e.g., a fluorophore or dye molecule) and produce fluorescence. Although the excitation wavelength is typically specified as a single wavelength, e.g. 620nm, the skilled person will understand that this specification refers to a wavelength range centered around the specified wavelength or excitation filter bandpass. For example, in some cases, light of a specified excitation wavelength includes light of a specified wavelength.+ -. 2nm,.+ -. 5nm,.+ -. 10nm,.+ -. 20nm,.+ -. 40nm,.+ -. 80nm or greater. In some cases, the excitation wavelength used may or may not coincide with the maximum absorption peak of the fluorescent indicator.

As used herein, the term "emission wavelength" refers to the wavelength of light emitted by a fluorescent indicator (e.g., a fluorophore or dye molecule) upon excitation with light of the appropriate wavelength. Although the emission wavelength is typically designated as a single wavelength, e.g., 670nm, those skilled in the art will appreciate that this specification refers to a wavelength range centered around the designated wavelength or emission filter bandpass. In some cases, the light of the designated emission wavelength includes light of the designated wavelength.+ -. 2nm,.+ -. 5nm,.+ -. 10nm,.+ -. 20nm,.+ -. 40nm,.+ -. 80nm or greater. In some cases, the emission wavelength used may or may not coincide with the maximum emission peak of the fluorescent indicator.

As used herein, a fluorescent is "specific" if it originates from a fluorophore that anneals or otherwise binds to a surface, e.g., a fluorescently labeled nucleic acid sequence that has a region that is reverse-complementary to and anneals to a corresponding segment of an oligonucleotide adapter on a surface. This fluorescence is in contrast to fluorescence from fluorophores that are not tethered to the surface by such an annealing process, or in some cases, background fluorescence of the surface.

As used herein, a "nucleic acid" (also referred to as a "nucleic acid molecule," "polynucleotide," "oligonucleotide," ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is a linear polymer of two or more nucleotides, or variants or functional fragments thereof, linked by covalent internucleoside linkages. In the natural case of nucleic acids, the internucleoside linkage is typically a phosphodiester linkage. However, other examples optionally include other internucleoside linkages, such as phosphorothioate linkages, and may or may not include phosphate groups. Nucleic acids include double-and single-stranded DNA, as well as double-and single-stranded RNA, DNA/RNA hybrids, peptide Nucleic Acids (PNAs), hybrids between PNAs and DNA or RNA, and may also include other types of nucleic acid modifications.

As used herein, "nucleotide" refers to a nucleotide, nucleoside, or analog thereof. In some cases, the nucleotide is an N-or C-glycoside of a purine or pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose or a ribonucleoside containing D-ribose). Examples of other nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, and the like.

Fluorescence imaging considered as an information conduit: a useful generalization of the role played by fluorescence imaging systems in typical genomic assay techniques (including nucleic acid sequencing applications) is as an information conduit, where a photon signal enters one end of the conduit, such as an objective lens for imaging, and localization specific information about the fluorescence signal appears at the other end of the conduit, such as at the location of an image sensor. As more information is transferred through this pipe, it is inevitable that some content will be lost during this transfer and never recovered. An example of this is where too many labeled molecules (or clonally amplified clusters of molecules) are present in a small area of the substrate surface to be clearly resolved in the image; at the location of the image sensor, it is difficult to distinguish photon signals generated by neighboring molecular clusters, thereby increasing the probability of attributing signals to erroneous clusters and causing detection errors.

Optical imaging module design: the optical imaging module is thus designed with the aim of maximizing the flow of information content through the detection conduit and minimizing detection errors. A number of key design elements need to be addressed in the design process, including:

1) The density of physical features on the surface of the substrate to be imaged is matched to the overall image quality of the optical imaging system and the pixel sampling frequency of the image sensor used. Mismatch of these parameters may lead to information loss or sometimes even to erroneous information, e.g. spatial aliasing may occur when the pixel sampling frequency is below twice the optical resolution limit.

2) The size of the area to be imaged is matched to the overall image quality of the optical imaging system and the focus quality over the entire field of view.

3) The optical collection efficiency, modulation transfer function, and image sensor performance characteristics of the optical system design are matched to the expected fluorescent photon flux of the input excitation photon flux, dye efficiency (related to dye extinction coefficient and fluorescent quantum yield), while taking into account background signal and system noise characteristics.

4) Spectral content is separated to the greatest extent to reduce cross-talk between fluorescent imaging channels.

5) By repositioning the sample or optics between image captures of different fields of view, the image acquisition steps are effectively synchronized to minimize downtime of the imaging system (or maximize the duty cycle) and thereby maximize the overall throughput of the image capture process.

The present disclosure describes a system approach to addressing each of the design elements outlined above and creating component level specifications for an imaging system.

Improved optical resolution and image quality to improve or maximize information transmission and throughput: one non-limiting design practice may be to start with an optical resolution that is required for: two adjacent features specified in relation to the number of line pairs X/mm (lp/mm) are distinguished and converted into corresponding Numerical Apertures (NA). Numerical aperture requirements can then be used to evaluate the resulting effects on modulation transfer function and image contrast.

The standard Modulation Transfer Function (MTF) describes the spatial frequency response of the image contrast (modulation) transferred through the optical system; image contrast decreases as a function of spatial frequency and increases with increasing NA. This function limits the contrast/modulation that a given NA can achieve. In addition, wavefront errors can negatively impact the MTF, and thus the actual system MTF rather than diffraction limited optics predictions need to be used to improve or optimize the optical system design. Note that while design practices may primarily consider the MTF of the objective lens, as used herein, MTF refers to the overall system MTF (including the complete optical path from the coverslip to the image sensor).

In genomic testing applications, the target to be imaged is a high density "dot" array (random distribution or pattern) ON the surface, and the minimum modulation transfer value required for downstream analysis can be determined to resolve two adjacent dots and distinguish between four possible states (e.g., ON-OFF, ON-ON, OFF-ON, and OFF-OFF). For example, assume that the dots are small enough to approximate a point light source. It is assumed that the detection task is to determine whether two adjacent points separated by a distance d are ON or OFF (in other words, light or dark), and determine the contrast-to-noise ratio (CNR) of the fluorescence signal generated by a point at the sample plane (or object plane) as C _{Sample of} Then under ideal conditions the CNR C of the readout signal of two adjacent spots at the image sensor plane _{Image processing apparatus} Can be approximately about C _{Image processing apparatus} ＝C _{Sample of} * MTF (1/d), where MTF (1/d) is the MTF value at spatial frequency= (1/d).

In a typical design, the value of C may need to be at least 4, so that a simple thresholding method may be used to avoid misclassification of the fluorescent signal. Assuming that the Gaussian distribution of fluorescence signal intensities is around the average, at C _{Image processing apparatus} >4, the expected error in correctly classifying the fluorescent signal (e.g., ON or OFF) is <0.035%. Using proprietary high CNR sequencing and surface chemistry methods (e.g., as described in U.S. patent application No. 16/363,842), a sample plane CNR (C) of greater than 12 (or even higher) for clusters of clonally amplified, labeled oligonucleotide molecules tethered to a substrate surface can be achieved when measured for sparse fields (i.e., when the surface density of clusters or spots is low), where the value of MTF is near 100% _{Sample of} ) Values. Assume a sample plane CNR value C _{Sample of} >12, and target classification error rate<0.1% (hence, C) _{Image processing apparatus} >4) In some embodiments, the minimum value of M (1/d) may be determined to be M (1/d) =4/12-33%. Thus, a modulation transfer function threshold of at least 33% may be used to preserve the information content of the transferred image.

Design practices may relate the minimum separation distance d of two features or points to the optical resolution requirement (expressed as X (lp/mm) as described above), d= (1 mm)/X, i.e., d is the minimum separation distance between two features or points that can be fully resolved by the optical system. In some designs disclosed herein, where the goal of the design analysis is to increase or maximize relevant information transfer, then the design criteria may be relaxed to d= (1 mm)/X/a, where 2> a >1. For the same optical resolution X lp/mm, the value of d (minimum resolvable point separation distance at the sample plane) is reduced, thereby enabling the use of higher feature densities.

Design practice uses the nyquist criterion to determine the minimum spatial sampling frequency at the sample plane, where the spatial sampling frequency S ≡ 2*X (and where X is the optical resolution of the imaging system expressed as X lp/mm as described above). As the system spatial sampling frequency approaches the nyquist criterion (which is often the case), imaging system resolution greater than S can lead to aliasing because the optical sensor cannot adequately sample the higher frequency information resolved by the optical system.

In some designs disclosed herein, an oversampling scheme based on the relationship s=b×y (where b+.2 and Y is the true optical system MTF limit) can be used to further improve the information transfer capability of the imaging system. As described above, X (lp/mm) corresponds to the actual non-zero (> 33%) minimum modulation transfer value, while Y (lp/mm) is the limit of optical resolution, so the modulation is 0 at Y (lp/mm). Thus, in the disclosed design, Y (lp/mm) may advantageously be significantly greater than X. For values of B.gtoreq.2, the disclosed design is oversampled for sample object frequency X, i.e., S.gtoreq.B.gtY >2*X.

The above relationship may be used to determine the system magnification and may provide an upper limit for the image sensor pixel size. The selection of the image sensor pixel size is matched to the optical quality of the system and the spatial sampling frequency required to reduce aliasing. The lower limit of the image sensor pixel size may be determined based on the photon flux, as the relative noise contribution increases as the pixel becomes smaller.

However, other design methods are also possible. For example, reducing the NA to less than 0.6 (e.g., 0.5 or less) may provide increased depth of field. This increased depth of field may enable dual surface imaging, where two surfaces of different depths may be imaged simultaneously with or without refocusing. As described above, decreasing NA may decrease optical resolution. In some embodiments, the use of a higher excitation beam power, e.g., 1W or higher, may be employed to generate a strong signal. Inherently high contrast samples (i.e., including sample surfaces exhibiting strong foreground signals and significantly reduced background signals) may also be used to facilitate acquisition of high contrast to noise ratio (CNR) images, e.g., having CNR values >20, which provide improved signal discrimination for base detection in nucleic acid sequencing applications and the like. In some optical system designs disclosed herein, sample carrier structures (e.g., flow cells with hydrophilic surfaces) are used to reduce background noise.

In various embodiments, the disclosed optical system provides a large field of view (FOV). For example, some optical imaging systems including, for example, objective lenses and tube lenses may be provided with FOV greater than 2mm or 3 mm. In some cases, the optical imaging system provides reduced magnification, for example, less than 10x magnification. In some embodiments, this reduced magnification may facilitate large FOV designs. The optical resolution of such a system is still sufficient, although the magnification is reduced, since detector arrays with small pixel sizes or pitches can be used. In some implementations, an image sensor including a pixel size that is less than twice the optical resolution provided by an optical imaging system (e.g., objective lens and tube lens) may be used to satisfy the nyquist theorem.

Other designs are also possible. In some optical designs configured to provide dual-surface imaging, where two surfaces at different depths may be imaged simultaneously, an optical imaging system (e.g., objective and/or tube lens) is configured to reduce optical aberrations to image more of the two surfaces (e.g., two planes) at the two respective depths than at other locations (e.g., other planes) at other depths. In addition, the optical imaging system may be configured to reduce aberrations to image the two surfaces (e.g., two planes) at the two respective depths through a transmissive layer (e.g., a glass layer (e.g., a cover slip)) on the sample carrier structure and through a solution (e.g., an aqueous solution) containing or in contact with the sample on at least one of the two surfaces.

Multichannel fluorescence imaging module and system: in some cases, the imaging modules or systems disclosed herein may include a fluoroscopic imaging module or system. In some cases, the fluorescence imaging systems disclosed herein can include a single fluorescence excitation light source (for providing excitation light at a single wavelength or within a single excitation wavelength range) and an optical path configured to deliver excitation light to a sample (e.g., a fluorescently labeled nucleic acid molecule or cluster thereof disposed on a substrate surface). In some cases, the fluorescence imaging systems disclosed herein can include a single fluorescence emission imaging and detection channel, such as an optical path configured to collect fluorescence emitted by a sample and deliver an image of the sample (e.g., an image of a substrate surface having fluorescent-labeled nucleic acid molecules or clusters thereof disposed thereon) to an image sensor or other light detection device. In some cases, a fluorescence imaging system may include two, three, four, or more than four fluorescence excitation light sources and/or light paths configured to deliver excitation light at (or within) two, three, four, or more than four excitation wavelengths. In some cases, the fluorescence imaging systems disclosed herein can include two, three, four, or more than four fluorescence emission imaging and detection channels configured to collect fluorescence emitted by a sample at two, three, four, or more than four emission wavelengths (or within two, three, four, or more than four emission wavelength ranges) and deliver an image of the sample (e.g., an image of a substrate surface having fluorescent-labeled nucleic acid molecules or clusters thereof disposed thereon) to two, three, four, or more than four image sensors or other photodetecting devices.

Dual surface imaging: in some cases, the imaging systems disclosed herein, including fluorescence imaging systems, may be configured to acquire high resolution images of a single sample carrier structure or substrate surface. In some cases, the imaging systems disclosed herein, including fluorescence imaging systems, may be configured to acquire high resolution images of two or more sample carrier structures or substrate surfaces (e.g., two or more surfaces of a flow cell). In some cases, the high resolution images provided by the disclosed imaging systems can be used to monitor reactions (e.g., nucleic acid hybridization, amplification, and/or sequencing reactions) occurring on two or more surfaces of a flow cell as various reagents flow through the flow cell or around the flow cell matrix. Fig. 1A and 1B provide schematic diagrams of such a dual surface carrier structure for presenting a sample site for imaging by the imaging system disclosed herein. Fig. 1A: illustrations of imaging the front and rear interior surfaces of the flow cell. Fig. 1B: graphical representations of imaging of the anterior and posterior outer surfaces of a substrate. FIG. 1A shows a dual surface carrier structure, such as a flow cell, that includes internal flow channels through which analytes or reagents can flow. The flow channels may be formed between the first and second layers, the top and bottom layers, and/or the front and back layers, such as between the first and second plates, the top and bottom plates, and/or the front and back plates as shown. The one or more plates may comprise glass plates, such as coverslips, and the like. In some embodiments, the layer comprises borosilicate glass, quartz, or plastic. The inner surfaces of these top and bottom layers provide walls of the flow channels that help to restrict the flow of analytes or reagents through the flow channels of the flow cell. In some designs, these inner surfaces are flat. Similarly, the top and bottom layers may be flat. In some designs, at least one additional layer (not shown) is disposed between the top and bottom layers. The additional layer may have one or more channels cut therein that help define one or more flow channels and control the flow of analytes or reagents within the flow channels. Additional discussion of sample carrier structures (e.g., flow cells) can be found below.

FIG. 1A schematically illustrates a plurality of fluorescing sample sites on first and second interior surfaces, top and bottom interior surfaces, and/or front and back interior surfaces of a flow cell. In some embodiments, reactions may occur at these sites to bind the sample such that fluorescence is emitted from these sites (note that FIG. 1A is schematic and not drawn to scale; for example, the size and spacing of the fluorescing sample sites may be smaller than shown).

Fig. 1B shows another dual-surface carrier structure having two surfaces containing fluorescing sample sites to be imaged. The sample carrier structure comprises a matrix having a first outer surface and a second outer surface, a top outer surface and a bottom outer surface, and/or a front outer surface and a back outer surface. In some designs, these outer surfaces are flat. In various embodiments, the analyte or reagent flows over these first and second outer surfaces. FIG. 1B schematically illustrates a plurality of fluorescing sample sites on first and second outer surfaces, top and bottom outer surfaces, and/or front and back outer surfaces of a sample carrier structure. In some embodiments, reactions may occur at these sites to bind the sample such that fluorescence is emitted from these sites (note that FIG. 1B is schematic and not drawn to scale; for example, the size and spacing of the fluorescing sample sites may be smaller than shown).

In some cases, the fluorescence imaging modules and systems described herein may be configured to image such fluorescing sample sites on the first and second surfaces at different distances from the objective lens. In some designs, only one of the first surface or the second surface is focused at a time. Thus, in such designs, one of the surfaces is imaged at a first time and the other surface is imaged at a second time. The focal point of the fluorescence imaging module may be changed after imaging one of the surfaces to image the other surface with comparable optical resolution because the images of the two surfaces are not in focus at the same time. In some designs, an optical compensation element may be introduced into the optical path between the sample carrier structure and the image sensor to image one of the two surfaces. In such a fluoroscopic imaging configuration, the depth of field may not be large enough to include both the first surface and the second surface. In some embodiments of the fluorescence imaging modules described herein, both the first surface and the second surface may be imaged at the same time, i.e., simultaneously. For example, the fluorescence imaging module may have a depth of field large enough to include two surfaces. In some cases, such increased depth of field may be provided by, for example, reducing the numerical aperture of the objective lens (or microscope objective lens), as will be discussed in more detail below.

As shown in fig. 1A and 1B, imaging optics (e.g., an objective lens) may be positioned a suitable distance (e.g., a distance corresponding to a working distance) from the first surface and the second surface to form in-focus images of the first surface and the second surface on the image sensor of the detection channel. As shown in the examples of fig. 1A and 1B, the first surface may be between the objective lens and the second surface. For example, as shown, the objective lens is disposed over both the first surface and the second surface, and the first surface is disposed over the second surface. The first surface and the second surface are for example at different depths. The first surface and the second surface are at different distances from any one or more of the fluorescent imaging module, the illumination and imaging module, the imaging optics, or the objective lens. The first surface and the second surface are spaced apart from each other, the first surface being spaced above the second surface. In the example shown, the first and second surfaces are planar and are separated from each other along a direction perpendicular to the first and second planes. Also in the example shown, the objective lens has an optical axis, and the first and second surfaces are separated from each other in the direction of the optical axis. Similarly, the separation between the first surface and the second surface may correspond to a longitudinal distance, such as along the optical path of the excitation beam and/or along the optical axis through the fluorescent imaging module and/or the objective lens. Thus, the two surfaces may be separated from each other by a distance in a longitudinal (Z) direction, which may be along the central axis of the excitation beam and/or the direction of the optical axis of the objective lens and/or the fluorescence imaging module. In some embodiments, the spacing may correspond to a flow channel within a flow cell, for example.

In various designs, the objective lens (possibly in combination with another optical component, such as a tube lens) has a depth of field and/or depth of focus that is at least as large as the longitudinal spacing (in the Z-direction) between the first surface and the second surface. Thus, the objective lens may simultaneously form in-focus images of both the first surface and the second surface on the image sensor of the one or more detection channels, alone or in combination with further optical components, wherein these images have comparable optical resolution. In some embodiments, the imaging module may or may not need to refocus to capture images of both the first and second surfaces with comparable optical resolution. In some embodiments, the compensation optics need not be moved into or out of the optical path of the imaging module to form in-focus images of the first and second surfaces. Similarly, in some embodiments, one or more optical elements (e.g., lens elements) in the imaging module (e.g., objective lens and/or tube lens) need not be moved longitudinally, e.g., along the first optical path and/or the second optical path (e.g., along the optical axis of the imaging optics), to form a focused image of the first surface as compared to the position of the one or more optical elements when used to form a focused image of the second surface. However, in some embodiments, the imaging module includes an autofocus system configured to simultaneously focus the first surface and the second surface. In various embodiments, the samples are focused to sufficiently resolve sample sites that are closely spaced together in the lateral direction (e.g., X and Y directions). Thus, in various embodiments, no optical element enters the optical path between the sample carrier structure (e.g., between translation stages supporting the sample carrier structure) and the image sensor (or photodetector array) in the at least one detection channel to form an in-focus image of the fluorescing sample site on the first surface of the sample carrier structure and the second surface of the sample carrier structure. Similarly, in various embodiments, there is no optical compensation for forming a focused image of the fluorescing sample sites on the first surface of the sample carrier structure on the image sensor or photodetector array, as opposed to forming a focused image of the fluorescing sample sites on the second surface of the sample carrier structure on the image sensor or photodetector array. In addition, in certain embodiments, no optical element in the optical path between the sample carrier structure (e.g., between translation stages supporting the sample carrier structure) and the image sensor in the at least one detection channel is differently adjusted to form an in-focus image of the fluorescing sample site on the first surface of the sample carrier structure compared to forming an in-focus image of the fluorescing sample site on the second surface of the sample carrier structure. Similarly, in some various embodiments, no optical element in the optical path between the sample carrier structure (e.g., between translation stages supporting the sample carrier structure) and the image sensor in the at least one detection channel is moved a different amount or a different direction to form a focused image of the fluorescing sample site on the first surface of the sample carrier structure on the image sensor than the image sensor. Any combination of features is possible. For example, in some embodiments, in-focus images of the upper and lower interior surfaces of the flow cell may be obtained without moving an optical compensator into or out of the optical path between the flow cell and the at least one image sensor and without moving one or more optical elements (e.g., objective and/or tube lenses) of the imaging system along the optical path therebetween (e.g., optical axis). For example, in-focus images of the upper and lower interior surfaces of the flow cell may be obtained without moving one or more optical elements of the tube lens into or out of the optical path or without moving one or more optical elements of the tube lens along the optical path therebetween (e.g., the optical axis).

Any one or more of the fluorescence imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two locations (e.g., two planes corresponding to two surfaces on a flow cell or other sample carrier structure, e.g., where a fluorescing sample site is located). Any one or more of the fluorescence imaging module, illumination optical path, imaging optical path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at selected locations or planes relative to other locations or planes, such as at the first and second surfaces containing fluorescing sample sites on a dual surface flow cell. For example, any one or more of the fluorescent imaging module, illumination light path, imaging light path, objective lens, or tube lens may be designed to reduce or minimize optical aberrations at two depths or planes at different distances from the objective lens compared to aberrations associated with other depths or planes at other distances from the objective lens. For example, the optical aberrations for imaging the first and second surfaces may be smaller than elsewhere in the region ranging from about 1mm to about 10mm from the objective lens. Additionally, in some cases, any one or more of the fluorescent imaging module, the illumination light path, the imaging light path, the objective lens, or the tube lens may be configured to compensate for optical aberrations caused by transmission of the emitted light through one or more portions of the sample carrier structure (e.g., a layer comprising one of the surfaces on which the sample is attached and a solution that may be in contact with the sample). The layer (e.g., a cover slip or a wall of a flow cell) may comprise, for example, glass, quartz, plastic, or other transparent material having a refractive index and introducing optical aberrations.

Thus, the imaging performance may be substantially the same when imaging the first surface and the second surface. For example, the Optical Transfer Function (OTF) and/or Modulation Transfer Function (MTF) may be substantially the same for imaging of the first surface and the second surface. One or both of these transfer functions may be, for example, within 20%, within 15%, within 10%, within 5%, within 2.5% or within 1% of each other, or any range formed by any of these values, when averaged over one or more specified spatial frequencies or over a range of spatial frequencies. Thus, the imaging performance index may be substantially the same for imaging the upper or lower inner surface of the flow cell without moving an optical compensator in or out of the optical path between the flow cell and the at least one image sensor and without moving one or more optical elements (e.g., objective and/or tube lenses) of the imaging system along the optical path therebetween (e.g., optical axis). For example, the imaging performance index may be substantially the same for imaging the upper or lower interior surface of the flow cell without moving one or more optical elements of the tube lens into or out of the optical path or without moving one or more optical elements of the tube lens along the optical path therebetween (e.g., the optical axis). Additional discussion of MTF is contained below and in U.S. provisional application No. 62/962,723, filed on 1/17 of 2020, the entire contents of which are incorporated herein by reference.

Those skilled in the art will appreciate that in some cases, the disclosed imaging modules or systems may be stand-alone optical systems designed to image a sample or substrate surface. In some cases, they may include one or more processors or computers. In some cases, they may include one or more software packages that provide instrument control functions and/or image processing functions. In some cases, in addition to optical components such as light sources (e.g., solid state lasers, dye lasers, diode lasers, arc lamps, tungsten halogen lamps, etc.), lenses, prisms, mirrors, dichroic reflectors, beam splitters, optical filters, bandpass filters, light guides, optical fibers, apertures, and image sensors (e.g., complementary Metal Oxide Semiconductor (CMOS) image sensors and cameras, charge Coupled Device (CCD) image sensors and cameras, etc.), they may also include mechanical and/or opto-mechanical components such as X-Y translation stages, X-Y-Z translation stages, piezoelectric focusing mechanisms, electro-optic phase plates, etc. In some cases, they may serve as modules, components, sub-components, or subsystems of a larger system designed for, for example, genomic applications (e.g., genetic testing and/or nucleic acid sequencing applications). For example, in some cases they may serve as modules, components, sub-assemblies, or sub-systems for larger systems that also include light-tight and/or other environmental control enclosures, temperature control modules, flow cells and boxes, fluid control modules, fluid dispensing robots, cassette and/or microplate handling (pick-and-place) robots, one or more processors or computers, one or more local and/or cloud-based software packages (e.g., instrument/system control software packages, image processing software packages, data analysis software packages), data storage modules, data communication modules (e.g., bluetooth, wiFi, intranet or internet communication hardware and related software), display modules, and the like, or any combination thereof. These additional components of larger systems (e.g., systems designed for genomic applications) are discussed in more detail below.

Fig. 2A and 2B illustrate a non-limiting example of an illumination and imaging module 100 for multichannel fluorescence imaging, with a dichroic beam splitter for transmitting an excitation beam to a sample and receiving and redirecting the resulting fluorescence emissions by reflection to four detection channels configured to detect four different corresponding wavelengths or bands of fluorescence emissions. Fig. 2A: top isometric view. Fig. 2B: bottom isometric view. The illumination and imaging module 100 includes an objective lens 110, an illumination source 115, a plurality of detection channels 120, and a first dichroic filter 130, which may include a dichroic reflector or beam splitter. An autofocus system may be included in some designs, which may include an autofocus laser 102, for example, that projects a spot, the size of which is monitored to determine when the imaging system is in focus. Some or all of the components of the illumination and imaging module 100 may be coupled to the substrate 105.

The illumination or light source 115 may include any suitable light source (discussed in more detail below) configured to generate light of at least a desired excitation wavelength. The light source may be a broadband light source that emits light within one or more excitation wavelength ranges (or bands). The light source may be a narrow band light source that emits light in one or more narrower wavelength ranges. In some cases, the light source may produce a single isolated wavelength (or line) or multiple isolated wavelengths (or lines) corresponding to the desired excitation wavelength. In some cases, the line may have some very narrow bandwidth. Example light sources that may be suitable for use in illumination source 115 include, but are not limited to, incandescent filaments, xenon arc lamps, mercury vapor lamps, light emitting diodes, laser sources (e.g., laser diodes), or solid state lasers or other types of light sources. As described below, in some designs, the light source may include a polarized light source, such as a linearly polarized light source. In some embodiments, the light source is oriented such that s-polarized light is incident on one or more surfaces of the one or more optical components, such as the dichroic reflective surfaces of the one or more dichroic filters.

Illumination source 115 may also include one or more additional optical components, such as lenses, filters, optical fibers, or any other suitable transmissive or reflective optics, to output an excitation light beam having suitable characteristics to first dichroic filter 130. For example, beam shaping optics may be included to, for example, receive light from a light emitter in a light source and produce a beam of light and/or provide desired beam characteristics. Such optics may, for example, comprise a collimating lens configured to reduce divergence of light and/or to increase collimation and/or to collimate light.

In some embodiments, a plurality of light sources are included in the illumination and imaging module 100. In some such embodiments, different light sources may produce light having different spectral characteristics, e.g., to excite different fluorescent dyes. In some embodiments, light generated by different light sources may be directed to coincide and form a concentrated excitation beam. The composite excitation beam may be comprised of excitation beams from each light source. The composite excitation beam will have a greater optical power than the individual beams that overlap to form the composite beam. For example, in some embodiments including two light sources that generate two excitation light beams, a composite excitation light beam formed from two separate excitation light beams may have an optical power that is the sum of the optical powers of the separate light beams. Similarly, in some embodiments, three, four, five, or more light sources may be included, and each of these light sources may output an excitation beam that together form a composite beam having an optical power that is the sum of the optical powers of the respective beams.

In some embodiments, the light source 115 outputs a sufficiently large amount of light to produce a sufficiently intense fluorescent emission. Stronger fluorescence emission can increase the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of images acquired by the fluorescence imaging module. In some embodiments, the power of the light source and/or the output of the excitation light beam (including the composite excitation light beam) derived therefrom may range from about 0.5W to about 5.0W, or more (as will be discussed in more detail below).

Referring again to fig. 2A and 2B, the first dichroic filter 130 is disposed relative to the light source to receive light therefrom. The first dichroic filter may include a dichroic mirror, a dichroic reflector, a dichroic beam splitter, or a dichroic beam combiner configured to transmit light in a first spectral region (or wavelength range) and reflect light having a second spectral region (or wavelength range). The first spectral region may include one or more spectral bands, for example, in the ultraviolet and blue wavelength ranges. Similarly, the second spectral region may include one or more spectral bands, e.g., one or more spectral bands extending from green to red and infrared wavelengths. Other spectral regions or wavelength ranges are also possible.

In some embodiments, the first dichroic filter may be configured to transmit light from the light source to a sample carrier structure, such as a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or carrier structure. The sample carrier structure supports and positions a sample, such as a composition comprising fluorescently labeled nucleic acid molecules or their complementary sequences, relative to the illumination and imaging module 100. Thus, the first light path extends from the light source to the sample via the first dichroic filter. In various embodiments, the sample carrier structure comprises at least one surface on which the sample is disposed or to which the sample is bonded. In some cases, the sample may be disposed in or bound to: different localized areas or sites on at least one surface of the sample carrier structure.

In some cases, the carrier structure may include two surfaces that are at different distances from the objective lens 110 (i.e., at different positions or depths along the optical axis of the objective lens 110) on which the sample is disposed. As described below, for example, the flow cell may include a fluid channel formed at least in part by first and second (e.g., upper and lower) interior surfaces, and the sample may be disposed at a localized site on the first interior surface, the second interior surface, or both interior surfaces. The first and second surfaces may be separated by an area corresponding to the fluid channel through which the solution flows, and thus at different distances or depths relative to the objective lens 110 of the illumination and imaging module 100.

The objective lens 110 may be included in a first optical path between the first dichroic filter and the sample. The objective lens may be configured, for example, to have a focal length, a working distance, and/or be positioned to focus light from a light source onto a surface of a sample, such as a microscope slide, capillary, flow cell, microfluidic chip, or other substrate or carrier structure. Similarly, the objective lens 110 may be configured to have a suitable focal length, working distance, and/or be positioned to collect light reflected, scattered, or emitted from the sample (e.g., fluorescent emissions) and form an image of the sample.

(e.g., a fluorescence image).

In some embodiments, the objective 110 may comprise a microscope objective, such as an off-the-shelf objective. In some embodiments, the objective lens 110 may comprise a custom objective lens. Examples of custom objectives and/or custom objective-barrel lens combinations are described below and in U.S. provisional application No. 62/962,723 (incorporated herein by reference in its entirety) filed on 1 month 17 2020. The objective lens 110 may be designed to reduce or minimize optical aberrations at two locations, e.g., two planes corresponding to two surfaces of a flow cell or other sample carrier structure. The objective lens 110 may be designed to reduce optical aberrations at selected locations or planes (e.g., the first and second surfaces of a dual surface flow cell) relative to other locations or planes in the optical path. For example, the objective lens 110 may be designed to reduce optical aberrations at two depths or planes at different distances from the objective lens compared to optical aberrations associated with other depths or planes at other distances from the objective lens. For example, in some cases, the optical aberrations for the first and second surfaces of the imaging flow cell may be smaller than the aberrations shown elsewhere in the region spanning 1 to 10mm from the objective front surface. In addition, the custom objective 110 may in some cases be configured to compensate for optical aberrations caused by transmission of fluorescent emitted light through one or more portions of the sample carrier structure, such as a layer comprising one or more flow cell surfaces (on which the sample is disposed), or a layer comprising a solution filling a flow channel of the flow cell. These layers may comprise, for example, glass, quartz, plastic, or other transparent materials that have refractive indices and may introduce optical aberrations.

In some embodiments, the objective lens 110 may have a Numerical Aperture (NA) of 0.6 or greater (as discussed in more detail below). Such numerical apertures may provide reduced depth of focus and/or depth of field, improved background discrimination, and increased imaging resolution.

In some embodiments, the objective lens 110 may have a Numerical Aperture (NA) of 0.6 or less (as discussed in more detail below). Such a numerical aperture may provide increased depth of focus and/or depth of field. Such increased depth of focus and/or depth of field may increase the ability to image planes that are separated by a distance that separates, for example, the first surface and the second surface of the dual-surface flow cell.

As described above, the flow cell may comprise, for example, a first layer and a second layer comprising a first inner surface and a second inner surface, respectively, separated by a fluid channel through which an analyte or reagent may flow. In some embodiments, the objective 110 and/or the illumination and imaging module 100 may be configured to provide a sufficiently large depth of field and/or depth of focus to image the first and second inner surfaces of the flow cell sequentially (by refocusing the imaging module between imaging the first and second surfaces) or simultaneously (by ensuring a sufficiently large depth of field and/or depth of focus) with comparable optical resolution. In some cases, the depth of field and/or depth of focus may be at least equal to or greater than a distance separating a first surface and a second surface of a flow cell to be imaged (e.g., a first inner surface and a second inner surface of the flow cell). In some cases, the first and second surfaces, e.g., the first and second inner surfaces of a dual surface flow cell or other sample carrier structure, may be separated, e.g., by a distance in the range of about 10 μm to about 700 μm or more (as will be discussed in more detail below). Thus, in some cases, the depth of field and/or depth of focus may be in the range of about 10 μm to about 700 μm, or greater (as will be discussed in more detail below).

In some designs, compensation optics (e.g., an "optical compensator" or "compensator") may be moved into or out of an optical path in the imaging module through which light collected by, for example, the objective lens 110 is transferred to the image sensor to enable the imaging module to image the first and second surfaces of the dual surface flow cell. The imaging module may be configured to, for example, image the first surface when the compensation optics are included in the optical path between the objective lens and an image sensor or photodetector array configured to capture an image of the first surface. In such a design, the imaging module may be configured to image the second surface when compensation optics are removed or not included from the optical path between the objective lens 110 and the image sensor or photodetector array configured to capture the second surface. The need for an optical compensator may be more pronounced when using an objective lens 110 having a high Numerical Aperture (NA) value (e.g., for numerical aperture values of at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1.0, or higher). In some embodiments, the optical compensation optics (e.g., an optical compensator or compensator) includes a refractive optical element (e.g., a lens), a sheet of optically transparent material (e.g., glass), or a quarter-wave plate or half-wave plate in the case of polarized light beams, or the like. Other configurations may be employed to enable the first surface and the second surface to be imaged at different times. For example, one or more lenses or optical elements may be configured to move in and out or translate along an optical path between the objective lens 110 and the image sensor.

However, in some designs, the objective 110 is configured to provide a sufficiently large depth of focus and/or depth of field to enable the first and second surfaces to be imaged with comparable optical resolution without such compensating optics moving into and out of the optical path in the imaging module, e.g., between the objective and the image sensor or photodetector array. Similarly, in various designs, the objective 110 is configured to provide a sufficiently large depth of focus and/or depth of field to enable the first and second surfaces to be imaged with comparable optical resolution without moving optics, e.g., without translating one or more lenses or other optical components along an optical path in the imaging module (e.g., an optical path between the objective and an image sensor or photodetector array). Examples of such objectives are described in more detail below.

In some implementations, the objective lens (or microscope objective lens) 110 may be configured to have a reduced magnification. The objective lens 110 may be configured, for example, such that the fluorescent imaging module has a magnification of less than 2 times to less than 10 times (as will be discussed in more detail below). This reduced magnification may change the design constraints so that other design parameters may be implemented. For example, the objective lens 110 may also be configured such that the fluorescent imaging module has a large field of view (FOV), ranging, for example, from about 1.0mm to about 5.0mm (e.g., in diameter, width, length, or longest dimension), as will be discussed in more detail below.

In some embodiments, the objective lens 110 may be configured to provide the fluorescent imaging module with a field of view as described above such that the FOV has a diffraction limited performance, e.g., aberrations less than 0.15 waves in at least 60%, 70%, 80%, 90% or 95% of the field of view, as will be discussed in more detail below.

In some embodiments, the objective lens 110 may be configured to provide the fluorescent imaging module with a field of view as described above such that the FOV has a diffraction limited performance, e.g., a stehl ratio greater than 0.8 in at least 60%, 70%, 80%, 90% or 95% of the field of view, as will be discussed in more detail below.

Referring again to fig. 2A and 2B, a first dichroic beam splitter or beam combiner is arranged in the first optical path between the light source and the sample to illuminate the sample with one or more excitation light beams. The first dichroic beamsplitter or combiner is also in one or more second optical paths from the sample to different optical channels for detecting fluorescent emissions. Thus, the first dichroic filter 130 couples a first optical path of the excitation light beam emitted by the illumination source 115 and a second optical path of the emission light emitted by the sample specimen to respective optical channels in which the light is directed to respective image sensors or photo detector arrays for capturing images of the sample.

In various embodiments, the first dichroic filter 130 (e.g., a first dichroic reflector or beam splitter or beam combiner) has a passband selected to transmit light from the illumination source 115 only within a specified wavelength band or possibly within multiple wavelength bands (including the desired excitation wavelength (s)). For example, the first dichroic beamsplitter 130 includes a reflective surface having a dichroic reflector with a spectral transmittance response, i.e., configured to transmit light output by the light source having at least some wavelengths, which forms part of the excitation beam, for example. The spectral transmittance response may be configured to not transmit (e.g., but reflect) light of one or more other wavelengths, such as light of one or more other fluorescence emission wavelengths. In some implementations, the spectral transmittance response may also be configured to not transmit (e.g., but reflect) light of one or more other wavelengths output by the light source. Thus, the first dichroic filter 130 may be used to select which wavelength or wavelengths of light output by the light source reach the sample. In contrast, the dichroic reflector in the first dichroic beamsplitter 130 has a spectral reflectance response that reflects light having one or more wavelengths corresponding to the desired fluorescent emission from the sample and possibly reflects light output from the light source having one or more wavelengths that is not intended to reach the sample. Thus, in some embodiments, the dichroic reflector has a spectral transmittance comprising one or more pass bands for transmitting light to be incident on the sample; and one or more stop bands that reflect light outside of the pass band, e.g., light of one or more emission wavelengths, and possibly one or more wavelengths output by the light source that are not intended to reach the sample. Also, in some embodiments, the dichroic reflector has a spectral reflectance that includes one or more spectral regions (the one or more spectral regions are configured to reflect one or more emission wavelengths and possibly one or more wavelengths output by the light source that are not intended to reach the sample) and includes one or more regions that transmit light outside of these reflection regions. The dichroic reflector included in first dichroic filter 130 may include a reflective filter, such as an interference filter (e.g., a quarter-wavelength stack) configured to provide an appropriate spectral transmission and reflection profile. Fig. 2A and 2B also show a dichroic filter 105, which may comprise, for example, a dichroic beam splitter or combiner, which may be used to guide the autofocus laser 102 through the objective and to the sample carrier structure.

Although the imaging module 100 shown in fig. 2A and 2B and described above is configured such that the excitation light beam is transmitted by the first dichroic filter 130 to the objective 110, in some designs, the illumination source 115 may be arranged relative to the first dichroic filter 130 and/or the first dichroic filter may be configured (e.g., oriented) such that the excitation light beam is reflected by the first dichroic filter 130 to the objective 110. Similarly, in some such designs, the first dichroic filter 130 is configured to transmit fluorescent emissions from the sample, and possibly light of one or more wavelengths output from the light source that is not intended to reach the sample. As will be discussed below, designs that transmit, rather than reflect, fluorescent emissions may potentially reduce wavefront errors in the detected emissions and/or may have other advantages. In either case, in various embodiments, the first dichroic reflector 130 is disposed in the second optical path to receive fluorescent emissions from the sample, at least some of which continue to the detection channel 120.

Fig. 3A and 3B illustrate the optical path within the multi-channel fluorescence imaging module of fig. 2A and 2B, which includes a dichroic beam splitter for transmitting an excitation beam to a sample and receiving and redirecting the resulting fluorescence emissions by reflection to four detection channels for detecting the four different corresponding wavelengths or bands of fluorescence emissions. Fig. 3A: a top view. Fig. 3B: a side view. In the example shown in fig. 2A and 3A, the detection channel 120 is arranged to receive fluorescent emissions from the sample specimen, which are transmitted by the objective lens 110 and reflected by the first dichroic filter 130. As mentioned above and described in more detail below, in some designs, the detection channel 120 may be arranged to receive a portion of the emitted light transmitted by the first dichroic filter, rather than reflected. In either case, the detection channel 120 may include optics for receiving at least a portion of the emitted light. For example, the detection channel 120 may include one or more lenses, such as a tube lens, and may include one or more image sensors or detectors, such as a photodetector array (e.g., a CCD or CMOS sensor array) for imaging or otherwise generating signals based on received light. The tube lens may, for example, include one or more lens elements configured to form an image of the sample onto the sensor or photodetector array to capture an image thereof. Additional discussion of detection channels is contained below and in U.S. provisional application No. 62/962,723, filed on 1/17 of 2020, the entire contents of which are incorporated herein by reference. In some cases, in conjunction with appropriate sampling schemes (including over-sampling or under-sampling), improved optical resolution may be achieved using image sensors with relatively high sensitivity, small pixels, and high pixel counts.

Fig. 3A and 3B are ray traces showing the optical paths of the illumination and imaging module 100 of fig. 2A and 2B. Fig. 3A corresponds to a top view of the illumination and imaging module 100. Fig. 3B corresponds to a side view of the illumination and imaging module 100. The illumination and imaging module 100 shown in these figures includes four detection channels 120. However, it will be appreciated that the disclosed illumination and imaging modules may equally be implemented in a system comprising more or less than four detection channels 120. For example, the multi-channel system disclosed herein may be implemented with as few as one detection channel 120, or as many as two detection channels 120, three detection channels 120, four detection channels 120, five detection channels 120, six detection channels 120, seven detection channels 120, eight detection channels 120, or more than eight detection channels 120 without departing from the spirit or scope of the present disclosure.

The non-limiting example of the imaging module 100 shown in fig. 3A and 3B includes four detection channels 120, a first dichroic filter 130 (which reflects the emitted light beam 150), a second dichroic filter (e.g., a dichroic beam splitter) 135 (which splits the light beam 150 into a transmissive portion and a reflective portion), and two channel-specific dichroic filters (e.g., dichroic beam splitters) 140 (which further split the transmissive and reflective portions of the light beam 150 between the respective detection channels 120). The dichroic reflective surfaces in

dichroic beamsplitters

135 and 140 used to split beam 150 between detection channels are shown arranged at 45 degrees with respect to the central beam axis of beam 150 or the optical axis of the imaging module. However, as described below, angles less than 45 degrees may be employed and may provide advantages such as steeper transitions from passband to stopband.

The different detection channels 120 include an imaging device 124, and the imaging device 124 may include an image sensor or photodetector array (e.g., a CCD or CMOS detector array). The different detection channels 120 further comprise optics 126, such as a lens (e.g., one or more tube lenses, each comprising one or more lens elements), arranged to focus a portion of the emitted light entering the detection channels 120 on a focal plane coincident with the plane of the photodetector array 124. Optics 126 (e.g., a tube lens) in combination with objective 110 are configured to form an image of the sample on photodetector array 124 to capture an image of the sample, e.g., after the sample is bound to a surface on a flow cell or other sample carrier structure. Thus, such an image of the sample may comprise a plurality of fluorescence emission points or areas within the spatial extent of the sample carrier structure, wherein the sample is emitting fluorescence. The objective lens 110, along with optics 126 (e.g., a tube lens), may provide a field of view (FOV) that includes a portion of the sample or the entire sample. Similarly, the photodetector array 124 of the different detection channels 120 may be configured to capture an image of the full field of view (FOV) provided by the objective lens and tube lens or a portion thereof. In some embodiments, the photodetector array 124 of some or all of the detection channels 120 may detect emitted light emitted by a sample disposed on a surface of a sample carrier structure, such as a flow cell or a portion thereof, and record electronic data representing an image thereof. In some embodiments, the photodetector array 124 of some or all of the detection channels 120 can detect features in the emitted light emitted by the sample without capturing and/or storing an image of the sample disposed on the flow cell surface and/or an image of the full field of view (FOV) provided by the objective lens and optics 126 and/or 122 (e.g., elements of a tube lens). In some embodiments, the FOV of the disclosed imaging module (e.g., provided by the combination of objective lens 110 and optics 126 and/or 122) may be in the range between about 1mm and 5mm (e.g., in terms of diameter, width, length, or longest dimension), for example, as described below. The FOV may be selected, for example, to provide a balance between magnification and resolution of the imaging module and/or to be selected based on one or more characteristics of the image sensor and/or the objective lens. For example, a relatively smaller FOV may be provided in combination with smaller and faster imaging sensors to achieve high throughput.

Referring again to fig. 3A and 3B, in some embodiments, optics 126 (e.g., a tube lens) in the detection channel may be configured to reduce optical aberrations in an image acquired using optics 126 in conjunction with objective lens 110. In some embodiments including multiple detection channels for imaging at different emission wavelengths, the optics 126 (e.g., tube lenses) for the different detection channels have different designs to reduce aberrations for the individual emission wavelengths for which the particular channel is configured for imaging. In some implementations, the optics 126 (e.g., tube lens) may be configured to reduce aberrations when imaging a particular surface (e.g., plane, object plane, etc.) on a sample carrier structure including a fluorescing sample disposed thereon as compared to other locations (e.g., other planes in the object space). Similarly, in some embodiments, optics 126 (e.g., a tube lens) may be configured to reduce aberrations when imaging first and second surfaces (e.g., first and second planes, first and second object planes, etc.) on a dual-surface sample carrier structure (e.g., a dual-surface flow cell) having fluorescent sample sites disposed thereon, as compared to other locations (e.g., other planes in the object space). For example, optics 126 (e.g., tube lenses) in the detection channel may be designed to reduce aberrations at two depths or planes located at different distances from the objective lens compared to aberrations associated with other depths or planes located at other distances from the objective lens. For example, the optical aberrations for imaging the first and second surfaces may be smaller than elsewhere in the region about 1mm to about 10mm from the objective lens. Additionally, in some embodiments, the custom optics 126 (e.g., tube lens) in the detection channel may be configured to compensate for aberrations due to transmission of emitted light through one or more portions of the sample carrier structure, such as a layer including one of the surfaces on which the sample is disposed and possibly a solution adjacent to and in contact with the surface on which the sample is disposed. The layer comprising one of the surfaces on which the sample is arranged may comprise, for example, glass, quartz, plastic or other transparent material having a refractive index and introducing optical aberrations. For example, in some embodiments, custom optics 126 (e.g., a tube lens) in the detection channel may be configured to compensate for optical aberrations caused by the sample carrier structure (e.g., a cover slip or flow cell wall) or other sample carrier structure component and possibly the solution adjacent to and in contact with the surface on which the sample is disposed.

In some implementations, the optics 126 (e.g., tube lenses) in the detection channel are configured to have a reduced magnification. The optics 126 (e.g., tube lenses) in the detection channel may be configured, for example, such that the fluorescence imaging module has a magnification of less than, for example, 10 times, as will be discussed further below. This reduced magnification may change the design constraints so that other design parameters may be implemented. For example, optics 126 (e.g., a tube lens) may also be configured such that the fluorescent imaging module has a large field of view (FOV), e.g., of at least 1.0mm or more (e.g., in terms of diameter, width, length, or longest dimension), as will be discussed further below.

In some implementations, the optics 126 (e.g., tube lenses) may be configured to provide the fluorescent imaging module with a field of view as described above such that the FOV has less than 0.15 wave aberration in at least 60%, 70%, 80%, 90%, or 95% of the field of view, as will be discussed further below.

Referring again to fig. 3A and 3B, in various embodiments, the sample is located at or near the focal position 112 of the objective lens 110. As described above with reference to fig. 2A and 2B, a light source, such as a laser source, provides an excitation beam to the sample to induce fluorescence. The objective lens 110 collects at least a portion of the fluorescent emissions as emitted light. The objective lens 110 transmits the emitted light towards the first dichroic filter 130, which first dichroic filter 130 reflects a part or all of the emitted light as a light beam 150, which light beam 150 is incident on the second dichroic filter 135 and reaches different detection channels, each comprising an optical device 126, which forms an image of the sample (e.g. a plurality of fluorescing sample sites on the surface of the sample carrier structure) on the photodetector array 124.

As described above, in some embodiments, the sample carrier structure comprises a flow cell, such as a dual surface flow cell, having two surfaces (e.g., two inner surfaces, a first surface and a second surface, etc.) that contain sample sites that emit fluorescent emissions. The two surfaces may be separated from each other by a distance in the longitudinal (Z) direction along the direction of the central axis of the excitation beam and/or the optical axis of the objective lens. The separation may for example correspond to a flow channel within a flow cell. Analytes or reagents can flow through the flow channel and contact the first and second interior surfaces of the flow cell, such that they can be contacted with the binding composition such that fluorescent emissions are emitted from the plurality of sites on the first and second interior surfaces. Imaging optics (e.g., objective lens 110) may be positioned at a suitable distance from the sample (e.g., a distance corresponding to a working distance) to form an in-focus image of the sample on one or more detector arrays 124. As described above, in various designs, the objective lens 110 (possibly in combination with the optics 126) may have a depth of field and/or depth of focus at least as large as the longitudinal spacing between the first and second surfaces. Thus, the objective 110 (of each detection channel) and optics 126 may simultaneously form images of both the first flow cell surface and the second flow cell surface on the photodetector array 124, with the images of both the first and second surfaces being in focus and of comparable optical resolution (or focusing may be performed by only minor refocusing of the object to obtain images of both the first and second surfaces of comparable optical resolution). In various embodiments, the compensation optics need not be moved into or out of the optical path of the imaging module (e.g., into or out of the first optical path and/or the second optical path) to form in-focus images of the first surface and the second surface with comparable optical resolution. Similarly, in various embodiments, one or more optical elements (e.g., lens elements) in the imaging module (e.g., objective lens 110 or optics 126) need not be moved longitudinally, e.g., along the first optical path and/or the second optical path, to form a focused image of the first surface, as compared to the position of the one or more optical elements when used to form a focused image of the second surface. In some embodiments, the imaging module includes an autofocus system configured to rapidly and sequentially refocus the imaging module on the first surface and/or the second surface such that the image has comparable optical resolution. In some embodiments, the objective 110 and/or the optics 126 are configured such that both the first flow cell surface and the second flow cell surface are simultaneously in focus with comparable optical resolution without moving the optical compensator into or out of the first optical path and/or the second optical path, and without moving one or more lens elements (e.g., the objective 110 and/or the optics 126 (e.g., a tube lens)) longitudinally along the first optical path and/or the second optical path. In some embodiments, images of the first surface and/or the second surface acquired sequentially (e.g., by refocusing between the surfaces) or simultaneously (e.g., without refocusing between the surfaces) using the novel objective and/or tube lens designs disclosed herein may be further processed using suitable image processing algorithms to enhance the effective optical resolution of the images such that the images of the first surface and the second surface have comparable optical resolution. In various embodiments, the sample plane is sufficiently focused to resolve sample sites on the first flow cell surface and/or the second flow cell surface, the sample sites being closely spaced in the lateral direction (e.g., in the X and Y directions).

As described above, the dichroic filters may include interference filters that use optical coatings having different refractive indices and specific thicknesses to selectively transmit and reflect different wavelengths of light based on thin film interference principles. Thus, the spectral response (e.g., transmission and/or reflection spectra) of a dichroic filter implemented within a multichannel fluorescence imaging module may depend, at least in part, on the angle of incidence or range of angles of incidence of the excitation beam and/or the light of the emission beam onto the dichroic filter. Such an effect may be particularly pronounced with respect to the dichroic filters of the detection light path (e.g.,

dichroic filters

135 and 140 of fig. 3A and 3B).

Fig. 4 is a graph showing the relationship between dichroic filter performance and incident beam Angle (AOI). In particular, the graph of fig. 4 shows the effect of incident angle on the transition width or spectral span of a dichroic filter, which corresponds to the wavelength range in which the spectral response (e.g., transmission spectrum and/or reflection spectrum) transitions between the passband and stopband regions of the dichroic filter. Thus, a transmissive edge (or reflective edge) having a relatively small spectral span (e.g., a small Δλ value in the graph of fig. 4) corresponds to a steeper transition between the passband and the stopband region or between the transmissive and reflective regions (or vice versa), while a transmissive edge (or reflective edge) having a relatively large spectral span (e.g., a large Δλ value in the graph of fig. 4) corresponds to a less steep transition between the passband and the stopband region. In various embodiments, a steeper transition between passband and stopband regions is generally desirable. Furthermore, it may also be desirable to have an increased uniformity or relatively uniform transition width across all or most of the field of view and/or beam area.

Accordingly, a fluorescence imaging module in which the dichroic mirror is disposed at a 45 degree angle with respect to the central beam axis of the emitted light or the optical axis of the optical path (e.g., the optical path of the objective and/or tube lens) may have a transition width of about 50nm for an exemplary dichroic filter, as shown in fig. 4. Because the emitted light beam is not collimated and has some degree of divergence, the fluorescence imaging module may have a range of incidence angles of about 5 degrees between opposite sides of the light beam. Thus, as shown in fig. 4, different portions of the emitted light beam may be incident on the dichroic filter of the subchannel at various angles of incidence, 40 degrees to 50 degrees. The relatively large range of angles of incidence corresponds to a transition width range of about 40nm to about 62 nm. The relatively large range of angles of incidence thus results in an increase in the transition width of the dichroic filters in the imaging module. Thus, the performance of the multi-channel fluorescence imaging module can be improved by providing a smaller angle of incidence over the entire beam, thereby making the transmission edge steeper and allowing better differentiation between different fluorescence emission bands.

Fig. 5 is a graph showing the relationship between beam footprint size (DBS) and incident beam angle (DBS angle) on a dichroic filter. In some cases, a smaller beam footprint may be desirable. For example, a smaller beam footprint allows the use of smaller dichroic filters to split the beam into different wavelength ranges. The use of smaller dichroic filters in turn reduces manufacturing costs and increases the ease with which suitable flat dichroic filters can be manufactured. As shown in fig. 5, any angle of incidence greater than 0 degrees (perpendicular to the surface of the dichroic filter) results in an elliptical beam footprint with an area greater than the cross-sectional area of the beam. An angle of incidence of 45 degrees will result in a larger beam footprint on the dichroic reflector that is greater than 1.4 times the cross-sectional area of the beam at zero degrees of incidence.

Fig. 6A and 6B schematically illustrate a non-limiting example configuration of a dichroic filter and detection channels in a multi-channel fluorescence imaging module, where the dichroic filter has a reflective surface that is tilted such that the angle between the incident light beam (e.g., the central angle) and the reflective surface of the dichroic filter is less than 45. Fig. 6A: schematic of a multi-channel fluorescence imaging module comprising four detection channels. Fig. 6B: a detailed view of the angle of incidence (AOI) of the light beam on the dichroic mirror is shown. As shown, the dichroic mirror is disposed at an angle of less than 45 degrees with respect to the optical axis of the central beam axis or optical path of the emitted light (e.g., the optical path of the objective lens and/or tube lens). Fig. 6A depicts an imaging module 500 comprising a plurality of

detection channels

520a, 520b, 520c, 520 d. Fig. 6B is a detailed view of a portion of imaging module 500 within circle 5B as shown in fig. 6A. As will be described in more detail, the configuration shown in fig. 6A and 6B is comprised of a number of aspects that can lead to significant improvements over conventional multichannel fluorescence imaging module designs. However, in some cases, the fluorescence imaging modules and systems of the present disclosure may be implemented with one or a subset of the features described with respect to fig. 6A and 6B without departing from the spirit or scope of the present disclosure.

The imaging module 500 depicted in fig. 6A comprises an objective lens 510 and four

detection channels

520a, 520b, 520c and 520d, which four

detection channels

520a, 520b, 520c and 520d are arranged to receive and/or image the emitted light transmitted by the objective lens 510. A first dichroic filter 530 is provided to couple the excitation and detection light paths. In contrast to the designs shown in fig. 2A and 2B and fig. 3A and 3B, a first dichroic filter 530 (e.g., a dichroic beam splitter or combiner) is configured to reflect light from the light source to the objective 510 and the sample, and transmit fluorescent emissions from the sample to the

detection channels

520a, 520B, 520c, and 520d. The second dichroic filter 535 splits the emitted light beam between the at least two

detection channels

520a, 520b by transmitting the first portion 550a and reflecting the second portion 550 b. Additional

dichroic filters

540a, 540b are provided to further split the emitted light. Dichroic filter 540a transmits at least a portion of first portion 550a of the emitted light and reflects portion 550c to third detection channel 520c. Dichroic filter 540b transmits at least a portion of second portion 550b of the emitted light and reflects portion 550d to fourth detection channel 520d. Although imaging module 500 is depicted as having four detection channels, in various embodiments imaging module 500 may include more or fewer detection channels, with a correspondingly greater or lesser number of dichroic filters to provide a portion of the emitted light to each detection channel as appropriate. For example, in some embodiments, features of imaging module 500 may be implemented with similar advantageous effects in a simplified imaging module that includes only two

detection channels

520a, 520b and omits additional

dichroic filters

540a, 540 b. In some embodiments, only one detection channel may be included. Alternatively, three or more detection channels may be employed.

The

detection channels

520a, 520B, 520c, 520d shown in fig. 6A may include some or all of the same or similar components as those of the detection channel 120 shown in fig. 2A-3B. For example, the

different detection channels

520a, 520b, 520c, 520d may include one or more image sensors or photodetector arrays, and may include transmissive optics and/or reflective optics, such as one or more lenses (e.g., tube lenses), that focus light received by the detection channels onto their respective image sensors or photodetector arrays.

The objective lens 510 is arranged to receive the emitted light emitted from the sample by the fluorescence. In particular, the first dichroic filter 530 is arranged to receive the emitted light collected and transmitted by the objective 510. As discussed above and shown in fig. 6A, in some designs, an illumination source such as a laser source (e.g., illumination source 115 of fig. 2A and 2B) is arranged to provide an excitation beam incident on first dichroic filter 530 such that first dichroic filter 530 reflects the excitation beam into the same objective 510 that transmits the emitted light, e.g., in an epi-fluorescence configuration. In some other designs, the illumination source may be directed to the sample by other optical components along different optical paths that do not include the same objective 510. In such a configuration, the first dichroic filter 530 may be omitted.

Similarly, as discussed above and shown in fig. 6A, detection optics (e.g., including

detection channels

520a, 520b, 520c, 520d and any optical components, such as

dichroic filters

535, 540a, 540b, along the optical path between objective 510 and

detection channels

520a, 520b, 520c, 520 d) may be disposed on the transmission path of first dichroic filter 530, rather than on the reflection path of first dichroic filter 530. In one exemplary embodiment, the objective lens 510 and detection optics are arranged such that the objective lens 510 transmits the beam 550 of emitted light directly towards the second dichroic filter 535. By the presence of the first dichroic filter 530 along the path of the light beam 550 of emitted light, the wavefront quality of the emitted light may be slightly reduced (e.g., by applying some wavefront error to the light beam 550). However, the wavefront error introduced by the light beam transmitted through the dichroic reflector of the dichroic beamsplitter is typically much smaller (e.g., an order of magnitude smaller) than the wavefront error of the light beam reflected from the dichroic reflective surface of the dichroic beamsplitter. Thus, by placing the detection optics along the transmitted beam path of the first dichroic filter 530, rather than along the reflected beam path, the wavefront quality and subsequent imaging quality of the emitted light in the multi-channel fluorescence imaging module may be substantially improved.

Still referring to fig. 6A, in the detection optics of imaging module 500,

dichroic filters

535, 540a, and 540b are provided to split the beam 550 of emitted light between

detection channels

520a, 520b, 520c, 520 d. For example,

dichroic filters

535, 540a, and 540b split light beam 550 based on wavelength such that a first wavelength or wavelength band of emitted light may be received by first detection channel 520a, a second wavelength or wavelength band of emitted light may be received by second detection channel 520b, a third wavelength or wavelength band of emitted light may be received by third detection channel 520c, and a fourth wavelength or wavelength band of emitted light may be received by fourth detection channel 520 d. In some embodiments, the detection channel may receive multiple individual wavelengths or wavelength bands.

In contrast to the multi-channel fluorescence imaging module designs shown in fig. 2A and 2B and fig. 3A and 3B, imaging module 500 has

dichroic filters

535, 540a, and 540B arranged at an angle of incidence of less than 45 degrees with respect to the central beam axis of the incident light beam. As shown in fig. 6B, the

different beams

550, 550a, 550B have respective central beam axes 552, 552a, 554B. In various embodiments, the

central beam axis

552, 552a,552b is centered in a cross-section of the beam orthogonal to the direction of propagation of the beam. These central beam axes 552, 552a,552b may correspond to the optical axis of the objective lens and/or optics within separate channels, e.g., the optical axis of a respective tube lens.

Additional rays

554, 554a, 554B of each

beam

550, 550a, 550B are shown in fig. 6B to indicate the diameter of each

beam

550, 550a, 550B. The beam diameter may be defined as the full width of, for example, half the maximum diameter, d4σ (i.e., 4σ, where σ is the standard deviation of the horizontal or vertical edge distribution of the beam, respectively), or any other suitable definition of the second moment width or beam diameter.

The central beam axis 552 of the emitted beam 550 may be used as a reference point to define the angle of incidence of the beam 550 on the second dichroic filter 535. Thus, the "angle of incidence" (AOI) of a light beam 550 may be the angle between the central beam axis 552 of the incident light beam 550 and a line N perpendicular to the surface (e.g., dichroic reflective surface) on which the light beam is incident. When the beam 550 of emitted light is incident on the dichroic reflective surface of the second dichroic filter 535 at an incident angle AOI, the second dichroic filter 535 transmits a first portion 550a of the emitted light (e.g., a portion having wavelengths within the passband region of the second dichroic filter 535) and reflects a second portion 550b of the emitted light (e.g., a portion having wavelengths within the stopband region of the second dichroic filter 535). The first portion 550a and the second portion 550b may each be similarly described with respect to the

central beam axes

552a, 552 b. As described above, an optical axis may alternatively or additionally be used.

In fig. 6A and 6B, the second dichroic filter 535 is arranged such that the central beam axis 552 of the beam 550 is incident at an angle of incidence of 30 degrees. Similarly, the further

dichroic filters

540a, 540b are arranged such that the

central beam axes

552a, 552b of the first and

second portions

550a, 550b of the light beam 550 are also incident at an angle of incidence of 30 degrees. However, in various embodiments, these angles of incidence may be other angles less than 45 degrees. In some cases, for example, the angle of incidence may be in the range of about 20 degrees to about 45 degrees, as will be discussed further below. Moreover, the angle of incidence on each of the

dichroic filters

535, 540a, 540b need not be the same. In some embodiments, some or all of the

dichroic filters

535, 540a, 540b may be arranged such that their incident light beams 550, 550a, 550b have different angles of incidence. As described above, the angle of incidence may be relative to the optical axis of optics within the imaging module (e.g., objective lens and/or optics in the detection channel (e.g., tube lens)) and the dichroic reflective surfaces in the respective dichroic beamsplitter. The same range of incidence angles and values apply in the case when the optical axis is used to specify AOI.

The light beams 550, 550a, 550b of the emitted light in the fluorescence imaging module system are typically divergent light beams. As described above, the beam of emitted light can have a beam divergence that is large enough such that a beam region within the beam diameter is incident on the dichroic filter at an angle of incidence that differs by as much as 5 degrees or more with respect to the central beam axis and/or optical axis of the optics. In some designs, the objective lens 510 may be configured, for example, to have an f-number or numerical aperture selected to produce a smaller beam diameter for a given field of view of the microscope. In one example, the f-number or numerical aperture of objective lens 510 may be selected such that the entire diameter of

light beams

550, 550a, 550b is incident on

dichroic filters

535, 540a, 540b at an incident angle within, for example, 1 degree, 1.5 degrees, 2 degrees, 2.5 degrees, 3 degrees, 3.5 degrees, 4 degrees, 4.5 degrees, or 5 degrees of the incident angle of

central beam axis

552, 552a, 552 b.

In some embodiments, the focal length of an objective lens suitable for producing such a narrow beam diameter may be longer than those typically employed in fluorescent microscopes or imaging systems. For example, in some embodiments, the focal length of the objective lens may be in the range of 20mm to 40mm, as will be discussed further below. In one example, an objective lens 510 having a focal length of 36mm can produce a beam 550 characterized by a divergence that is sufficiently small that light across the diameter of the beam 550 is incident on the second dichroic filter 535 at an angle within 2.5 degrees of the angle of incidence of the central beam axis.

Fig. 7 and 8 provide graphs showing improved dichroic filter performance due to aspects of the imaging module configurations of fig. 6A and 6B (or any of the imaging module configurations disclosed herein). The graph in fig. 7 is similar to that of fig. 4 and shows the effect of the angle of incidence on the transition width of the dichroic filter (e.g., the spectral span of the transmissive edge). Fig. 7 shows an example in which dichroic filters (e.g.,

dichroic filters

535, 540a, and 540 b) and dichroic reflective surfaces therein are oriented such that their incident light beams have an incident angle of 30 degrees instead of 45 degrees. Fig. 7 shows how the reduced angle of incidence significantly improves the steepness and uniformity of the transition width across the beam diameter. For example, when a 45 degree angle of incidence at the central beam axis results in a transition width range of about 40nm to about 62nm, a 30 degree angle of incidence at the central beam axis results in a transition width range of about 16nm to about 30 nm. In this example, the average transition width decreases from about 51nm to about 23nm, indicating a steeper transition between the pass band and stop band. Moreover, the transition width variation across the beam diameter is reduced from the 22nm range to the 14nm range by nearly 40%, indicating a more uniform transition steepness across the beam region.

Fig. 8 illustrates additional advantages that may be realized by selecting an appropriate f-number or numerical aperture for the objective lens to reduce beam divergence in any of the imaging module configurations disclosed herein. In some embodiments, a longer focal length is used. In the example of fig. 8, the objective lens 510 has a focal length of 36mm with a suitable numerical aperture (e.g., less than 5) that reduces the range of angles of incidence within the beam 550 from 30 degrees ± 5 degrees to 30 degrees ± 2.5 degrees. With this design, the transition width can be reduced to a range of about 19nm to about 26nm. When compared to the improved system of fig. 7, the transition width variation across the beam diameter is further reduced to the 7nm range, indicating a reduction of approximately 70% relative to the transition width range shown in fig. 4, although the average transition width is substantially the same (e.g., spectral span is approximately 23 nm).

Referring again to fig. 5, reducing the angle of incidence from 45 degrees to 30 degrees at the central beam axis is further advantageous because it reduces the beam spot size on the dichroic filter. As in fig. 5, an angle of incidence of 45 degrees results in a beam footprint area on the dichroic filter that is greater than 1.4 times the beam cross-sectional area. However, an angle of incidence of 30 degrees results in an area of the beam footprint on the dichroic filter that is only about 1.15 times the cross-sectional area of the beam. Thus, reducing the angle of incidence at the

dichroic filters

535, 540a, 540b from 45 degrees to 30 degrees results in a reduction of the area of the beam footprint on the

dichroic filters

535, 540a, 540b by about 18%. The reduction in beam footprint area allows the use of smaller dichroic filters.

The schematic diagrams of fig. 9A-1, 9A-2, 9B-1, and 9B-2 illustrate reduced surface distortions caused by the imaging module configuration of fig. 6A and 6B. Fig. 9A-1 and 9A-2 show the effect of fold angle on image quality degradation due to the addition of 1 wave PV sphere power to the last mirror. 9B-1 and 9B-2 show the effect of fold angle on image quality degradation due to adding 0.1 wave PV spherical power to the last mirror referring now collectively to FIGS. 9A-1, 9A-2, 9B-1, 9B-2, the reduction of the angle of incidence from 45 degrees to 30 degrees may also provide improved performance with respect to surface distortions caused by dichroic filters in any of the imaging module configurations disclosed herein, as shown by the improvement in the modulation transfer function. In general, the amount of surface deformation increases with larger area optical elements. If a larger area is used on the dichroic filter, a larger amount of surface distortion is encountered, thereby introducing more wavefront error into the beam. Fig. 9A-1 and 9A-2 show the effect of fold angle on image quality degradation due to the addition of 1 Peak Valley (PV) spherical power to the last mirror. Fig. 9B-1 and 9B-2 show the effect of fold angle on image quality degradation due to the addition of 0.1 wave PV sphere power to the last mirror. As shown in fig. 9A-1, 9A-2, 9B-1, and 9B-2, reducing the incident angle to 30 degrees can significantly reduce the effect of surface deformation, thereby achieving diffraction limited performance of the proximity detection optics.

In some embodiments of the disclosed imaging modules, the polarization state of the excitation beam may be utilized to further improve the performance of the multichannel fluorescence imaging modules disclosed herein. Referring back to fig. 2A, 2B, and 6A, for example, some embodiments of the multi-channel fluorescence imaging module disclosed herein have an epi-fluorescence configuration in which a first

dichroic filter

130 or 530 combines the optical paths of the excitation and emission beams such that both excitation and emission light are transmitted through the

objective lens

110, 510. As described above, illumination source 115 may include a light source, such as a laser or other light source that provides light that forms an excitation beam. In some designs, the light source comprises a linearly polarized light source, and the excitation beam may be linearly polarized. In some designs, polarizing optics are included to polarize light and/or rotate the polarization of the light. For example, a polarizer (such as a linear polarizer) may be included in the optical path of the excitation beam to polarize the excitation beam. In some designs, a retarder (e.g., a half-wave retarder or a plurality of quarter-wave retarders or retarders with other amounts of retardation) may be included to rotate the linear polarization.

When a linearly polarized excitation beam is incident on any dichroic filter or other planar interface, it may be p-polarized (e.g., have an electric field component parallel to the plane of incidence), s-polarized (e.g., have an electric field component perpendicular to the plane of incidence), or may have a combination of p-polarized and s-polarized states within the beam. The p-polarization state or s-polarization state of the excitation light beam may be selected and/or changed by selecting the orientation of illumination source 115 and/or one or more components thereof relative to first

dichroic filters

130, 530 and/or relative to any other surface with which the excitation light beam is to interact. In some embodiments where the light source outputs linearly polarized light, the light source may be configured to provide s-polarized light. For example, the light source may comprise an emitter such as a solid state laser or a laser diode, which may be rotated about its optical axis or the central axis of the light beam to orient the linearly polarized light output therefrom. Alternatively or additionally, retarders may be employed to rotate the linear polarization about the optical axis or central axis of the beam. As described above, in some embodiments, a polarizer disposed in the optical path of the excitation beam may polarize the excitation beam, for example, when the light source does not output polarized light. For example, in some designs, a linear polarizer is disposed in the optical path of the excitation beam. The polarizer can be rotated to provide the proper orientation of linear polarization to provide s-polarized light.

In some designs, the linear polarization is rotated about the optical axis or central axis of the light beam such that the s-polarization is incident on the dichroic reflector of the dichroic beamsplitter. The transition between the pass band and stop band is steeper when s-polarized light is incident on the dichroic reflector of the dichroic beamsplitter, as opposed to when p-polarized light is incident on the dichroic reflector of the dichroic beamsplitter.

Fig. 10A-10B provide schematic diagrams illustrating improved excitation filter performance (e.g., steeper transitions between pass band and surrounding stop band) due to the use of s-polarization of the excitation beam. Fig. 10A: the transmission spectrum of an exemplary bandpass dichroic filter at angles of incidence of 40 degrees and 45 degrees, where the incident light beam is linearly polarized and p-polarized relative to the plane of the dichroic filter. Fig. 10B: the orientation of the light source relative to the dichroic filter is changed such that the incident light beam is s-polarized relative to the plane of the dichroic filter, resulting in substantially steeper edges between the pass band and the stop band. As shown in fig. 10A and 10B, using either the p-polarization state or the s-polarization state of the excitation beam can significantly affect the narrowband performance of any excitation filter (e.g., first dichroic filter 130, 530). Fig. 10A shows the transmission spectrum of an exemplary bandpass dichroic filter between 610nm and 670nm at angles of incidence of 40 degrees and 45 degrees, where the incident beam is linearly polarized and p-polarized with respect to the plane of the dichroic filter. As shown in fig. 10B, the orientation of the light source relative to the dichroic filter is changed such that the incident light beam is s-polarized relative to the plane of the dichroic filter, resulting in substantially steeper edges between the passband and stopband of the dichroic filter. Thus, the illumination and

imaging modules

100, 500 disclosed herein may advantageously have the illumination source 115 oriented relative to the first

dichroic filter

130, 530 such that the excitation light beam is s-polarized relative to the plane of the first

dichroic filter

130, 530. As described above, in some embodiments, a polarizer (such as a linear polarizer) may be used to polarize the excitation beam. The polarizer may be rotated to provide an orientation of linearly polarized light corresponding to s-polarized light. Also as described above, in some embodiments, other methods of rotating linearly polarized light may be used. For example, an optical retarder (such as a half-wave retarder or a variety of quarter-wave retarders) may be used to rotate the polarization direction. Other arrangements are also possible.

As discussed elsewhere herein, reducing the Numerical Aperture (NA) of the fluorescent imaging module and/or the objective lens may increase the depth of field to enable comparable imaging of both surfaces. 11A-11B illustrate Modulation Transfer Functions (MTFs) of the exemplary dual surface imaging systems disclosed herein having a Numerical Aperture (NA) of 0.3. Fig. 11A: a first surface. Fig. 11B: a second surface. Figures 11A-16B show how the MTF is more similar on a first surface and a second surface separated by 1mm glass for a smaller numerical aperture than for a larger numerical aperture.

Fig. 11A and 11B show MTFs at the first surface (fig. 11A) and the second surface (fig. 11B) for NA of 0.3.

Fig. 12A and 12B show MTFs at the first (fig. 12A) and second (fig. 12B) surfaces for NA of 0.4. Fig. 12A: a first surface. Fig. 12B: a second surface.

Fig. 13A and 13B show MTFs at 0.5 for NA at the first (fig. 13A) and second (fig. 13B) surfaces. Fig. 13A: a first surface. Fig. 13B: a second surface.

Fig. 14A and 14B show MTFs at 0.6 for NA at the first (fig. 14A) and second (fig. 14B) surfaces. Fig. 14A: a first surface. Fig. 14B: a second surface.

Fig. 15A and 15B show MTFs at the first (fig. 15A) and second (fig. 15B) surfaces for NA of 0.7. Fig. 15A: a first surface. Fig. 15B: a second surface.

Fig. 16A and 16B show MTFs at 0.8 for NA at the first (fig. 16A) and second (fig. 16B) surfaces. Fig. 16A: a first surface. Fig. 16B: a second surface. The first and second surfaces in each of these figures correspond to, for example, the top and bottom surfaces of the flow cell.

17A-17B provide graphs of calculated Style ratios (i.e., the ratio of peak light intensities focused or collected by an optical system to peak light intensities focused or collected by an ideal optical system and a point light source) for imaging a second flow cell surface through a first flow cell surface. Fig. 17A shows a plot of the stell ratio for imaging the second flow cell surface through the first flow cell surface as a function of intermediate fluid layer thickness (fluid channel height) for different objective lenses and/or optical system numerical apertures. As shown, the stehl ratio decreases as the spacing between the first and second surfaces increases. Thus, as the separation between the two surfaces increases, one of the surfaces will have a reduced image quality. The reduction in imaging performance of the second surface is reduced with increasing separation distance between the two surfaces for imaging systems having smaller numerical apertures than for imaging systems having larger numerical apertures. FIG. 17B shows a plot of the Style ratio as a function of numerical aperture for an intermediate aqueous layer of 0.1mm thickness and imaged through a first flow cell surface to a second flow cell surface. The loss of imaging performance at higher numerical apertures may be due to an increase in optical aberrations caused by the fluid used for second surface imaging. As NA increases, the increased optical aberrations introduced by the fluid used for second surface imaging can greatly reduce image quality. However, in general, reducing the numerical aperture of an optical system reduces the achievable resolution. By providing an increased sample plane (or object plane) contrast-to-noise ratio, the loss of image quality can be at least partially offset, for example, by using chemical reagents for nucleic acid sequencing applications that enhance fluorescent emission of labeled nucleic acid clusters and/or reduce background fluorescent emission. In some cases, for example, a sample carrier structure comprising a hydrophilic matrix material and/or a hydrophilic coating may be used. In some cases, such hydrophilic substrates and/or hydrophilic coatings may reduce background noise. Additional discussion of sample carrier structures, hydrophilic surfaces, and coatings, and methods for enhancing contrast-to-noise ratio (e.g., for nucleic acid sequencing applications) can be found below.

In some embodiments, any one or more of the fluorescent imaging system, the illumination and imaging module 100, the imaging optics (e.g., optics 126), the objective lens, and/or the tube lens are configured to have a reduced magnification, e.g., less than 10 times magnification, as will be discussed further below. This reduced magnification may adjust the design constraints so that other design parameters may be implemented. For example, any one or more of the fluorescence microscope, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens, or tube lens may also be configured such that the fluorescence imaging module has a large field of view (FOV), e.g., a field of view of at least 3.0mm or more (e.g., in terms of diameter, width, height, or longest dimension), as will be discussed further below. Any one or more of the fluorescence imaging system, illumination and imaging module 100, imaging optics (e.g., optics 126), objective lens, and/or tube lens may be configured to provide a fluorescence microscope with a field of view such that the FOV has less than, for example, 0.1 wave of aberration over at least 80% of the field of view. Similarly, any one or more of the fluoroscopic imaging system, the illumination and imaging module 100, the imaging optics (e.g., optics 126), the objective lens, and/or the tube lens may be configured such that the fluoroscopic imaging module has such a FOV and is diffraction limited, or diffraction limited on such FOV.

As described above, in various embodiments, the disclosed optical system provides a large field of view (FOV). In some embodiments, obtaining an increased FOV is facilitated in part by using a larger image sensor or photodetector array. The photodetector array may, for example, have an active area in which the diagonal is at least 15mm or greater, as will be discussed further below. As described above, in some embodiments, the disclosed optical imaging systems provide reduced magnification, e.g., less than 10 times magnification, which may facilitate large FOV designs. Despite the reduced magnification, the optical resolution of the imaging module may still be sufficient because detector arrays having small pixel sizes or pitches may be used. The pixel size and/or pitch may be, for example, about 5 μm or less, as will be discussed in more detail below. In some embodiments, the pixel size is less than twice the optical resolution provided by the optical imaging system (e.g., objective lens and tube lens) to meet the nyquist theorem. Thus, the pixel size and/or pitch of the image sensor may be such that the spatial sampling frequency of the imaging module is at least twice the optical resolution of the imaging module. For example, the spatial sampling frequency of the photodetector array may be at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, or at least 5 times the optical resolution of the fluorescence imaging module (e.g., illumination and imaging module, objective and tube lenses, objective and optics 126 in the detection channel, sample carrier structure or platform (configured to support the sample carrier platform), and imaging optics between the photodetector array), or any spatial sampling frequency in the range between any of these values.

Although a broad range of features are discussed herein with respect to fluorescence imaging modules, any of the features and designs described herein may be applied to other types of optical imaging systems, including but not limited to bright-field and dark-field imaging, and may be applied to luminescence or phosphorescence imaging.

Dual wavelength excitation/four-channel imaging system: fig. 18 shows a dual excitation wavelength/four channel imaging system for dual-sided imaging applications, comprising an objective lens and tube lens combination scanning in a direction perpendicular to the optical axis to provide large area imaging, for example by tiling multiple images to create a composite image, the total field of view (FOV) of which is much larger than each individual image. The system comprises two excitation light sources, e.g. lasers or laser diodes, operating at different wavelengths, and an autofocus laser. The two excitation beams and the autofocus laser beam are combined using a series of mirrors and/or dichroic reflectors and then delivered to the upper or lower inner surface of the flow cell by an objective lens. Fluorescence emitted by the labeled oligonucleotides (or other biomolecules) tethered to one of the flow cell surfaces is collected by the objective lens, transmitted through the tube lens, and directed to one of the four imaging sensors by a series of intermediate dichroic reflectors according to the wavelength of the emitted light. The autofocus laser light reflected from the flow cell surface is collected by the objective lens, transmitted through the tube lens, and directed to the autofocus sensor by a series of intermediate dichroic reflectors. The system allows the objective/tube lens combination to maintain accurate focus as it scans in a direction perpendicular to the optical axis of the objective (e.g., by adjusting the relative distance between the flow cell surface and the objective using a precision linear actuator, translation stage, or focus adjustment mechanism mounted on the turret of the microscope to reduce or minimize the reflected spot size on the autofocus image sensor). Dual wavelength excitation is used in combination with a four-channel (i.e., four wavelength) imaging capability to provide high throughput imaging of the upper (near) and lower (far) interior surfaces of the flow cell.

Multiplexing optical read heads:

in some cases, miniaturized versions of any of the imaging modules described herein may be assembled to create a multiplexed read head that can be horizontally translated in one or more directions relative to a sample surface (e.g., an inner surface of a flow cell) to simultaneously image portions of the surface. A non-limiting example of a multiplexed read head is described recently in U.S. published patent application 2020/013975 A1.

For example, in some cases, a miniaturized imaging module may include a "microfluorometer" that includes an illumination or excitation light source (e.g., an LED or laser diode) (or a tip of an optical fiber connected to an external light source), one or more lenses for collimating or focusing illumination or excitation light, one or more dichroic reflectors, one or more filters, one or more mirrors, beamsplitters, prisms, apertures, etc., one or more objective lenses, one or more custom tube lenses (for achieving duplex imaging with minimal focus adjustment, as described elsewhere herein), one or more image sensors, or any combination thereof, as described elsewhere herein. In some cases, miniaturized imaging modules (e.g., "microfluorometers") may also include autofocus mechanisms, microprocessors, power and data transmission connectors, light-tight housings, and the like. The resulting miniaturized imaging module may thus comprise an integrated imaging package or unit having a small form factor. In some cases, the shortest dimension (e.g., width or diameter) of the miniaturized imaging module may be less than 5cm, less than 4.5cm, less than 4cm, less than 3.5cm, less than 3cm, less than 2.5cm, less than 2cm, less than 1.8cm, less than 1.6cm, less than 1.4cm, less than 1.2cm, less than 1cm, less than 0.8cm, or less than 0.6cm. In some cases, the longest dimension (e.g., height or length) of the miniaturized imaging module may be less than 16cm, less than 14cm, less than 12cm, less than 10cm, less than 9cm, less than 8cm, less than 7cm, less than 5cm, less than 4.5cm, less than 4cm, less than 3.5cm, less than 3cm, less than 2.5cm, less than 2cm, less than 1.8cm, less than 1.6cm, less than 1.4cm, less than 1.2cm, or less than 1cm. In some cases, one or more individual miniaturized imaging modules within the multiplexed read head may include an autofocus mechanism.

In some cases, a multiplexed read head as described herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 miniaturized imaging modules or assemblies of microfluorometers held in fixed positions relative to each other. In some cases, the optical design specifications and performance attributes (e.g., numerical aperture, field of view, depth of field, image resolution, etc.) of each miniaturized imaging module or microfluorometer may be the same, as described elsewhere herein for other versions of the disclosed imaging module. In some cases, the plurality of individual miniaturized imaging modules may be arranged in a linear arrangement including 1, 2, 3, 4 rows and/or columns or more than 4 rows and/or columns. In some cases, the plurality of individual miniaturized imaging modules may be arranged in, for example, a hexagonal close-packed arrangement. In some cases, the plurality of individual miniaturized imaging modules may be arranged in a circular or spiral arrangement, a randomly distributed arrangement, or any other arrangement known to those skilled in the art.

43A-43B provide non-limiting schematic diagrams of multiplexed read heads as disclosed herein. FIG. 43A shows a side view of a multiplexed read head in which two rows of individual microfluorometers (as viewed from the end) having a common optical design specification (e.g., numerical aperture, field of view, working distance, etc.) are configured to image a common surface (e.g., a first interior surface of a flow cell). FIG. 43B shows a top view of the same multiplexed read head showing overlapping imaging paths acquired by the individual microfluorometers of the multiplexed read head as the multiplexed read head translates relative to the flow cell (and vice versa). In some cases, as shown in fig. 43B, the fields of view of the individual microfluorometers may overlap. In some cases, they may not overlap. In some cases, the multiplexed read head may be designed to align and image predetermined features (e.g., individual fluid channels) within the flow cell.

44A-44B provide a non-limiting schematic of a multiplexed read head in which a first subset of the plurality of individual miniaturized imaging modules is configured to image a first sample plane (e.g., a first interior surface of a flow cell) and a second subset of the plurality of individual miniaturized imaging modules is configured to simultaneously image a second sample plane (e.g., a second interior surface of a flow cell). FIG. 44A illustrates a side view of a multiplexed read head in which a first subset of individual microfluorometers is configured to image, for example, a first or upper interior surface of a flow cell and a second subset is configured to image a second surface (e.g., a second or lower interior surface of the flow cell). FIG. 44B illustrates a top view of the multiplexed read head of FIG. 44A showing imaging paths acquired by the individual microfluorometers of the multiplexed read head. Also, in some cases, the fields of view of the individual microfluorometers in a given subset may overlap. In some cases, they may not overlap. In some cases, the multiplexed read head may be designed such that each miniaturized imaging module of the first subset and the second subset is aligned with and images a predetermined feature (e.g., each fluid channel) within the flow cell.

Improved or optimized objective and/or tube lenses for thicker coverslips: existing design practices include the design of objective lenses and/or the use of commonly used off-the-shelf microscope objectives to optimize image quality when images are acquired through thin (e.g., <200 μm thick) microscope coverslips. When used to image both sides of a fluid channel or flow cell, the additional height of the gap between the two surfaces (i.e., the height of the fluid channel; typically about 50 μm to 200 μm) can introduce optical aberrations in the image captured for the non-optimal side of the fluid channel, resulting in reduced optical resolution. This is mainly because the additional gap height is significant compared to the optimal coverslip thickness (typical fluid channel or gap heights are 50-200 μm, relative to the coverslip thickness <200 μm). Another common design practice is to use an additional "compensation" lens in the optical path when imaging is to be performed on the non-optimal side of the flow channel or flow cell. The "compensation" lens and the mechanisms required to move it in and out of the optical path (so that both sides of the flow cell can be imaged) further increase system complexity and imaging system downtime, and may reduce image quality due to vibrations and the like.

In the present disclosure, the imaging system is designed to be compatible with flow cell consumables that include thicker coverslips or flow cell walls (thickness. Gtoreq.700 μm). The objective design can be improved or optimized for a coverslip equal to the true coverslip thickness plus half the effective gap thickness (e.g., 700 μm +1/2 x fluid channel (gap) height). This design significantly reduces the effect of gap height on the image quality of the two surfaces of the fluid channel and balances the optical quality of the images of the two surfaces because the gap height is small relative to the total thickness of the coverslip and therefore has a reduced effect on the optical quality.

Other advantages of using thicker coverslips include improved control of thickness tolerance errors during manufacturing and reduced likelihood of coverslips deforming from thermal and installation induced stresses. Thickness errors and deformations of the cover slip can adversely affect the imaging quality of both the top and bottom surfaces of the flow cell.

To further improve the quality of dual surface imaging for sequencing applications, our optical system design focuses on improving or optimizing the MTF (e.g., by improving or optimizing objective and/or tube lens designs) over a medium to high spatial frequency range that is best suited for imaging and resolving small spots or clusters.

An improved or optimized tube lens design for use in combination with commercially available off-the-shelf objectives: for low cost sequencer designs, it is preferable to use off-the-shelf objectives, which are commercially available, because of their relatively low price. However, as mentioned above, low cost off-the-shelf objectives are optimized primarily for thin coverslips with a thickness of about 170 μm. In some cases, the disclosed optical system may utilize a tube lens design that compensates for thicker flow cell coverslips while enabling high image quality for both interior surfaces of the flow cell in dual surface imaging applications. In some cases, the tube lens designs disclosed herein are capable of high quality imaging of both inner surfaces of a flow cell without moving an optical compensator into or out of the optical path between the flow cell and an image sensor, without moving one or more optical elements or components of the tube lens along the optical path, and without moving one or more optical elements or components of the tube lens into or out of the optical path.

Fig. 19 provides an optical ray trace for a low-light objective design that has been improved or optimized to image surfaces on opposite sides of a 0.17mm thick coverslip. The modulation transfer function diagram of the objective (shown in fig. 20) represents near diffraction limited imaging performance when used with a coverslip designed for a thickness of 0.17 mm.

Fig. 21 provides a plot of modulation transfer function as a function of spatial frequency for the same objective lens shown in fig. 19 when used to image a surface on the opposite side of a 0.3mm thick coverslip. The relatively small deviation in MTF values over the spatial frequency range of about 100 lines/mm to about 800 lines/mm (or periods/mm) suggests that the quality of the image obtained is reasonable even when using a cover slip of 0.3mm thickness.

Fig. 22 provides a plot of modulation transfer function as a function of spatial frequency for the same objective lens shown in fig. 19 when used to image a surface of an aqueous fluid layer 0.1mm thick spaced from the surface on the opposite side of a 0.3mm thick coverslip (i.e., under conditions encountered in duplex imaging of a flow cell when imaging a far surface). As can be seen from the graph of fig. 22, the deviation of the MTF curve from the ideal diffraction-limited case over the spatial frequency range of about 50lp/mm to about 900lp/mm indicates a decrease in imaging performance.

Fig. 23 and 24 provide graphs of modulation transfer functions as a function of spatial frequency for the upper (or near) inner surface (fig. 23) and the lower (or far) inner surface (fig. 24) of the flow cell when imaged through a 1.0mm thick coverslip using the objective lens shown in fig. 19 and when the upper and lower inner surfaces are separated by a 0.1mm thick aqueous fluid layer. It can be seen that the imaging performance of both surfaces is greatly reduced.

Fig. 25 provides a ray trace for a tube lens design that provides improved duplex imaging through a 1mm thick coverslip if used in conjunction with the objective lens shown in fig. 19. The optical design 700 comprising composite objectives (

lens elements

702, 703, 704, 705, 706, 707, 708, 709, and 710) and tube lenses (

lens elements

711, 712, 713, and 714) is improved or optimized for use with a flow cell comprising a thick cover slip (or wall), e.g., greater than 700 μm thick, and a fluid channel thickness of at least 50 μm, and transmitting an image of an inner surface from the flow cell 701 to an image sensor 715, wherein optical image quality is significantly improved and CNR is higher.

In some cases, a tube lens (or tube lens assembly) may include at least two optical lens elements, at least three optical lens elements, at least four optical lens elements, at least five optical lens elements, at least six optical lens elements, at least seven optical lens elements, at least eight optical lens elements, at least nine optical lens elements, at least ten optical lens elements, or more, wherein the number of optical lens elements, the surface geometry of each element, and their placement order in the assembly are improved or optimized to correct for optical aberrations caused by flow cell thick walls, and in some cases, allow one to use commercially available off-the-shelf objectives while still maintaining high quality bifacial imaging capabilities.

In some cases, as shown in fig. 25, the tube lens assembly may include a first asymmetric convex-convex lens 711, a second convex-flat lens 712, a third asymmetric concave-concave lens 713, and a fourth asymmetric convex-concave lens 714 in this order.

Fig. 26 and 27 provide graphs of modulation transfer functions as a function of spatial frequency for the upper (or near) inner surface (fig. 26) and the lower (or far) inner surface (fig. 27) of the flow cell when imaged through a 1.0mm thick coverslip using an objective lens (corrected for a 0.17mm coverslip) and the tube lens combination shown in fig. 25, and when the upper and lower inner surfaces are separated by a 0.1mm thick aqueous fluid layer. It can be seen that the imaging performance obtained is almost expected for diffraction-limited optical designs.

Fig. 28 provides a ray trace (left) for the tube lens design of the present disclosure that has been improved or optimized to provide high quality dual sided imaging performance. Since the tube lens is no longer infinity corrected, a properly designed zero lens (right) can be used in combination with the tube lens to compensate for the tube lens for manufacturing and testing purposes rather than infinity correction.

Adaptation or optimization of imaging channel specific tube lenses: in imaging system designs, it is possible to improve or optimize both the objective lens and the tube lens in the same wavelength region for all imaging channels. Typically, the same objective lens is shared by all imaging channels (see, e.g., fig. 18), and each imaging channel uses either the same tube lens or has tube lenses sharing the same design.

In some cases, the imaging systems disclosed herein may also include a tube lens for each imaging channel, where the tube lens has been independently improved or optimized for a particular imaging channel to improve image quality, e.g., to reduce or minimize distortion and field curvature, and to improve depth of field (DOF) performance for each channel. Since the wavelength range (or bandpass) of each particular imaging channel is much narrower than the combined wavelength range of all channels, the wavelength or channel-specific adaptation or optimization of the tube lenses used in the disclosed systems results in significant improvements in imaging quality and performance. This channel-specific adaptation or optimization results in an improvement of the image quality of the top and bottom surfaces of the flow cell in a duplex imaging application.

Double sided imaging without fluid in the flow cell: in order to provide optimal imaging performance for both the top and bottom interior surfaces of the flow cell, a motion actuated compensator is typically required to correct for optical aberrations caused by the fluid in the flow cell (typically comprising a fluid layer thickness of about 50-200 μm). In some cases of the disclosed optical system designs, the top interior surface of the flow cell may be imaged in the presence of fluid in the flow cell. Once the sequencing chemistry cycle is complete, fluid can be extracted from the flow cell to image the bottom interior surface. Therefore, in some cases, the image quality of the bottom surface can be maintained even without using a compensator.

Compensating for optical aberrations and/or vibrations using an electro-optic phase plate: in some cases, by using an electro-optic phase plate (or other corrective lens) in combination with an objective lens to eliminate optical aberrations due to the presence of fluid, dual surface image quality may be improved without removing fluid from the flow cell. In some cases, an electro-optic phase plate (or lens) may be used to eliminate vibration effects caused by mechanical motion of the motion-actuated compensator, and may provide faster image acquisition times and sequencing cycle times for genome sequencing applications.

Improved contrast to noise ratio (CNR), field of view (FOV), spectral separation, and timing design to increase or maximize information transmission and flux: another approach for increasing or maximizing information transfer in imaging systems designed for genomic applications is to increase the size of the field of view (FOV) and reduce the time required to image a particular FOV. For a typical large NA optical imaging system, it is common to obtain an area of approximately 1mm ² Wherein in the presently disclosed imaging system design a large FOV objective lens with a long working distance is specified to achieve a 2mm for ² Or larger areas.

In some cases, the disclosed imaging systems are designed for use in combination with proprietary low-binding substrate surfaces and DNA amplification methods that reduce fluorescent background caused by a variety of confounding signals, including but not limited to non-specific adsorption of fluorescent dyes to substrate surfaces, non-specific nucleic acid amplification products (e.g., nucleic acid amplification products present on substrate surfaces in regions between spots or features corresponding to clonally amplified clusters of nucleic acid molecules (i.e., specifically amplified clones), non-specific nucleic acid amplification products that may be present in amplified clones, nucleic acid strands prior to phasing and phasing, and the like). The use of low-binding substrate surfaces and DNA amplification methods (which reduce fluorescent background) in combination with the disclosed optical imaging system can significantly reduce the time required to image each FOV.

The presently disclosed system design may further reduce the required imaging time by imaging sequence improvement or optimization, wherein fluorescence images of multiple channels are acquired simultaneously or in overlapping timing, and wherein spectral separation of fluorescence signals is designed to reduce cross-talk between fluorescence detection channels and between excitation light and fluorescence signals.

The presently disclosed system design may further reduce the required imaging time by improving or optimizing the scan motion sequence. In a typical method, the object FOV is moved to a position below the objective lens using an X-Y translation stage, an autofocus step is performed (where the best focus position is determined), then the objective lens is moved to the determined focus position in the Z direction, and then an image is acquired. A series of fluoroscopic images are acquired by cycling through a series of target FOV positions. From the point of view of the information transmission duty cycle, the information is transmitted only during the fluorescent image acquisition portion of the cycle. In the presently disclosed imaging system design, a single step motion is performed in which all axes (X-Y-Z) are repositioned simultaneously, and an autofocus step is used to check for focus position errors. An additional Z motion command is issued only if the focus position error (difference between the focus plane position and the sample plane position) exceeds a certain limit, e.g. a specified error threshold. In combination with the high speed X-Y motion, this approach increases the duty cycle of the system, thereby increasing the imaging throughput per unit time.

Furthermore, by matching the designed optical collection efficiency, modulation transfer function, and image sensor performance characteristics with the expected fluorescent photon flux of the input excitation photon flux, dye efficiency (related to dye extinction coefficient and fluorescent quantum yield), while taking into account background signal and system noise characteristics, the time required to acquire high quality (high contrast to noise ratio (CNR) images) can be reduced or minimized.

The combination of efficient image acquisition with improved or optimized translation stage step size and settling time results in fast imaging times (i.e., total time required per field of view) and higher throughput imaging system performance.

With a large FOV and fast image acquisition duty cycle, the disclosed designs may also include specified image plane flatness, color focusing performance between fluorescence detection channels, sensor flatness, image distortion, and focus quality specifications.

By aligning the image sensors to different fluorescence detection channels, respectively, such that the best focal planes of each detection channel overlap, color focusing performance may be further improved. The design goal is to ensure that images over more than 90% of the field of view are acquired within ±100nm (or less) relative to the optimal focal plane of each channel, thereby increasing or maximizing the transmission of single point intensity signals. In some cases, the disclosed designs also ensure that images over 99% of the field of view are acquired within ±150nm (or less) relative to the optimal focal plane for each channel, and that more images over the entire field of view can be acquired within ±200nm (or less) relative to the optimal focal plane for each imaging channel.

Designing an illumination light path: another factor for improving signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and/or increasing flux is increasing the illumination power density of the sample. In some cases, the disclosed imaging system may include an illumination path design that utilizes a high power laser or laser diode coupled with a liquid light guide. The liquid light guide eliminates the speckle inherent to coherent light sources such as lasers and laser diodes. Furthermore, the coupling optics are designed as an entrance aperture for the underfill liquid light guide. The underfill of the liquid light guide into the aperture reduces the effective numerical aperture of the illumination beam entering the objective, thereby improving the light transmission efficiency through the objective onto the sample plane. With this design innovation, the illumination power density can be three times that of conventional designs over a large field of view (FOV).

In some cases, by utilizing angle-dependent discrimination of s-polarization and p-polarization, the illumination beam polarization can be oriented to reduce the amount of backscattered and back-reflected illumination light reaching the imaging sensor.

Structured illumination system: in some cases, the disclosed imaging modules and systems can include structured illumination optics designs to increase the effective spatial resolution of the imaging system, enabling the use of higher surface densities of clonally amplified target nucleic acid sequences (clusters) on the flow cell surface to increase sequencing throughput. Structured Illumination Microscopes (SIMs) utilize spatially structured (i.e., periodic) light patterns to illuminate the sample plane and rely on the generation of interference patterns known as moire fringes. Multiple images are acquired under slightly different illumination conditions, for example by shifting and/or rotating the pattern of structured illumination, to produce moire fringes. The mathematical deconvolution of the resulting interference signal can reconstruct a super-resolution image with a spatial resolution up to about a double improvement over using diffraction-limited imaging optics [ Lutz (2011), "Biological Imaging by Superresolution Light Microscopy", Comprehensive Biotechnology (second) A plate), volume 1, pages 579-589, elsevier; feiner-Gracia, et al (2018), "15-Advanced Optical Microscopy Techniques for the Investigation of Cell-Nanoparticle Interactions",Smart Nanoparticles for Biomedicine: Micro and Nano Technologiespages 219-236, elsevier; nylk, et al (2019), "Light-Sheet Fluorescence Microscopy With Structured Light",Neurophotonics and Biomedical Spectroscopypages 477-501, elsevier]. An example of a structured illumination microscope imaging system is recently described in U.S. patent application publication 2020/0218052 to Hong.

Fig. 41 provides a non-limiting schematic view of an imaging system 4100, the imaging system 4100 comprising a branched structured illumination optical design as disclosed herein. A first branch (or arm) of the illumination light path of system 4100 includes, for example, a light source (light emitter) 4110A, an optical collimator 4120A for collimating light emitted by light source 4110A, a diffraction grating 4130A in a first orientation relative to the optical axis, a swivel window 4140A, and a lens 4150A. The second branch of the illumination light path of system 4100 includes, for example, a light source 4110B, an optical collimator 4120B for collimating light emitted by light source 4110B, a diffraction grating 4130B in a second orientation relative to the optical axis, a rotating window 4140B, and a lens 4150B. The

diffraction gratings

4130A and 4130B may project a pattern of light fringes onto the sample plane.

In some cases,

light sources

4110A and 4110B may be incoherent light sources (e.g., comprising one or more Light Emitting Diodes (LEDs)) or coherent light sources (e.g., comprising one or more lasers or laser diodes). In some examples,

light sources

4110A and 4110B may include optical fibers coupled to, for example, LEDs, lasers, or laser diodes that output light beams that are subsequently collimated by

respective collimating lenses

4120A and 4120B. In some cases,

light sources

4110A and 4110B may output light of the same wavelength. In some cases,

light sources

4110A and 4110B may output light of different wavelengths.

Light sources

4110A and 4110B may each be configured to output light of any of the wavelengths and/or wavelength ranges described elsewhere herein. During imaging, the

light sources

4110A and 4110B may be turned on or off using, for example, a high-speed shutter (not shown) located on the optical path or by pulsing the light sources at a predetermined frequency.

In the example shown in fig. 41, the first illumination arm of system 4100 comprises a fixed vertical grating 4130A for projecting a grating pattern (e.g., a vertical light stripe pattern) onto a sample plane (e.g., a first inner surface 4188 of flow cell 4187) in a first orientation, and the second illumination arm comprises a fixed horizontal grating 4130B for projecting a grating pattern (e.g., a horizontal light stripe pattern) onto the sample plane 4188 in a second orientation. Advantageously, in this non-limiting example, the diffraction grating of the imaging system 4100 does not require mechanical rotation or translation during imaging, which may provide improved imaging speed, system reliability, and system repeatability. In some cases, the diffraction gratings 4130A and/or 4130B may be rotated about their respective optical axes such that the angle between the light fringe patterns projected onto the sample plane is adjustable.

As shown in fig. 41, in some cases, the

diffraction gratings

4130A and 4130B may be transmissive diffraction gratings that include a plurality of diffraction elements (e.g., parallel slits or grooves) formed in a glass substrate or other suitable surface. In some cases, the grating may be implemented as a phase grating that provides a periodic variation of the refractive index of the grating material. In some cases, the grooves or feature spacing may be selected to cause light to be diffracted at a suitable angle and/or adjusted to the smallest resolvable feature size of the imaged sample for operation of the imaging system 4100. In other cases, the diffraction grating may be a reflective diffraction grating.

In the example shown in fig. 41, the orientations of the vertical and horizontal light stripe patterns are offset by about 90 degrees. In other cases, other orientations of the diffraction grating may be used to produce an offset of about 90 degrees. For example, the diffraction gratings may be oriented such that they project a pattern of light fringes that are offset ±45 degrees from the x-axis or y-axis of the sample plane (e.g., the first inner flow cell surface) 4188. Where the sample carrier surface (e.g., the inner surface 4188 of the flow cell 4187) includes regularly patterned features arranged on a rectangular grid, the configuration of the imaging system 4100 shown in fig. 41 may be particularly advantageous because the use of structured illumination methods to increase image resolution may be achieved using only two perpendicular grating orientations (e.g., a vertical grating orientation and a horizontal grating orientation).

In an example of system 4100,

diffraction gratings

4130A and 4130B may be configured to diffract an input illumination beam into a series of intensity maxima due to constructive interference according to the following relationship:

m=order=dsin (θ)/λ

Where d=the distance between slits or grooves in the diffraction grating, θ=the angle of incidence of the illumination light with respect to the normal of the diffraction grating surface, λ=the wavelength of the illumination light, and m=an integer value corresponding to the maximum of the intensity of the diffracted light, e.g. m=0, ±1, ±2, etc. In some cases, a particular order of diffracted illumination light, e.g., first order (m= ±1) light, may be projected onto a sample plane, e.g., inner flow cell surface 4188. In some cases, for example, the vertical grating 4130A may diffract the collimated light beam into a first order diffracted light beam (±1 order) that is focused onto the sample plane in a first orientation, and the horizontal grating 4130B may diffract the collimated light beam into a first order diffracted light beam that is focused onto the sample plane in a second orientation. In some cases, the zero order beam and/or all other higher order beams (e.g., m= ±2 or higher) may be blocked, i.e., filtered out of the illumination pattern projected onto the sample plane 4188, using, for example, a beam blocking element (not shown) (e.g., an order filter) that may be inserted into the optical path after the diffraction grating.

In the example of 4100, each branch of the structured illumination system includes an optical phase modulator or

phase shifter

4140A and 4140B to phase shift the diffracted light transmitted or reflected by each of the

diffraction gratings

4130A and 4130B. During structured imaging, the optical phase of each diffracted beam may be offset by some fraction (e.g., 1/2, 1/4, etc.) of the pitch (X) of each stripe of the structured pattern. In the example of fig. 41, the

phase modulators

4140A and 4140B may be implemented as, for example, rotating optical phase plates actuated by a rotary actuator or other actuator mechanism to rotate and modulate the optical path length of each diffracted beam. For example, the optical phase plate 4140A may be rotated about a vertical axis to move the image projected by the vertical grating 4130A on the sample plane 4188 left and right, and the optical phase plate 4140B may be rotated about a horizontal axis to move the image projected by the horizontal grating 4130B on the sample plane 4188 in a vertical direction.

In other embodiments, other types of phase modulators that change the optical path length of the diffracted light may be used (e.g., wedges mounted on a linear translation stage, etc.). In addition, although the

optical phase modulators

4140A and 4140B are illustrated as being placed after the

diffraction gratings

4130A and 4130B, in other embodiments they may be placed at other locations in the illumination path. In some cases, a single optical phase modulator may operate in two different directions to produce different patterns of light fringes, or a single motion may be used to adjust the position of the single optical phase modulator to adjust the optical path lengths of both arms of the illumination optical path simultaneously.

In the example shown in fig. 41, an optical assembly 4160 may be used to combine light from the two illumination light paths. The optical assembly 4160 may include, for example, partially silvered mirrors, dichroic mirrors (depending on the wavelength of the light output by the

light sources

4110A and 4110B), mirrors containing a pattern of holes or a patterned reflective coating that allows the light from the two arms of the illumination system to combine in a non-destructive or near-non-destructive manner (e.g., without significant light power loss other than a small absorption by the reflective coating), polarizing beamsplitters (where the

light sources

4110A and 4110B are configured to produce polarized light), and the like. The optical assembly 4160 may be positioned so as to spatially resolve the desired diffraction orders of the light reflected or transmitted by each diffraction grating and block unwanted orders of light. In some cases, the optical assembly 4160 may pass the first order light output by the first illumination light path and reflect the first order light output by the second illumination light path. In some cases, the structured illumination pattern on the sample surface 4188 may be switched from a vertical orientation (e.g., using diffraction grating 4130A) to a horizontal orientation (e.g., using diffraction grating 4130B) by turning on or off each light source, or by turning on and off a shutter in the light path of the light source. In other cases, the structured illumination pattern may be switched by using an optical switch to change the illumination light path used to illuminate the sample plane.

Referring again to fig. 41, a lens 4170, a half mirror or dichroic mirror 4180, and an objective lens 4185 can be used to focus the structured illumination light onto a sample surface 4188 (e.g., a first inner surface of a flow cell 4187). Light emitted, reflected, or scattered by the sample surface 4188 is then collected by the objective lens 4185, transmitted through the mirror 4180, and imaged by the image sensor or camera 4195. As described, the mirror 4180 may be a dichroic mirror for reflecting structured illumination light received from each branch of the illumination light path into the objective 4185 for projection onto the sample plane 4188 and passing light emitted by the sample plane 4188 (e.g., fluorescence light, which emits light at a wavelength different from that of the excitation light) for imaging onto the image sensor 4195.

In some cases, the system 4100 may optionally include a custom tube lens 4190, as described elsewhere herein, such that the focus of the imaging system may be moved from the first inner surface 4188 to the second inner surface 4189 of the flow cell 4187 to achieve dual surface imaging with little adjustment. In some cases, the lens 4170 may include a custom tube lens, as described elsewhere herein, such that the focal point of the illumination light path may be moved from the first inner surface 4188 to the second inner surface 4189 of the flow cell 4187 to achieve dual surface imaging with little adjustment. In some cases, the lens 4170 may be implemented to articulate along the optical axis to adjust the focus of the structured illumination pattern on the sample plane. In some cases, system 4100 may include an autofocus mechanism (not shown) to adjust the focus of illumination light and/or the focus of the image at the plane of image sensor 4195. In some cases, the system 4100 shown in fig. 41 may provide high optical efficiency because of the absence of polarizers in the optical path. Depending on the numerical aperture of objective 4185, the use of unpolarized light may or may not have a significant impact on the contrast of the illumination pattern.

For simplicity, some optical components of the imaging system 4100 may have been omitted from fig. 41 and the foregoing discussion. Although system 4100 is shown in this non-limiting example as a single channel detection system, in other cases it may also be implemented as a multi-channel detection system (e.g., using two different image sensors and appropriate optics and light sources emitting at two different wavelengths). Furthermore, although the illumination light path of system 4100 is illustrated in this non-limiting example as including two branches, in some cases it may be implemented to include, for example, three branches, four branches, or more than four branches, each of which includes a diffraction grating that is fixed or adjustably oriented relative to each other.

In some cases, alternative illumination path optical designs may be used to create structured illumination. For example, in some cases, a single large rotating optical phase modulator may be placed after the optical assembly 4160 and used in place of the

optical phase modulators

4140A and 4140B to modulate the phases of the two diffracted beams output by the vertical diffraction grating 4130A and the horizontal diffraction grating 4130B. In some cases, instead of being parallel with respect to the optical axis of one of the diffraction gratings, the axis of rotation of a single rotating optical compensator may be offset 45 degrees (or by another angle) from the optical axis of each of the vertical and horizontal diffraction gratings to allow phase shifting in both illumination directions. In some cases, a single rotating optical phase modulator may be replaced by a wedge-shaped optical component that rotates, for example, about a nominal beam axis.

In another alternative illumination light path design, the

diffraction gratings

4130A and 4130B may be mounted on respective linear motion stages such that they may be translated to change the light path length (and thus the phase) of the light reflected or transmitted by the

diffraction gratings

4130A and 4130B. The axes of motion of the linear motion stages may be perpendicular to or otherwise offset from the orientation of their respective diffraction gratings to provide translation of the fringe pattern of the diffraction gratings along the sample plane 4188. Suitable translation stages may include, for example, cross roller bearing stages, linear motors, high precision linear encoders, and/or other linear actuator techniques to provide precise linear translation of the diffraction grating.

FIG. 42 provides a non-limiting example of a workflow for acquiring and processing imaging using structured illumination to enhance spatial resolution of an imaging system. In some cases, the workflow shown in fig. 42 may be performed to image the entire sample plane (e.g., the interior surface of a flow cell is imaged by image tiling), or to image a single region of a larger sample plane. The vertical 4130A and horizontal 4130B diffraction gratings of the system 4100 shown in fig. 41 may be used to project illumination light stripe patterns onto sample planes having different known orientations and/or different known phase shifts. For example, the imaging system 4100 may generate horizontal and vertical illumination patterns using the vertical grating 4130A and the horizontal grating 4130B, respectively, while the

optical phase modulators

4140A and 4140B may be set to three different positions to produce the three phase shifts shown for each orientation.

During operation, a grating light stripe pattern may be projected onto a sample plane (e.g., a flow cell surface) using a first illumination condition (e.g., a particular orientation and phase shift setting of a diffraction grating). After capturing the image using the first illumination condition, one or more additional images acquired using one or more phase-shifted illumination patterns (e.g., 1, 2, 3, 4, 5, 6, or more than 6 additional images acquired using 1, 2, 3, 4, 5, 6, or more than 6 phase-shifted illumination patterns) may be acquired. If the imaging system includes a second branch of the illumination light path, the image acquisition process may be repeated using the second illumination condition as a starting point (e.g., a second particular orientation and phase shift setting of the diffraction grating), and the image acquisition process may be repeated. In some cases, at least 5 different phase-shifted light fringe patterns may be used to acquire images for at least three different orientations of the diffraction grating (e.g., 60 degrees apart relative to each other). If no more images are acquired using a diffraction grating or a different orientation of the phase-shifted illumination light stripe pattern, an image reconstruction algorithm may be used to process the acquired images and produce a reconstructed super-resolution image. In some cases, at least 1, 2, 3, 4, 5, 6, or more than 6 different phase-shifted light stripe patterns are used in each orientation to acquire images for at least 1, 2, 3, 4, 5, 6, or more than 6 different orientations of the diffraction grating.

A potential disadvantage of acquiring multiple images for reconstructing a single super-resolution image is the time required to adjust the orientation and/or relative phase shift of the projected light stripe pattern and the exposure time required to acquire each image, as well as downstream image processing. Therefore, an optical design that minimizes the time required to change the orientation and relative phase of the diffraction grating and an efficient image reconstruction algorithm are preferred. In some cases, fewer images may be required to reconstruct, for example, a super-resolution image of a flow cell surface comprising discrete fluorescently labeled clusters of amplified target nucleic acid sequences tethered to a low non-specific binding surface as described elsewhere herein, than would normally be required to reconstruct a higher resolution image of a conventional sample (e.g., a stained tissue sample).

Referring again to fig. 42, for example, where images are tiled to create a higher resolution image of the entire flow cell surface, the above-described loop may be repeated for different areas of a given flow cell surface. In some cases, if, for example, the second flow cell surface is to be imaged, the above-described cycle may be repeated after the focus of the imaging system is adjusted.

Other super resolution imaging techniques: in some cases, the disclosed imaging systems may include the use of alternative super-resolution imaging techniques, such as light-sensitive positioning microscopy (PALM), fluorescent light-sensitive positioning microscopy (FPALM), and/or random optical reconstruction microscopy (stop). See, e.g., lutz, et al (2011), "Biological Imaging by Superresolution Light Microscopy",comprehensive Biotechnology (second version), volume 1, pages 579-589, elsevier), which is based on statistically curve fitting the intensity distribution observed in an image of the Point Spread Function (PSF) of a single molecule to a gaussian distribution function. The gaussian distribution function is then used to define the position of the molecules in the sample plane with a much higher accuracy than is allowed by classical resolution limits. The same method can be used to image small discrete subsets of fluorescently labeled molecules (e.g., clonal amplified clusters of target nucleic acid sequences tethered to a low non-specific binding surface on a sample carrier or to the interior surface of a flow cell).

The spatial accuracy or resolution obtained using these methods depends on the number of photons collected from the molecule before it is photobleaching and the background noise level [ Lutz, et al (2011), supra ]. If the background noise is negligible and at least 10,000 photons per molecule can be collected, the positional accuracy is demonstrated to be 1-2nm. In some cases, for example, using the polymer-nucleotide conjugates (e.g., each conjugate comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 labels) comprising a plurality of fluorescent labels (to ensure high photon counting) by affinity sequencing methods described elsewhere herein, optionally in combination with low non-specific binding surfaces (to ensure very low background signals) disclosed elsewhere herein, which can facilitate the use of these super-resolution imaging techniques for gene testing and sequencing applications. Spatial accuracy or resolution decreases with decreasing number of photons collected, but even in case of collecting only moderate number of photons, positioning accuracy or resolution of 20nm is possible. In some cases, a 10-fold or higher increase in lateral spatial resolution may be achieved. In some cases, image resolutions better than 500nm, 400nm, 300nm, 200nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm, or 10nm may be achieved.

A second fundamental principle of such imaging is to image a small number of spatially separated fluorescent molecules within a sample at any given time.

In some cases, the ability to control the fluorescence emission of a small, dispersed subset of fluorescent molecules in the sample plane is critical to facilitating super-resolution imaging. In the case of fluorescent light-sensitive localization microscopes (FPALMs) and light-sensitive localization microscopes (PALM), for example, the use of a photoactivatable green fluorescent protein (PA-GFP) as a marker may allow the controlled induction of a subset of fluorescence in a sample using a short pulse of 405nm light to convert PA-GFP from a dark non-fluorescent state to a 488nm excitable fluorescent state, thereby producing a spatially separated subset of fluorescent molecules that can be imaged [ Lutz, et al (2011), supra ]. In the case of random optical reconstruction microscopy (STORM), for example, the light conversion properties of cyanine dyes for Cy5-Cy3 can be used in a similar manner to enable random induction of Cy5 fluorescence from a small subset of molecules in the sample (e.g., a small subset of molecules spatially separated by at least a plurality of resolution units) at any given time. In some cases, for example, when combined with the methods described elsewhere herein by affinity sequencing, the polymer-nucleotide conjugates can comprise a photoactivatable green fluorescent protein (PA-GFP) or subdomain thereof, or a portion thereof. In some cases, the polymer-nucleotide conjugates can comprise a mixture of conjugates in which a first moiety is labeled with, for example, a Cy3 label and a second moiety is labeled with, for example, a Cy5 label. In some cases, the polymeric nucleotide conjugates can comprise a mixture of Cy3 and Cy5 labels, for example, within the same conjugate.

Super-resolution images [ Lutz, et al (2011), supra ] are reconstructed from a gaussian fit sum of all molecules or features (e.g., labeled nucleic acid clusters) imaged in a time stack of acquired images, where the intensities correspond to the positional uncertainty of the position of each molecule or subset of molecules. Such a dataset is unique in the ability to render images with different positioning accuracy or resolution. In some cases, imaging modules comprising Total Internal Reflection Fluorescence (TIRF) optical imaging designs may be advantageous in implementing the use of these super-resolution imaging techniques because the evanescent wave for exciting fluorescence is confined within an axial dimension of less than 200nm from the sample carrier or flow cell surface, thereby suppressing the background fluorescence signal. In some cases, the imaging system may include an objective lens with a higher numerical aperture than that used by other imaging module designs disclosed herein. The use of a higher numerical aperture objective lens may be advantageous for achieving evanescent wave excitation and efficient photon capture from the fluorescent probe. In some cases, wide field imaging using a single photon sensitive EM-CCD camera or other type of image sensor may enable simultaneous imaging of many molecules or subsets of molecules (e.g., clusters of nucleic acid sequences) per frame, thereby improving throughput of image acquisition.

In some cases, the data acquisition time required to acquire sufficient images to achieve adequate feature definition and resolution may be reduced by improving the sensitivity and speed of the imaging system by: the signal is increased by using the reagents and low non-specific binding surfaces by affinity sequencing disclosed herein while reducing or eliminating background, as well as using improved image reconstruction algorithms.

Evaluating image quality: for any of the embodiments of the optical imaging designs disclosed herein, imaging performance or imaging quality may be assessed using any of a variety of performance metrics known to those skilled in the art. Examples include, but are not limited to, measurement of Modulation Transfer Function (MTF) at one or more specified spatial frequencies, defocus, spherical aberration, chromatic aberration, coma, astigmatism, field curvature, image distortion, contrast-to-noise ratio (CNR), or any combination thereof.

In some cases, the disclosed optical designs for dual-sided imaging (e.g., the disclosed objective lens designs, tube lens designs, combined use of electro-optic phase plates with objective lenses, etc., alone or in combination) can significantly improve the image quality of the upper (near) and lower (far) interior surfaces of the flow cell such that the difference in imaging performance index for imaging the upper and lower interior surfaces of the flow cell is less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% for any of the imaging performance indices listed above (whether alone or in combination).

In some cases, the disclosed optical designs for dual-sided imaging (e.g., including the disclosed tube lens designs, combining an electro-optic phase plate with an objective lens, etc.) can significantly improve image quality such that for any of the imaging performance metrics listed above (whether alone or in combination), the image quality performance metrics for dual-sided imaging provide at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% improvement in the imaging performance metrics for dual-sided imaging compared to conventional systems (including, e.g., objective lens, motion actuated compensator (which moves out of or into the optical path when imaging the near or far inner surface of the flow cell), and image sensor). In some cases, a fluorescence imaging system including one or more of the disclosed tube lens designs provides at least equal or more improved imaging performance metrics for dual-sided imaging as compared to conventional systems including an objective lens, a motion actuated compensator, and an image sensor. In some cases, a fluorescence imaging system including one or more of the disclosed tube lens designs provides at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% imaging performance index improvement for dual-sided imaging as compared to conventional systems including an objective lens, a motion actuated compensator, and an image sensor.

Imaging module specification:

excitation light wavelength: in any of the disclosed optical imaging module designs, the light source of the disclosed imaging module may generate visible light, such as green and/or red light. In some cases, the light source alone or in combination with one or more optical components (e.g., excitation optical filters and/or dichroic beam splitters) can produce excitation light of about 350nm, 375nm, 400nm, 425nm, 450nm, 475nm, 500nm, 525nm, 550m,575nm, 600nm, 625nm, 650nm, 6755 nm, 700nm, 725nm, 750nm, 775nm, 800nm, 825nm, 850nm, 875nm, or 900 nm. Those skilled in the art will recognize that the excitation wavelength may have any value within this range, for example about 620nm.

Excitation light bandwidth: in any of the disclosed optical imaging module designs, the light source alone or in combination with one or more optical components (e.g., excitation optical filters and/or dichroic beam splitters) can produce light at a specified excitation wavelength over a bandwidth of + -2 nm, + -5 nm, + -10 nm, + -20 nm, + -40 nm, + -80 nm, or more. Those skilled in the art will recognize that the excitation bandwidth may have any value within this range, for example about 18nm.

Light source power output: in any of the disclosed optical imaging module designs, the power range of the output of the light source and/or the excitation light beam (including the composite excitation light beam) obtained therefrom may be about 0.5W to about 5.0W, or greater (as will be discussed in more detail below). In some cases, the output of the light source and/or the power of the excitation beam obtained therefrom may be at least 0.5W, at least 0.6W, at least 0.7W, at least 0.8W, at least 1W, at least 1.1W, at least 1.2W, at least 1.3W, at least 1.4W, at least 1.5W, at least 1.6W, at least 1.8W, at least 2.0W, at least 2.2W, at least 2.4W, at least 2.6W, at least 2.8W, at least 3.0W, at least 3.5W, at least 4.0W, at least 4.5W, or at least 5.0W. In some embodiments, the output of the light source and/or the power of the excitation light beam (including the composite excitation light beam) obtained therefrom may be at most 5.0W, at most 4.5W, at most 4.0W, at most 3.5W, at most 3.0W, at most 2.8W, at most 2.6W, at most 2.4W, at most 2.0W, at most 1.8W, at most 1.6W, at most 1.5W, at most 1.4W, at most 1.3W, at most 1.2W, at most 1.1W, at most 0.8W, at most 0.7W, at most 0.6W, or at most 0.5W. Any of the lower and upper values described in this paragraph may be combined to form the ranges encompassed by the present disclosure, for example, in some cases the output of the light source and/or the power of the excitation beam (including the composite excitation beam) obtained therefrom may range from about 0.8W to about 2.4W. Those skilled in the art will recognize that the output of the light source and/or the power of the excitation beam (including the composite excitation beam) derived therefrom may have any value within this range, for example, about 1.28W.

Light source output power and CNR: in some embodiments of the disclosed optical imaging module designs, the output power of the light source and/or the power of one or more excitation light beams (including composite excitation light beams) derived therefrom is sufficient, in combination with an appropriate sample, to provide a contrast-to-noise ratio (CNR) of at least 5, at least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, or at least 50 or more, or any CNR within any range formed by any of these values, in the image obtained by the illumination and imaging module.

Fluorescent emission band: in some cases, the disclosed fluorescence optical imaging modules can be configured to detect fluorescence emissions generated by any of a variety of fluorophores known to those of skill in the art. Examples of suitable fluorescent dyes for use in, for example, genotyping and nucleic acid sequencing applications (e.g., by conjugating nucleotides, oligonucleotides or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof, including cyanine derivatives cyanine dye 3 (Cy 3), cyanine dye 5 (Cy 5), cyanine dye 7 (Cy 7), and the like.

Fluorescence emission wavelength: in any of the disclosed optical imaging module designs, the detection channel or imaging channel of the disclosed optical system may include one or more optical components, such as an emission optical filter and/or a dichroic beam splitter, configured to collect emitted light at about 350nm, 375nm, 400nm, 425nm, 450nm, 475nm, 500nm, 525nm, 550m, 575nm, 600nm, 625nm, 650nm, 675nm, 700nm, 725nm, 750nm, 775nm, 800nm, 825nm, 850nm, 875nm, or 900 nm. Those skilled in the art will recognize that the emission wavelength may have any value within this range, for example, about 825nm.

Fluorescence emission bandwidth: in any of the disclosed optical imaging module designs, the detection channel or imaging channel may include one or more optical components, such as an emission optical filter and/or a dichroic beam splitter, configured to collect light of a specified emission wavelength over a bandwidth of ±2nm, ±5nm, ±10nm, ±20nm, ±40nm, ±80nm, or more. Those skilled in the art will recognize that the excitation bandwidth may have any value within this range, for example, about ±18nm.

Numerical aperture: in some cases, in any of the disclosed optical system designs, the numerical aperture of the objective lens and/or the optical imaging module (e.g., including the objective lens and/or the tube lens) may be in the range of about 0.1 to about 1.4. In some cases, the numerical aperture may be at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, or at least 1.4. In some cases, the numerical aperture may be at most 1.4, at most 1.3, at most 1.2, at most 1.1, at most 1.0, at most 0.9, at most 0.8, at most 0.7, at most 0.6, at most 0.5, at most 0.4, at most 0.3, at most 0.2, or at most 0.1. Any of the lower and upper values described in this paragraph may be combined to form a range included in the present disclosure, for example, in some cases, the numerical aperture may range from about 0.1 to about 0.6. Those skilled in the art will recognize that the numerical aperture may have any value within this range, for example, about 0.55.

Optical resolution: in some cases, the minimum resolvable spot (or feature) separation distance at the sample plane that may be achieved by any of the disclosed optical system designs may range from about 0.5 μm to about 2 μm, depending on the numerical aperture of the objective lens and/or optical system (e.g., including the objective lens and/or tube lens). In some cases, the minimum resolvable spot separation distance at the sample plane may be at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 1.0 μm. In some cases, the minimum resolvable spot separation distance may be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm, at most 0.6 μm, or at most 0.5 μm. Any of the lower and upper values described in this paragraph may be combined to form the ranges encompassed in the present disclosure, for example, in some cases the minimum resolvable spot separation distance may be in the range of about 0.8 μm to about 1.6 μm. Those skilled in the art will recognize that the minimum resolvable spot separation distance may have any value within this range, for example, about 0.95 μm.

Optical resolution of the first and second surfaces at different depths: in some cases, in any of the optical modules or systems disclosed herein, the use of the novel objective lens and/or tube lens designs disclosed herein can impart comparable optical resolution to the first and second surfaces (e.g., the upper and lower interior surfaces of the flow cell) with or without refocusing between acquiring images of the first and second surfaces. In some cases, the optical resolution of the images of the first and second surfaces thus obtained may have 20%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 1% of each other, or any value within this range.

Magnification ratio: in some cases, the magnification of the objective lens and/or tube lens, and/or the optical system (e.g., including the objective lens and/or tube lens) in any of the disclosed optical configurations may be in the range of about 2 to about 20. In some cases, the optical system magnification may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, or at least 20-fold. In some cases, the optical system magnification may be up to 20 times, up to 15 times, up to 10 times, up to 9 times, up to 8 times, up to 7 times, up to 6 times, up to 5 times, up to 4 times, up to 3 times, or up to 2 times. Any of the lower and upper values described in this paragraph may be combined to form a range included in the present disclosure, for example, in some cases, the optical system magnification may be in a range of about 3 to about 10 times. Those skilled in the art will recognize that the optical system magnification may have any value within this range, for example, about 7.5 times.

Focal length of objective lens: in some embodiments of the disclosed optical designs, the focal length of the objective lens may be in the range of 20mm to 40mm. In some cases, the focal length of the objective lens may be at least 20mm, at least 25mm, at least 30mm, at least 35mm, or at least 40mm. In some cases, the focal length of the objective lens may be at most 40mm, at most 35mm, at most 30mm, at most 25mm, or at most 20mm. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the present disclosure, for example, in some cases, the focal length of the objective lens may be in the range of 25mm to 35 mm. Those skilled in the art will recognize that the focal length of the objective lens may have any value within the range of values specified above, for example, about 37mm.

Working distance of objective lens: in some embodiments of the disclosed optical designs, the working distance of the objective lens may be in the range of about 100 μm to 30mm. In some cases, the working distance may be at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1mm, at least 2mm, at least 4mm, at least 6mm, at least 8mm, at least 10mm, at least 15mm, at least 20mm, at least 25mm, or at least 30mm. In some cases, the working distance may be at most 30mm, at most 25mm, at most 20mm, at most 15mm, at most 10mm, at most 8mm, at most 6mm, at most 4mm, at most 2mm, at most 1mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm. Any of the lower and upper values described in this paragraph may be combined to form a range encompassed in the present disclosure, for example, in some cases, the working distance of the objective lens may be in the range of 500 μm to 2 mm. Those skilled in the art will recognize that the working distance of the objective lens may have any value within the range of values specified above, for example, about 1.25mm.

For objective lenses optimized by thick coverslip imaging: in some examples of the disclosed optical designs, the design of the objective lens may be improved or optimized for cover slips of different flow cell thickness. For example, in some cases, the objective lens may be designed to have optimal optical performance for coverslips having a thickness of about 200 μm to about 1,000 μm. In some cases, the objective lens may be designed to have optimal performance for a coverslip having a thickness of at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, or at least 1,000 μm. In some cases, the objective may be designed to have optimal performance for coverslips having a thickness of at most 1,000 μm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, or at most 200 μm. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the present disclosure, for example, in some cases, the objective lens may be designed to have optimal optical performance for a cover slip having a thickness of about 300 μm to about 900 μm. Those skilled in the art will recognize that the objective lens can be designed to have optimal optical performance for the following coverslips: the cover slip may have any value within this range, for example, about 725 μm.

Depth of field and depth of focus: in some cases, the depth of field and/or depth of focus of any of the disclosed imaging module (e.g., including objective lens and/or tube lens) designs may range from about 10 μm to about 800 μm, or more. In some cases, the depth of field and/or depth of focus may be at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200 μm, at least 250 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, or at least 800 μm, or greater. In some cases, the depth of field and/or depth of focus is at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 250 μm, at most 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, at most 10 μm, or less. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the disclosure, e.g., in some cases, the depth of field and/or depth of focus may range from about 100 μm to about 175 μm. Those skilled in the art will recognize that the depth of field and/or depth of focus may have any value within the ranges of values specified above, for example, about 132 μm.

Field of view (FOV): in some embodiments, the FOV of any of the disclosed imaging module designs (e.g., provided by the combination of objective lens and detection channel optics (e.g., tube lens)) may be in the range of, for example, about 1mm to about 5mm (e.g., in terms of diameter, width, length, or longest dimension). In some cases, the FOV may be at least 1.0mm, at least 1.5mm, at least 2.0mm, at least 2.5mm, at least 3.0mm, at least 3.5mm, at least 4.0mm, at least 4.5mm, or at least 5.0mm (e.g., in diameter, width, length, or longest dimension). In some cases, the FOV may be at most 5.0mm, at most 4.5mm, at most 4.0mm, at most 3.5mm, at most 3.0mm, at most 2.5mm, at most 2.0mm, at most 1.5mm, or at most 1.0mm (e.g., in terms of diameter, width, length, or longest dimension). Any of the lower and upper values described in this paragraph may be combined to form the ranges encompassed by the present disclosure, for example, in some cases the FOV may range from about 1.5mm to about 3.5mm (e.g., in terms of diameter, width, length, or longest dimension). Those skilled in the art will recognize that the FOV may have any value within the range of values specified above, for example, about 3.2mm (e.g., in terms of diameter, width, length, or longest dimension).

Field of view (FOV) area: in some examples of the disclosed optical system designs, the area of the field of view may be about 2mm ² To about 5mm ² Within a range of (2). In some cases, the area of the field of view may be at least 2mm ² At least 3mm ² At least 4mm ² Or at least 5mm ² . In some cases, the field of view area may be at most 5mm ² At most 4mm ² At most 3mm ² Or at most 2mm ² . Any of the lower and upper values described in this paragraph may be combined to form a range encompassed within this disclosure, e.g., in some cases, the area of the field of view may be at about 3mm ² Up to about 4mm ² Within the range. Those skilled in the art will recognize that the area of the field of view may have any value within this range, for example, 2.75mm ² 。

Optimization of objective and/or tube lens MTF: in some cases, the objective lens and/or at least one tube lens designs in the disclosed imaging modules and systems are configured to optimize the modulation transfer function over a medium to high spatial frequency range. For example, in some cases, the design of the objective lens and/or at least one tube lens in the disclosed imaging modules and systems is configured to optimize the modulation transfer function in the sample plane over the following spatial frequency ranges: 500 cycles/mm to 900 cycles/mm, 700 cycles/mm to 1100 cycles/mm, 800 cycles/mm to 1200 cycles/mm, or 600 cycles/mm to 1000 cycles/mm.

Optical aberration and diffraction limited imaging performance: in some embodiments of any of the optical imaging module designs disclosed herein, the objective lens and/or tube lens may be configured to provide the imaging module with a field of view as described above such that the FOV has an aberration of less than 0.15 wave over at least 60%, 70%, 80%, 90% or 95% of the field of view. In some embodiments, the objective lens and/or tube lens may be configured to provide the imaging module with a field of view as described above such that the FOV has an aberration of less than 0.1 wave over at least 60%, 70%, 80%, 90% or 95% of the field of view. In some embodiments, the objective lens and/or tube lens may be configured to provide the imaging module with a field of view as described above such that the FOV has an aberration of less than 0.075 wave over at least 60%, 70%, 80%, 90%, or 95% of the field of view. In some embodiments, the objective lens and/or tube lens may be configured to provide the imaging module with a field of view as described above such that the FOV is diffraction limited over at least 60%, 70%, 80%, 90% or 95% of the field of view.

Incidence angle of light beam on dichroic reflector, beam splitter and beam combiner: in some examples of the disclosed optical designs, the angle of incidence of the light beam on the dichroic reflector, beam splitter, or beam combiner may range from about 20 degrees to about 45 degrees. In some cases, the angle of incidence may be at least 20 degrees, at least 25 degrees, at least 30 degrees, at least 35 degrees, at least 40 degrees, or at least 45 degrees. In some cases, the angle of incidence may be at most 45 degrees, at most 40 degrees, at most 35 degrees, at most 30 degrees, at most 25 degrees, or at most 20 degrees. Any of the lower and upper values described in this paragraph may be combined to form a range included in the present disclosure, for example, in some cases, the angle of incidence may be in a range of about 25 degrees to about 40 degrees. Those skilled in the art will recognize that the angle of incidence may have any value within the ranges of values specified above, for example, about 43 degrees.

Image sensor (photodetector array) size: in some cases, the disclosed optical system may include an image sensor having an active area with a diagonal in a range of about 10mm to about 30mm or more. In some cases, the diagonal of the active area of the image sensor is at least 10mm, at least 12mm, at least 14mm, at least 16mm, at least 18mm, at least 20mm, at least 22mm, at least 24mm, at least 26mm, at least 28mm, or at least 30mm. In some cases, the diagonal of the active area of the image sensor is at most 30mm, at most 28mm, at most 26mm, at most 24mm, at most 22mm, at most 20mm, at most 18mm, at most 16mm, at most 14mm, at most 12mm, or at most 10mm. Any of the lower and upper values described in this paragraph may be combined to form a range included in the present disclosure, for example, in some cases, the image sensor may have an effective area with a diagonal range of about 12mm to about 24 mm. Those skilled in the art will recognize that one or more image sensors may have an active area with a diagonal having any value (e.g., about 28.5 mm) within the range of values specified above.

Image sensor pixel size and pitch: in some cases, the pixel size and/or pitch selected for the image sensor used in the disclosed optical system designs may be in the range of about 1 μm to about 10 μm in at least one dimension. In some cases, the pixel size and/or pitch may be at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 6 μm, at least 7 μm, at least 8 μm, at least 9 μm, or at least 10 μm. In some cases, the pixel size and/or pitch may be at most 10 μm, at most 9 μm, at most 8 μm, at most 7 μm, at most 6 μm, at most 5 μm, at most 4 μm, at most 3 μm, at most 2 μm, or at most 1 μm. Any of the lower and upper values described in this paragraph may be combined to form a range encompassed by the present disclosure, for example, in some cases, the pixel size and/or pitch may be in the range of about 3 μm to about 9 μm. Those skilled in the art will recognize that the pixel size and/or pitch may have any value within this range, for example, about 1.4 μm.

Oversampling: in some examples of the disclosed optical designs, a spatial oversampling scheme is utilized in which the spatial sampling frequency is at least 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 times the optical resolution X (lp/mm).

Maximum translation stage speed: in some examples of the disclosed optical imaging module, the maximum translation stage speed in any one axis may be in the range of about 1mm/s to about 5mm/s. In some cases, the maximum translation stage speed may be at least 1mm/s, at least 2mm/s, at least 3mm/s, at least 4mm/s, or at least 5mm/s. In some cases, the maximum translation stage speed may be at most 5mm/s, at most 4mm/s, at most 3mm/s, at most 2mm/s, or at most 1mm/s. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the present disclosure, for example, in some cases, the maximum translation stage speed may be in the range of about 2mm/s to about 4 mm/s. Those skilled in the art will recognize that the maximum translation stage speed may have any value within this range, for example, about 2.6mm/s.

Maximum translation stage acceleration: in some examples of the disclosed optical imaging module, the maximum acceleration in any one axis of motion may be about 2mm/s ² To about 10mm/s ² Within the range. In some cases, the maximum acceleration may be at least 2mm/s ² At least 3mm/s ² At least 4mm/s ² At least 5mm/s ² At least 6mm/s ² At least 7mm/s ² At least 8mm/s ² At least 9mm/s ² Or at least 10mm/s ² . In some cases, the maximum acceleration may be at most 10mm/s ² At most 9mm/s ² At most 8mm/s ² At most 7mm/s ² At most 6mm/s ² At most 5mm/s ² At most 4mm/s ² At most 3mm/s ² Or at most 2mm/s ² . Any of the lower and upper values described in this paragraph may be combined to form a range encompassed within this disclosure, e.g., in some cases, the maximum acceleration may be at about 2mm/s ² To about 8mm/s ² Within a range of (2). Those skilled in the art will recognize that the maximum acceleration may have any value within this range, for example, about 3.7mm/s ² 。

Translation stage positioning repeatability: in some examples of the disclosed optical imaging module, the repeatability of positioning for any one axis may be in the range of about 0.1 μm to about 2 μm. In some cases, the repeatability of positioning may be at least 0.1 μm, at least 0.2 μm, at least 0.3 μm, at least 0.4 μm, at least 0.5 μm, at least 0.6 μm, at least 0.7 μm, at least 0.8 μm, at least 0.9 μm, at least 1.0 μm, at least 1.2 μm, at least 1.4 μm, at least 1.6 μm, at least 1.8 μm, or at least 2.0 μm. In some cases, the repeatability of the positioning may be at most 2.0 μm, at most 1.8 μm, at most 1.6 μm, at most 1.4 μm, at most 1.2 μm, at most 1.0 μm, at most 0.9 μm, at most 0.8 μm, at most 0.7 μm, at most 0.6 μm, at most 0.5 μm, at most 0.4 μm, at most 0.3 μm, at most 0.2 μm, or at most 0.1 μm. Any of the lower and upper values described in this paragraph may be combined to form a range encompassed by the present disclosure, for example, in some cases, the repeatability of positioning may be in the range of about 0.3 μm to about 1.2 μm. Those skilled in the art will recognize that the repeatability of positioning may have any value within this range, for example, about 0.47 μm.

FOV repositioning time: in some examples of the disclosed optical imaging module, the maximum time required to reposition the sample plane (field of view) relative to the optics, or vice versa, may be in the range of about 0.1 seconds to about 0.5 seconds. In some cases, the longest repositioning time (i.e., the scanning stage step size and settling time) may be at least 0.1 seconds, at least 0.2 seconds, at least 0.3 seconds, at least 0.4 seconds, or at least 0.5 seconds. In some cases, the longest relocation time may be at most 0.5 seconds, at most 0.4 seconds, at most 0.3 seconds, at most 0.2 seconds, or at most 0.1 seconds. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the present disclosure, for example, in some cases, the longest repositioning time may be in the range of about 0.2 seconds to about 0.4 seconds. Those skilled in the art will recognize that the longest relocation time may have any value within this range, for example, about 0.45 seconds.

Error threshold for auto-focus correction: in some examples of the disclosed optical imaging module, the specified error threshold for triggering autofocus correction may be in a range of about 50nm to about 200nm. In some cases, the error threshold may be at least 50nm, at least 75nm, at least 100nm, at least 125nm, at least 150nm, at least 175nm, or at least 200nm. In some cases, the error threshold may be at most 200nm, at most 175nm, at most 150nm, at most 125nm, at most 100nm, at most 75nm, or at most 50nm. Any of the lower and upper values described in this paragraph may be combined to form a range included within the disclosure, for example, in some cases, the error threshold may be in a range of about 75nm to about 150 nm. Those skilled in the art will recognize that the error threshold may have any value within this range, for example, about 105nm.

Image acquisition time: in some cases of the disclosed optical imaging module, the image acquisition time may be in the range of about 0.001 seconds to about 1 second. In some cases, the image acquisition time may be at least 0.001 seconds, at least 0.01 seconds, at least 0.1 seconds, or at least 1 second. In some cases, the image acquisition time may be at most 1 second, at most 0.1 second, at most 0.01 second, or at most 0.001 second. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the present disclosure, for example, in some cases, the image acquisition time may be in the range of about 0.01 seconds to about 0.1 seconds. Those skilled in the art will recognize that the image acquisition time may have any value within this range, for example, about 0.250 seconds.

Imaging time per FOV: in some cases, the imaging time per field of view may be in the range of about 0.5 seconds to about 3 seconds. In some cases, the imaging time of each FOV may be at least 0.5 seconds, at least 1 second, at least 1.5 seconds, at least 2 seconds, at least 2.5 seconds, or at least 3 seconds. In some cases, the imaging time per FOV may be at most 3 seconds, at most 2.5 seconds, at most 2 seconds, at most 1.5 seconds, at most 1 second, or at most 0.5 seconds. Any of the lower and upper values described in this paragraph may be combined to form a range included in the present disclosure, for example, in some cases, the imaging time may be in the range of about 1 second to about 2.5 seconds. Those skilled in the art will recognize that the imaging time may have any value within this range, for example, about 1.85 seconds.

Field of view flatness: in some cases, images over 80%, 90%, 95%, 98%, 99% or 100% of the field of view are acquired within ±200nm, ±175nm, ±150nm, ±125nm, ±100nm, ±75nm or ±50nm of the optimal focal plane for each fluorescence (or other imaging mode) detection channel.

Systems and system components for genomics and other applications: as described above, in some embodiments, the disclosed optical imaging modules may be used as modules, components, sub-assemblies, or sub-systems of a larger system configured for performing, for example, genomic applications (e.g., genetic testing and/or nucleic acid sequencing applications) or other chemical, biochemical, nucleic acid, cellular, or tissue analysis applications. FIG. 39 provides a non-limiting example of a block diagram for a sequencing system such as that disclosed herein. In addition to one, two, three, four, or more than four imaging modules disclosed herein (each imaging module may include one or more illumination light paths and/or one or more detection light paths (e.g., one or more detection channels configured to image fluorescence emissions over a particular wavelength range onto an image sensor)), such systems may include one or more X-Y translation stages, one or more X-Y-Z translation stages, flow cells or cassettes, fluid systems and fluid flow control modules, kits, temperature control modules, fluid dispensing robots, cassettes and/or microplate processing (pick-and-place) robots, opaque housings and/or environmental control chambers, one or more processors or computers, data storage modules, data communication modules (e.g., bluetooth, wiFi, intranet or internet communication hardware and related software), display modules, one or more local and/or cloud-based software packages (e.g., analysis/system control software packages, image processing software packages, data packages, or the like), or any combination thereof.

Translation stage: in some embodiments of the imaging and analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the systems can include one or more (e.g., one, two, three, four, or more than four) high precision X-Y (or in some cases X-Y-Z) translation stages for repositioning one or more sample carrier structures (e.g., one or more flow cells) relative to one or more imaging modules, e.g., such as to tile one or more images (each image corresponding to a field of view of the imaging module) to reconstruct a composite image of the entire flow cell surface. In some embodiments of the imaging systems and genomic analysis systems (e.g., nucleic acid sequencing systems) disclosed herein, the systems can include one or more (e.g., one, two, three, four, or more than four) high precision X-Y (or in some cases X-Y-Z) translation stages for repositioning one or more imaging modules relative to one or more sample carrier structures (e.g., flow cells), e.g., to tile one or more images (each image corresponding to a field of view of an imaging module) to reconstruct one or more composite images of the entire flow cell surface.

Suitable translation stages are commercially available from a number of suppliers, such as Parker Hannifin. Precision translation stage systems typically include a combination of components including, but not limited to, linear actuators, optical encoders, servo and/or stepper motors, and motor controllers or drive units. For the systems and methods disclosed herein, high precision and repeatability of platform movement is required to ensure accurate and reproducible positioning and imaging of, for example, fluorescent signals when disseminated reagent delivery and optical detection are repeated steps.

Thus, the systems disclosed herein may include an accuracy specifying that the translation stage is configured to position the sample carrier structure relative to the illumination and/or imaging optics (or vice versa). In one aspect of the disclosure, the accuracy of the one or more translation stages is between about 0.1 μm to about 10 μm. In other aspects, the precision of the translation stage is about 10 μm or less, about 9 μm or less, about 8 μm or less, about 7 μm or less, about 6 μm or less, about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.9 μm or less, about 0.8 μm or less, about 0.7 μm or less, about 0.6 μm or less, about 0.5 μm or less, about 0.4 μm or less, about 0.3 μm or less, about 0.2 μm or less, about 0.1 μm or less. Those skilled in the art will appreciate that in some cases, the positioning accuracy of the translation stage may fall within any range defined by any two of these values (e.g., about 0.5 μm to about 1.5 μm). In some cases, the positioning accuracy of the translation stage may have any value within the range of values encompassed by the segment, for example, about 0.12 μm.

Flow cell, microfluidic device and cartridge: the flow cell devices and flow cell cartridges disclosed herein may be used as components of systems designed for a variety of chemical, biochemical, nucleic acid, cellular or tissue analysis applications. In general, such systems may include one or more of the disclosed single capillary flow cell devices, multiple capillary flow cell devices, capillary flow cell cartridges, and/or one or more of the microfluidic devices and cartridges described herein. Additional description of the disclosed flow cell devices and cartridges can be found in PCT patent application publication WO 2020/118255, the entire contents of which are incorporated herein by reference.

In some cases, the systems disclosed herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 single capillary flow cell devices, multiple capillary flow cell devices, capillary flow cell cartridges, and/or microfluidic devices and cartridges. In some cases, a single capillary flow cell device, multiple capillary flow cell devices, and/or microfluidic devices and cartridges may be stationary components of the disclosed systems. In some cases, a single capillary flow cell device, multiple capillary flow cell devices, and/or microfluidic devices and cartridges may be removable, replaceable components of the disclosed systems. In some cases, a single capillary flow cell device, multiple capillary flow cell devices, and/or microfluidic devices and cartridges may be disposable or consumable components of the disclosed systems.

In some embodiments, the disclosed single capillary flow cell device (or single capillary flow cell cartridge) includes a single capillary, e.g., a glass or fused quartz capillary, the lumen of which forms a fluid flow path through which reagents or solutions can flow, and the inner surface of which can form a sample carrier structure to which a sample of interest is bound or tethered. In some embodiments, a multi-capillary flow cell device (or multi-capillary flow cell cartridge) disclosed herein can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 capillaries configured to perform an analytical technique that also includes imaging as a detection method.

In some cases, one or more capillaries may be packaged within a rack to form a conveniently operable cartridge, incorporating an adapter or connector for making external fluid connections, and may optionally include additional integrated functions, such as reagent reservoirs, waste reservoirs, valves (e.g., micro-valves), pumps (e.g., micro-pumps), and the like, or any combination thereof.

Fig. 29 illustrates one non-limiting example of a single glass capillary flow cell device that includes two fluid adaptors (one secured to each end of a one-piece glass capillary) designed to mate with standard OD fluid tubes to provide a convenient, exchangeable fluid connection with an external fluid control system. The fluid adapter may be attached to the capillary tube using any of a variety of techniques known to those skilled in the art, including but not limited to press fitting, adhesive bonding, solvent bonding, laser welding, and the like, or any combination thereof.

Typically, the capillaries used in the disclosed capillary flow cell devices and capillary flow cell cartridges will have at least one internal axially aligned fluid flow channel (or "lumen") that extends the entire length of the capillary. In some cases, the capillary tube may have two, three, four, five, or more than five internal axially aligned fluid flow passages (or "lumens").

A number of specified cross-sectional geometries for suitable capillaries (or lumens thereof) are consistent with the disclosure herein, including, but not limited to, circular, oval, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some cases, the capillary tube (or lumen thereof) may have any specified cross-sectional dimension or set of dimensions. For example, in some cases, the maximum cross-sectional dimension of the capillary lumen (e.g., diameter if the lumen is circular, or diagonal if the lumen is square or rectangular) may be in the range of about 10 μm to about 10mm. In some cases, the maximum cross-sectional dimension of the capillary lumen may be at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1mm, at least 2mm, at least 3mm, at least 4mm at least 5mm, at least 6mm, at least 7mm, at least 8mm, at least 9mm, or at least 10mm. In some aspects, the maximum cross-sectional dimension of the capillary lumen may be at most 10mm, at most 9mm, at most 8mm, at most 7mm, at most 6mm, at most 5mm, at most 4mm, at most 3mm, at most 2mm, at most 1mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of the lower and upper values described in this paragraph may be combined to form the ranges encompassed by the present disclosure, for example, in some cases the maximum cross-sectional dimension of the capillary lumen may be in the range of about 100 μm to about 500 μm. Those skilled in the art will recognize that the maximum cross-sectional dimension of the capillary lumen may have any value within this range, for example, about 124 μm.

In some cases, for example, where the lumen of one or more capillaries in a flow cell device or cartridge has a square or rectangular cross-section, the distance between a first inner surface (e.g., top surface or upper surface) and a second inner surface (e.g., bottom surface or lower surface), which defines the gap height or thickness of a fluid flow channel, can range from about 10 μm to about 500 μm. In some cases, the gap height may be at least 10 μm, at least 20 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, at least 100 μm, at least 125 μm, at least 150 μm, at least 175 μm, at least 200 μm, at least 225 μm, at least 250 μm, at least 275 μm, at least 300 μm, at least 325 μm, at least 350 μm, at least 375 μm, at least 400 μm, at least 425 μm, at least 450 μm, at least 475 μm, or at least 500 μm. In some cases, the gap height may be at most 500 μm, at most 475 μm, at most 450 μm, at most 425 μm, at most 400 μm, at most 375 μm, at most 350 μm, at most 325 μm, at most 300 μm, at most 275 μm, at most 250 μm, at most 225 μm, at most 200 μm, at most 175 μm, at most 150 μm, at most 125 μm, at most 100 μm, at most 90 μm, at most 80 μm, at most 70 μm, at most 60 μm, at most 50 μm, at most 40 μm, at most 30 μm, at most 20 μm, or at most 10 μm. Any of the lower and upper values described in this paragraph may be combined to form a range encompassed by the present disclosure, for example, in some cases, the gap height may be in the range of about 40 μm to about 125 μm. Those skilled in the art will recognize that the gap height may have any value within the range of values for the segment, for example, about 122 μm.

In some cases, the length of one or more capillaries used to make the disclosed capillary flow cell devices or cartridges may be in the range of about 5mm to about 5cm or more. In some cases, the length of the one or more capillaries can be less than 5mm, at least 1cm, at least 1.5cm, at least 2cm, at least 2.5cm, at least 3cm, at least 3.5cm, at least 4cm, at least 4.5cm, or at least 5cm. In some cases, the length of the one or more capillaries may be at most 5cm, at most 4.5cm, at most 4cm, at most 3.5cm, at most 3cm, at most 2.5cm, at most 2cm, at most 1.5cm, at most 1cm, or at most 5mm. Any of the lower and upper values described in this paragraph may be combined to form a range included in the present disclosure, for example, in some cases, the length of the one or more capillaries may be in the range of about 1.5cm to about 2.5 cm. Those skilled in the art will recognize that the length of the one or more capillaries may have any value within this range, for example, about 1.85cm. In some cases, the device or cartridge may include a plurality of two or more capillaries of the same length. In some cases, the device or cartridge may include a plurality of two or more capillaries of different lengths.

Capillaries for constructing the disclosed capillary flow cell devices or cartridges can be made from any of a variety of materials known to those skilled in the art, including, but not limited to, glass (e.g., borosilicate glass, soda lime glass, etc.), fused silica (quartz), polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high Density Polyethylene (HDPE), cyclic Olefin Polymer (COP), cyclic Olefin Copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomer (FFKM) as more chemically inert substitutes, or any combination thereof. PEI is between polycarbonate and PEEK in terms of cost and chemical compatibility. FFKM is also known as Kalrez.

The material or materials used to fabricate the capillaries are typically optically transparent to facilitate use with spectroscopic-based or imaging-based detection techniques. In some cases, the entire capillary will be optically transparent. Alternatively, in some cases, only a portion of the capillary (e.g., an optically transparent "window") will be optically transparent.

Any of a variety of techniques known to those skilled in the art may be used to fabricate capillaries for constructing the disclosed capillary flow cell devices and capillary flow cell cartridges, with the choice of fabrication technique generally depending on the choice of materials and vice versa. Examples of suitable capillary manufacturing techniques include, but are not limited to, extrusion, drawing, precision Computer Numerical Control (CNC) machining and boring, laser ablation, and the like.

In some embodiments, the capillaries used in the disclosed capillary flow cell devices and cartridges may be off-the-shelf commercial products. Examples of commercial suppliers that provide precision capillaries include Accu-Glass (St. Louis, MO; precision Glass capillaries), polymicro Technologies (Phoenix, AZ; precision Glass and fused quartz capillaries), friedrich & Dimmack, inc. (Millville, NJ; custom-made precision Glass capillaries), and Drummond Scientific (Broomall, PA; OEM Glass and plastic capillaries).

Fluid adaptors attached to capillaries of capillary flow cell devices and cartridges disclosed herein, as well as other components of capillary flow cell devices or cartridges, can be manufactured using any of a variety of suitable techniques (e.g., extrusion, injection molding, compression molding, precision CNC processing, etc.) and materials (e.g., glass, fused silica, ceramic, metal, polydimethylsiloxane, polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HOPE), cyclic Olefin Polymer (COP), cyclic Olefin Copolymer (COC), polyethylene terephthalate (PET), etc.), wherein the choice of manufacturing technique is also generally dependent on the choice of materials used, and vice versa.

FIG. 30 provides a non-limiting example of a capillary flow cell cartridge that includes two glass capillaries, a fluid adapter (in this example, two for each capillary), and a cartridge mount that mates with the capillaries and/or fluid adapter so that the capillaries remain in a fixed orientation relative to the cartridge. In some cases, the fluid adapter may be integrated with the cartridge base. In some cases, the cartridge may include additional adapters that mate with the capillary and/or capillary fluid adapters. As described elsewhere herein, in some cases, the cartridge may include additional functional components. In some cases, the capillary tube may be permanently mounted in the cartridge. In some cases, the cartridge base is designed to allow for interchangeable removal and replacement of one or more capillaries of the flow cell cartridge. For example, in some cases, the cartridge base may include a hinged "flip" configuration that allows it to be opened so that one or more capillaries may be removed and replaced. In some cases, the cassette mount is configured to be mounted on a stage of, for example, a fluorescence microscope or within a cassette holder of a fluorescence imaging module or instrument system of the present disclosure.

In some cases, the disclosed flow cell devices may include microfluidic devices (or "microfluidic chips") and cartridges, where the microfluidic devices are fabricated by forming fluidic channels in one or more layers of suitable materials, and include one or more fluidic channels (e.g., an "analysis" channel) configured to perform analytical techniques that also include imaging as a detection method. In some embodiments, a microfluidic device or cartridge disclosed herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 fluidic channels (e.g., an "analysis" fluidic channel) configured to perform an analytical technique that also includes imaging as a detection method. In some cases, the disclosed microfluidic devices may also include additional fluidic channels (e.g., for dilution or mixing of reagents), reagent reservoirs, waste reservoirs, adapters for making external fluidic connections, etc., to provide integrated "lab-on-a-chip" functionality within the device.

Non-limiting examples of microfluidic flow cell cartridges include: a chip having two or more parallel glass channels formed on the chip, a fluidic adapter coupled to the chip, and a cartridge mount mated to the chip and/or the fluidic adapter such that the chip is placed in a fixed orientation relative to the cartridge. In some cases, the fluid adapter may be integrated with the cartridge base. In some cases, the cartridge may include additional adapters that mate with the chip and/or fluid adapters. In some cases, the chip is permanently mounted in the cartridge. In some cases, the cartridge base is designed to allow for interchangeable removal and replacement of one or more chips in the flow cell cartridge. In some cases, the cartridge base may include a hinged "flip" configuration that allows it to be opened so that one or more chips may be removed and replaced. In some cases, the cassette mount is configured to be mounted on a stage of a microscope system or within a cassette holder of an imaging system, for example. In a non-limiting example, even though only one chip is described, it should be understood that more than one chip may be used in a microfluidic flow cell cartridge. The flow cell of the present disclosure may include a single microfluidic chip or multiple microfluidic chips. In some cases, a flow cell cartridge of the present disclosure can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 microfluidic chips. Packaging one or more microfluidic devices within a cartridge may facilitate ease of handling and proper positioning of the devices in an optical imaging system.

The fluidic channels within the disclosed microfluidic devices and cartridges can have a variety of cross-sectional geometries including, but not limited to, circular, oval, square, rectangular, triangular, rounded square, rounded rectangular, or rounded triangular cross-sectional geometries. In some cases, the fluid channels may have any specified cross-sectional dimension or set of dimensions. For example, in some cases, the height (e.g., gap height), width, or maximum cross-sectional dimension (e.g., diagonal if the fluid channel has a square, rounded square, rectangular, or rounded rectangular cross-section) of the fluid channel may be in the range of about 10 μm to about 10mm. In some aspects, the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel may be at least 10 μm, at least 25 μm, at least 50 μm, at least 75 μm, at least 100 μm, at least 200 μm, at least 300 μm, at least 400 μm, at least 500 μm, at least 600 μm, at least 700 μm, at least 800 μm, at least 900 μm, at least 1mm, at least 2mm, at least 3mm, at least 4mm, at least 5mm, at least 6mm, at least 7mm, at least 8mm, at least 9mm, or at least 10mm. In some aspects, the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel may be at most 10mm, at most 9mm, at most 8mm, at most 7mm, at most 6mm, at most 5mm, at most 4mm, at most 3mm, at most 2mm, at most 1mm, at most 900 μm, at most 800 μm, at most 700 μm, at most 600 μm, at most 500 μm, at most 400 μm, at most 300 μm, at most 200 μm, at most 100 μm, at most 75 μm, at most 50 μm, at most 25 μm, or at most 10 μm. Any of the lower and upper values described in this paragraph may be combined to form a range encompassed within this disclosure, for example, in some cases the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel may be in the range of about 20 μm to about 200 μm. Those skilled in the art will recognize that the height (e.g., gap height), width, or maximum cross-sectional dimension of the fluid channel may have any value within this range, for example, about 122 μm.

In some cases, the length of the fluidic channels in the disclosed microfluidic devices and cartridges may range from about 5mm to about 10cm or more. In some cases, the length of the fluid channel may be less than 5mm, at least 1cm, at least 1.5cm, at least 2cm, at least 2.5cm, at least 3cm, at least 3.5cm, at least 4cm, at least 4.5cm, at least 5cm, at least 6cm, at least 7cm, at least 8cm, at least 9cm, or at least 10cm. In some cases, the length of the fluid channel may be at most 10cm, at most 9cm, at most 8cm, at most 7cm, at most 6cm, at most 5cm, at most 4.5cm, at most 4cm, at most 3.5cm, at most 3cm, at most 2.5cm, at most 2cm, at most 1.5cm, at most 1cm, or at most 5mm. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the disclosure, for example, in some cases, the length of the fluid channel may be in the range of about 1.5cm to about 2.5 cm. Those skilled in the art will recognize that the length of the fluid channel may have any value within this range, for example, about 1.35cm. In some cases, a microfluidic device or cartridge may include multiple fluidic channels of the same length. In some cases, a microfluidic device or cartridge may include multiple fluidic channels of different lengths.

The disclosed microfluidic device will include at least one layer of material having one or more fluidic channels formed therein. In some cases, a microfluidic chip may include two layers bonded together to form one or more fluidic channels. In some cases, a microfluidic chip may include three or more layers that are bonded together to form one or more fluidic channels. In some cases, the microfluidic channel may have an open top. In some cases, the microfluidic channel may be fabricated within one layer (e.g., the top surface of the bottom layer) and may be sealed by bonding the top surface of the bottom layer to the bottom surface of the top layer of material. In some cases, microfluidic channels may be fabricated within one layer, for example as patterned channels, the depth of which extends through the entire thickness of the layer, then sandwiched between and bonded to two non-patterned layers to seal the fluidic channels. In some cases, microfluidic channels are fabricated by removing a sacrificial layer on the surface of a substrate. The method does not require etching away the bulk substrate (e.g., glass or silicon wafer). Instead, the fluid channels are located on the surface of the substrate. In some cases, microfluidic channels may be fabricated within or on the surface of a substrate, and then sealed by depositing a conformal film or layer on the surface of the substrate to form subsurface or buried fluidic channels in the chip.

A combination of microfabrication processes may be used to fabricate microfluidic chips. Because the devices are microfabricated, the matrix materials will typically be selected based on their compatibility with known microfabrication techniques (e.g., photolithography, wet chemical etching, laser ablation, laser irradiation, air abrasion techniques, injection molding, embossing, and other techniques). The matrix material is also typically selected to be compatible with the entire range of conditions to which the microfluidic device may be exposed, including extreme pH, temperature, salt concentration, and application of an electromagnetic (e.g., light) or electric field.

The disclosed microfluidic chip may be made from any of a variety of materials known to those skilled in the art, including, but not limited to, glass (e.g., borosilicate glass, soda lime glass, etc.), quartz glass (quartz), silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HOPE), cyclic Olefin Polymer (COP), cyclic Olefin Copolymer (COC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), etc.), polyetherimide (PEI), and perfluoroelastomer (FFKM) (as a substitute for higher chemical inertness), or any combination thereof. In some preferred cases, the matrix material may include a silica-based matrix, such as borosilicate glass and quartz, as well as other suitable materials.

The disclosed microfluidic devices may be fabricated using any of a variety of techniques known to those skilled in the art, wherein the choice of fabrication technique generally depends on the choice of materials used, and vice versa. Microfluidic channels on the chip may be constructed using techniques suitable for forming microstructures or micropatterns on the surface of a substrate. In some cases, the fluid channel is formed by laser irradiation. In some cases, the microfluidic channel is formed by focused femtosecond laser radiation. In some cases, microfluidic channels are formed by photolithography and etching, including, but not limited to, chemical etching, plasma etching, or deep reactive ion etching. In some cases, laser etching is used to form microfluidic channels. In some cases, the microfluidic channels are formed using direct write lithography. Examples of write-through lithography include electron beam write-through and focused ion beam milling.

In further preferred examples, the matrix material may comprise a polymeric material, such as a plastic (e.g., polymethyl methacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON) ^TM ) Polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone, etc. Such polymer matrices can be readily patterned or micromachined using micromachining techniques such as those described above. In some cases, the microfluidic chip may be made of a polymeric material, such as a micromachined master, using well-known molding techniques, such as injection molding, embossing, compression molding, or by polymerizing polymeric precursor materials in a mold (see, e.g., U.S. Pat. No. 5,512,131). In some cases, such polymer matrix materials are preferred because they are easy to manufacture, low cost and disposable, and they are generally inert to most extreme reaction conditions. As with flow cell devices made of other materials (e.g., glass), for example, flow cell devices made of these polymeric materials may include treated surfaces (e.g., derivatized or coated surfaces) to enhance their utility in microfluidic systems, as will be discussed in more detail below.

The fluid channels and/or fluid chambers of the microfluidic device are typically fabricated into the upper surface of the first substrate as microscale channels (e.g., grooves, recesses, etc.) using the microfabrication techniques described above. The first substrate includes a top side having a first planar surface and a bottom side. In a microfluidic device prepared according to the methods described herein, a plurality of fluidic channels (e.g., grooves and/or recesses) are formed on a first planar surface. In some cases, the fluid channels (e.g., grooves and/or notches) formed in the first planar surface (prior to bonding to the second substrate) have a bottom wall and side walls, with the top remaining open. In some cases, the fluid channels (e.g., grooves and/or notches) formed in the first planar surface (prior to bonding to the second substrate) have bottom walls and side walls, and the top remains closed. In some cases, the fluid channels (e.g., grooves and/or recesses) formed in the first planar surface (prior to bonding to the second substrate) have only sidewalls, and no top or bottom surfaces (i.e., the fluid channels span the entire thickness of the first substrate).

The fluid channels and chambers may be sealed by placing the first planar surface of the first substrate in contact with and bonded to the planar surface of the second substrate to form channels and/or chambers (e.g., interiors) of the device at the junction of the two components. In some cases, after bonding the first substrate to the second substrate, the structure may be further placed in contact with and bonded to a third substrate. In some cases, the third substrate may be placed in contact with a side of the first substrate that is not in contact with the second substrate. In some cases, the first substrate is disposed between the second substrate and the third substrate. In some cases, the second and third substrates may cover and/or seal grooves, notches, or apertures formed on the first substrate to form channels and/or chambers (e.g., interiors) of the device at junctions of these components.

The device may have openings oriented such that they are in fluid communication with at least one of a fluid channel and/or a fluid chamber formed in the interior of the device, thereby forming a fluid inlet and/or a fluid outlet. In some cases, the openings are formed in the first substrate. In some cases, the openings are formed on the first substrate and the second substrate. In some cases, the openings are formed on the first substrate, the second substrate, and the third substrate. In some cases, the opening is located on the top side of the device. In some cases, the opening is located on the bottom side of the device. In some cases, the opening is located at the first end and/or the second end of the device, and the channel extends in a direction from the first end to the second end.

The conditions under which the substrates can be bonded together are generally well understood by those skilled in the art, and such bonding of the substrates is typically performed by any of a variety of methods, the choice of which may vary depending on the nature of the substrate material used. For example, thermal bonding of substrates can be applied to many substrate materials, including, for example, glass or silica based substrates and some polymer based substrates. Such thermal bonding techniques typically involve mating the surfaces of the substrates to be bonded under conditions of elevated temperature and, in some cases, application of external pressure. The exact temperature and pressure used will generally vary depending on the nature of the material matrix used.

For example, for silica-based matrix materials, i.e. glass (borosilicate glass, pyrex) ^TM Soda lime glass, etc.), fused silica (quartz), etc., the substrates are typically thermally bonded at a temperature in the range of about 500 c to about 1400 c, and preferably about 500 c to about 1200 c. For example, soda lime glass is typically bonded at a temperature of about 550 ℃, while borosilicate glass is typically thermally bonded at a temperature of 800 ℃ or near 800 ℃. On the other hand, the quartz substrates are typically thermally bonded at a temperature of 1200 ℃ or near 1200 ℃. These bonding temperatures are typically achieved by placing the substrates to be bonded in a high temperature annealing furnace.

On the other hand, thermally bonded polymer matrices will typically use lower temperatures and/or pressures than silica-based matrices to prevent excessive melting and/or deformation of the matrix, such as flattening of the interior of the device (i.e., the fluid channel or chamber). Typically, such elevated temperatures for bonding to the polymer matrix will vary from about 80 ℃ to about 200 ℃, depending on the polymer material used, and preferably between about 90 ℃ to about 150 ℃. Because the temperature required to bond the polymer matrix is greatly reduced, such bonding can typically be performed without the need for a high temperature oven for bonding the silica-based matrix. As described in more detail below, this allows the heat source to be incorporated into a single integrated bonding system.

The bonding agent may also be used to bond the substrates together according to well known methods, which generally involve applying a layer of bonding agent between the substrates to be bonded and pressing them together until the bonding agent cures. According to these methods, various binders may be used, including, for example, commercially available UV curable binders. Alternative methods of bonding substrates together may also be used in accordance with the present invention, including, for example, sonic or ultrasonic welding and/or solvent welding of polymeric components.

Typically, a plurality of such microfluidic chips or devices will be fabricated simultaneously using, for example, "wafer-level" fabrication. For example, the polymer matrix may be embossed or molded into large separable pieces, which are then mated and bonded together. Individual devices or bonded substrates can then be separated from the larger sheet by cutting or dicing. Similarly, for silica-based substrates, individual devices may be fabricated from larger substrate wafers or plates, allowing for higher manufacturing process yields. In particular, a plurality of fluid channel structures may be fabricated on a first substrate wafer or plate, which is then covered with and bonded to a second substrate wafer or plate, and optionally, further covered with and bonded to a third substrate wafer or plate. The individual devices are then singulated from the larger substrate using known methods such as sawing, dicing, and breaking.

As described above, the top or second substrate is overlaid on the bottom or first substrate to seal the various channels and chambers. During bonding according to the methods of the present disclosure, the first substrate and the second substrate may be bonded using vacuum and/or pressure to maintain the two substrate surfaces in optimal contact. In particular, optimal contact of the bottom substrate with the top substrate may be maintained by, for example, matching the planar surface of the bottom substrate with the planar surface of the top substrate and by applying a vacuum through holes provided through the top substrate. Typically, applying vacuum to the holes in the top substrate is performed by placing the top substrate on a vacuum chuck, which typically includes a mounting table or surface with an integrated vacuum source. In the case of silica-based substrates, the bonded substrates are subjected to elevated temperatures to produce an initial bond, so that the bonded substrates can then be transferred into an annealing furnace without any offset relative to each other.

Alternative bonding systems for bonding with the devices described herein include, for example, a bonding agent dispensing system for applying a layer of bonding agent between two planar surfaces of a substrate. This may be accomplished by applying a layer of binder before the mating substrates, or by placing a quantity of binder on one edge of an adjacent substrate and allowing the wicking action of the two mating substrates to pull the binder through the space between the two substrates.

In some cases, the entire bonding system may include an automated system for placing the top and bottom substrates on a mounting surface and aligning them for subsequent bonding. Typically, such systems include a translation system for moving the mounting surface or one or more top and bottom substrates relative to each other. For example, a robotic system may be used to sequentially lift, translate and place each of the top and bottom substrates onto a mounting table and into an alignment structure. After the bonding process, such systems may also remove the finished product from the mounting surface and transfer these mated substrates to subsequent operations, such as a separation or dicing operation, an annealing oven for silica-based substrates, etc., and then place additional substrates thereon for bonding.

In some cases, the fabrication of microfluidic chips involves layering or laminating two or more layers of a substrate (e.g., patterned and non-patterned polymer sheets) to produce the chips. For example, in microfluidic devices, microfluidic features of the device are typically created by laser irradiation, etching, or otherwise fabricating features into the surface of the first layer. The second layer is then laminated or bonded to the surface of the first layer to seal these features and provide fluidic elements of the device, e.g., fluidic channels.

As described above, in some cases, one or more capillary flow cell devices or microfluidic chips may be mounted in a cartridge base to form a capillary flow cell cartridge or microfluidic cartridge. In some cases, the capillary flow cell cartridge or microfluidic cartridge may further include additional components integrated with the cartridge to provide enhanced performance for a particular application. Examples of additional components that may be integrated into the cartridge include, but are not limited to, adapters or connectors for fluid connection with other components of the system, fluid flow control components (e.g., micro-valves, micro-pumps, mixing manifolds, etc.), temperature control components (e.g., resistive heating elements, metal plates used as heat sources or sinks, piezoelectric (peltier) devices for heating or cooling, temperature sensors), or optical components (e.g., optical lenses, windows, filters, mirrors, prisms, optical fibers, and/or Light Emitting Diodes (LEDs) or other micro-light sources that may be used together to facilitate spectral measurement and/or imaging of one or more capillaries or fluid channels.

The fluidic adapter, cartridge mount, and other cartridge assemblies may be connected to the capillary, capillary flow cell device, microfluidic chip (or fluidic channels within the chip) using any of a variety of techniques known to those skilled in the art, including but not limited to press-fit, adhesive bonding, solvent bonding, laser welding, and the like, or any combination thereof. In some cases, the inlets and/or outlets of the microfluidic channels in the microfluidic chip are holes on the top surface of the chip, and the fluidic adapter may be attached to or coupled to the inlets and/or outlets of the microfluidic channels within the chip. In some cases, the cartridge may include additional adapters (i.e., in addition to the fluidic adapters) that mate with the chips and/or fluidic adapters and help position the chips within the cartridge. These adapters may be constructed using the same fabrication techniques and materials as outlined above for the fluid adapters.

The cartridge base (or "housing") may be made of a metallic and/or polymeric material, such as aluminum, anodized aluminum, polycarbonate (PC), acrylic (PMMA), or Ultem (PEI), although other materials are also consistent with the present disclosure. The housing may be manufactured using CNC machining and/or molding techniques and is designed such that one, two or more capillaries or microfluidic chips are constrained by the base in a fixed orientation to create one or more independent flow channels. The capillary tube or chip may be mounted in the mount using, for example, a press fit design or by mating with a compressible adapter made of silicone or fluoroelastomer. In some cases, two or more components of the cartridge base (e.g., the upper half and the lower half) are assembled using, for example, screws, clips, pliers, or other fasteners such that the two halves are separable. In some cases, two or more components of the cartridge base are assembled using, for example, a bonding agent, solvent bonding, or laser welding, such that the two or more components are permanently attached.

Flow cell surface coating: in some cases, one or more interior surfaces of capillary cavities or microfluidic channels in the disclosed flow cell devices may be coated using any of a variety of surface modification techniques or polymer coatings known to those skilled in the art. In some cases, the coating may be formulated to increase or maximize the number of available binding sites (e.g., tethered oligonucleotide adaptor/primer sequences) on one or more interior surfaces to increase or maximize the foreground signal, e.g., fluorescent signal generated by hybridization of the labeled nucleic acid molecule to the tethered oligonucleotide adaptor/primer sequences. In some cases, the coating may be formulated to reduce or minimize non-specific binding of fluorophores to other small molecules or labeled or unlabeled nucleotides, proteins, enzymes, antibodies, oligonucleotides, or nucleic acid molecules (e.g., DNA, RNA, etc.) to reduce or minimize background signals, such as background fluorescence resulting from non-specific binding of labeled biomolecules or autofluorescence of sample carrier structures. The combination of increased foreground and decreased background signals, which in some cases may be achieved by using the disclosed coatings, may thus provide improved signal-to-noise ratio (SNR) in spectral measurements or improved contrast-to-noise ratio (CNR) in imaging methods.

As will be discussed in more detail below, the disclosed hydrophilic polymer coated flow cell devices (optionally used in combination with improved hybridization and/or amplification protocols) produce solid phase bioassay reactions that exhibit: (i) negligible non-specific binding of proteins to other reaction components (thereby reducing or minimizing matrix background), (ii) negligible non-specific nucleic acid amplification products, and (iii) providing a tunable nucleic acid amplification reaction. Although described herein primarily in the context of nucleic acid hybridization, amplification, and sequencing assays, those skilled in the art will appreciate that the disclosed low-binding vectors can be used in any of a variety of other bioassay formats, including, but not limited to, sandwich immunoassays, enzyme-linked immunosorbent assays (ELISA), and the like.

In a preferred aspect, one or more layers of coating material may be applied to the interior flow cell device surface, wherein the number of layers and/or the material composition of each layer is selected to adjust one or more surface properties of the interior flow cell device surface, as described in U.S. patent application Ser. No. 16/363,842. Examples of surface properties that can be modulated include, but are not limited to, surface hydrophilicity/hydrophobicity, total coating thickness, surface density of chemically reactive functional groups, surface density of grafted linker molecules or oligonucleotide adaptors/primers, and the like. In some preferred applications, one or more surface properties of the capillary or channel lumens are adjusted, for example, (i) to provide very low non-specific binding of proteins, oligonucleotides, fluorophores, and other molecular components for chemical or biological analysis applications, including solid phase nucleic acid amplification and/or sequencing applications, (ii) to provide improved solid phase nucleic acid hybridization specificity and efficiency, and (iii) to provide improved solid phase nucleic acid amplification rate, specificity, and efficiency.

Any of a variety of molecules known to those skilled in the art (including but not limited to silanes, amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof) may be used to create one or more chemically modified layers on the surface of the internal flow cell device, wherein the choice of components used may be varied to alter one or more properties of the support surface, such as the surface density of the functional and/or tethered oligonucleotide primers, the hydrophilicity/hydrophobicity of the support surface, or three dimensional properties (i.e., the "thickness") of the support surface.

The attachment chemistry used to graft the first chemically modified layer to the inner surface of the flow cell (capillary or channel) generally depends on both the material from which the flow cell device is made and the chemistry of the layer. In some cases, the first layer may be covalently attached to the internal flow cell device surface. In some cases, the first layer may be non-covalently attached, e.g., adsorbed, to the surface, e.g., by non-covalent interactions, such as electrostatic interactions, hydrogen bonds, or van der Waals interactions between the surface of the first layer and the molecular components. In either case, the substrate surface may be treated prior to attaching or depositing the first layer. Any of a variety of surface treatment techniques known to those skilled in the art may be used to clean or treat the surface of the carrier. For example, piranha solution (sulfuric acid (H) ₂ SO ₄ ) And hydrogen peroxide (H) ₂ O ₂ ) A mixture of (c) acid-washed glass or silicon surfaces and/or cleaned using an oxygen plasma treatment process.

Silane chemistry constitutes a non-limiting method for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amine groups or carboxyl groups), which can then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths such as C6, cl2, C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to create any of the disclosed low-binding support surfaces include, but are not limited to, (3-aminopropyl) trimethoxysilane (APTMS), (3-aminopropyl) triethoxysilane (APTES), and any of a variety of PEG-silanes (e.g., having a molecular weight of 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., having free amino functionality), maleimide-PEG silanes, biotin-PEG silanes, and the like.

Examples of preferred polymers that can be used to create one or more layers of low non-specific binding materials in any of the disclosed support surfaces include, but are not limited to, polyethylene glycols (PEG) of various molecular weights and branching structures, streptavidin, polyacrylamide, polyesters, dextran, polylysine, and polylysine copolymers, or any combination thereof. Examples of conjugation chemistries that may be used to graft the material of one or more layers (e.g., the polymer layer) to the support surface and/or crosslink the layers to each other include, but are not limited to, biotin-streptavidin interactions (or variants thereof), his tag-Ni/NTA conjugation chemistries, methoxy ether conjugation chemistries, carboxylate conjugation chemistries, amine conjugation chemistries, NHS esters, maleimides, thiols, epoxies, azides, hydrazides, alkynes, isocyanates, and silanes.

In some cases, the number of layers of polymer or other chemical layers on the surface of the internal flow cell device may be in the range of 1 to about 10 or greater than 10. In some cases, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the number of layers may be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of the lower and upper values described in this paragraph may be combined to form a range encompassed by the present disclosure, e.g., in some cases, the number of layers may be in the range of about 2 to about 4. In some cases, one or more layers may all comprise the same material. In some cases, each layer may comprise a different material. In some cases, the plurality of layers may comprise a plurality of materials.

One or more of the layers of the multi-layer surface may comprise branched polymers or may be linear. Examples of suitable branched polymers include, but are not limited to: branched PEG, branched polyvinyl alcohol (branched PVA), branched poly (vinylpyridine), branched poly (vinylpyrrolidone) (branched PVP), branched), poly (acrylic acid) (branched PAA), branched polyacrylamide, branched poly (N-isopropylacrylamide) (branched PNIPAM), branched poly (methyl methacrylate) (branched PMA), branched poly (2-hydroxyethyl methacrylate) (branched PHEMA), branched poly (oligo (ethylene glycol) monomethyl ether methacrylate (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched polyglucoside, and dextran.

In some cases, the branched polymer used to create one or more layers of any of the multi-layer surfaces disclosed herein can include at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches. Molecules typically exhibit a "power of 2" number of branches, e.g., 2, 4, 8, 16, 32, 64, or 128 branches.

In some cases, after deposition of one or more layers, such as a polymer layer, the resulting functional end groups remote from the surface may include, but are not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide, and disilane.

The molecular weight of the linear, branched, or multi-branched polymer used to form one or more layers of any of the multi-layer surfaces disclosed herein can be at least 500 daltons, at least 1,000 daltons, at least 1,500 daltons, at least 2,000 daltons, at least 2,500 daltons, at least 3,000 daltons, at least 3,500 daltons, at least 4,000 daltons, at least 4,500 daltons, at least 5,000 daltons, at least 7,500 daltons, at least 10,000 daltons, at least 12,500 daltons, at least 15,000 daltons, at least 17,500 daltons, at least 20,000 daltons, at least 25,000 daltons, at least 30,000 daltons, at least 35,000 daltons, at least 40,000 daltons, at least 45,000 daltons, or at least 50,000 daltons. In some cases, the molecular weight of the linear, branched, or multi-branched polymer used to form one or more layers of any of the multi-layer surfaces disclosed herein can be at most 50,000 daltons, at most 45,000 daltons, at most 40,000 daltons, at most 35,000 daltons, at most 30,000 daltons, at most 25,000 daltons, at most 20,000 daltons, at most 17,500 daltons, at most 15,000 daltons, at most 12,500 daltons, at most 10,000 daltons, at most 7,500 daltons, at most 5,000 daltons, at most 4,500 daltons, at most 4,000 daltons, at most 3,500 daltons, at most 3,000 daltons, at most 2,500 daltons, at most 2,000 daltons, at most 1,500 daltons, at most 1,000 daltons, or at most 500 daltons. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed within this disclosure, for example, in some cases the molecular weight of the linear, branched, or multi-branched polymer used to form any one or more of the layers of any of the multi-layer surfaces disclosed herein can be in the range of about 1,500 daltons to about 20,000 daltons. Those skilled in the art will recognize that the molecular weight of the linear, branched, or multi-branched polymer used to produce one or more layers of any of the multi-layer surfaces disclosed herein can have any number within this range, for example, about 1,260 daltons.

In some cases, two or more layers may be covalently coupled or internally crosslinked to each other to improve the stability of the resulting surface. In some cases, for example, where at least one layer of the multi-layer surface comprises a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the previous layer may be in the range of about one covalent bond per molecule and about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, or more than 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may be at most 32, at most 30, at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, for example, in some cases the number of covalent bonds between a branched polymer molecule of a new layer and a molecule of a previous layer can range from about 4 to about 16. Those skilled in the art will recognize that the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may have any number within this range, for example, about 11 in some cases, or an average value of about 4.6 in other cases.

Any reactive functional groups remaining after the material layer is coupled to the internal flow cell device surface can be selectively blocked by coupling small inert molecules using high yield coupling chemistry. For example, where amine coupling chemistry is used to attach a new material layer to a previous layer, any remaining amine groups can then be acetylated or deactivated by coupling with a small amino acid (e.g., glycine).

To scale the binding site surface density, e.g., oligonucleotide adapter/primer surface density and increase the dimension for hydrophilic or amphiphilic surfaces, matrices have been developed that include multiple layers of coatings of PEG and other hydrophilic polymers. The adaptor/primer loading density on the surface can be significantly increased by using hydrophilic and amphiphilic surface layering methods (including, but not limited to, the polymer/copolymer materials described below). Traditional PEG coating methods use monolayer primer deposition, which has been tested and reported for single molecule sequencing applications, but do not produce high copy numbers in nucleic acid amplification applications. As described herein, "layering" may be accomplished using any compatible polymer or monomer subunits using conventional crosslinking methods such that a surface comprising two or more highly crosslinked layers may be built up sequentially. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some cases, the different layers may be cross-linked to each other by various conjugation reactions including, but not limited to, biotin-streptavidin binding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, ionic interactions between positively and negatively charged polymers. In some cases, the high adaptor/primer density material may be built up in solution and then layered on the surface by multiple steps.

Exemplary PEG multilayers include PEG (8-arm, 16-arm, 8-arm) on PEG-amine-APTES. Similar concentrations were observed for 3 layers of multi-arm PEG (8 arm, 16 arm, 8 arm) and (8 arm, 64 arm, 8 arm) on PEG-amine-APTES exposed to 8uM primer and 3 layers of multi-arm PEG (8 arm ) using star PEG-amine instead of 16 arm and 64 arm. PEG multilayers having comparable first, second, and third PEG layers are also contemplated.

In some cases, the resulting surface density of binding sites on the surface of the internal flow cell device, e.g., oligonucleotide adaptors/primer surface density, can be in the range of about 100 primer molecules/μm ² To about 1,000,000 primer molecules/μm ² Within a range of (2). In some of the cases where the number of the cases, the surface density of binding sites can be at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules/μm ² . In some cases, the surface density of binding sites may be at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000. Up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to at most 15,000, at most 10,000, at most 9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, at most 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, or at most 100 molecules/μm ² . Any of the lower and upper values described in this paragraph can be combined to form a range encompassed within this disclosure, e.g., in some cases, the surface density of binding sites can be in the range of about 10,000 molecules to about 100,000 molecules/μm ² Within a range of (2). Those skilled in the art will recognize that the surface density of binding sites can have any value within this range, for example, in some cases about 3,800 molecules/μm ² In other cases about 455,000 molecules/μm ² . In some cases, as will be discussed further below for nucleic acid sequencing applications, the surface density of template library nucleic acid sequences (e.g., sample DNA molecules) that are initially hybridized to the adapter or primer sequences tethered to the surface of the internal flow cell device may be less than or equal to the surface density indicated for the binding sites. In some cases, as will also be discussed further below, the surface density of clonally amplified template library nucleic acid sequences that hybridize to adapter or primer sequences on the surface of the internal flow cell device may span the same range or different ranges as indicated by the surface density of tethered oligonucleotide adapters or primers.

As outlined above, the localized surface density of binding sites on the surface of the internal flow cell device does not exclude that the whole surface isThe density varies, and thus the surface may include binding sites with a density of, for example, 500,000/um ² And further comprises at least a second region having a substantially different local surface density.

In some cases, capture probes, e.g., oligonucleotide primers (or other biomolecules, such as enzymes or antibodies) having different base sequences and base modifications, can be tethered to one or more layers of the resulting surface at various surface densities. In some cases, for example, both the surface functional group density and the capture probe concentration used for coupling may be varied to target a certain capture probe surface density range. In addition, the surface density of the capture probes can be controlled by diluting the capture probes with other "inert" molecules bearing the same reactive functional groups for coupling to the surface. For example, amine-labeled oligonucleotide probes may be diluted with amine-labeled polyethylene glycol in a reaction with NHS ester-coated surfaces to reduce final primer density. In the case of oligonucleotide adaptors/primers, probe sequences having linkers of different length between the hybridization region and the surface attachment functionality can also be used to alter the surface density. Examples of suitable linkers include poly (thymidylate) (poly-T) and poly (poly-A) chains (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.) at the 5' end of the primer. To measure or estimate the capture probe surface density, a fluorescently labeled capture probe can be tethered to the surface, then a fluorescent reading is taken, and then compared to a calibration solution containing a known concentration of fluorophores.

In some cases, the degree of hydrophilicity (or "wettability" in the case of aqueous solutions) of the disclosed support surfaces (e.g., inner flow cell device surfaces) can be assessed, for example, by measuring water contact angles, wherein a droplet of water is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some cases, the static contact angle may be determined. In some cases, the advancing or receding contact angle may be determined. In some cases, the water contact angle of the hydrophilic, low-binding support surfaces disclosed herein can be in the range of about 0 degrees to about 50 degrees. In some cases, the hydrophilic, low-binding support surfaces disclosed herein can have a water contact angle of no more than 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed any value within this range, for example, 40 degrees. Those skilled in the art will recognize that a given hydrophilic, low-binding support surface of the present disclosure may exhibit a water contact angle having any number within this range, for example, about 27 degrees. In some cases, the disclosed low non-specific binding surfaces have a water contact angle of less than 45 degrees. In some cases, the disclosed low non-specific binding surfaces have a water contact angle of less than 35 degrees.

As noted, the hydrophilic coated internal flow cell device surfaces of the present disclosure exhibit reduced non-specific binding of proteins, nucleic acids, fluorophores, and other components of biological and biochemical assay methods. The extent of non-specific binding exhibited by a given support surface (e.g., the inner flow cell device surface) can be assessed qualitatively or quantitatively. For example, in some cases, under a standard set of conditions, the surface may be exposed to a fluorescent dye (e.g., cyanine dye 3 (Cy 3), cyanine dye 5 (Cy 5), etc.), a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and/or a fluorescently labeled protein (e.g., polymerase), followed by a designated wash procedure and fluorescence imaging to serve as a qualitative tool for comparing non-specific binding on a carrier comprising different surface preparations. In some cases, under a standard set of conditions, the surface may be exposed to a fluorescent dye, a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and/or a fluorescently labeled protein (e.g., a polymerase), followed by a designated wash procedure and fluorescence imaging as quantitative tools for comparing non-specific binding on a support comprising different surface preparations-provided that fluorescent imaging is performed using appropriate calibration standards under conditions where the fluorescent signal is linearly related (or related in a predictable manner) to the number of fluorophores on the support surface (e.g., under conditions where signal saturation and/or self-quenching of the fluorophores is not problematic). In some cases, other techniques known to those skilled in the art, such as radioisotope labeling and counting methods, can be used to quantitatively evaluate the extent of non-specific binding exhibited by the different carrier surface preparations of the present disclosure.

In some cases, the degree of non-specific binding exhibited by the disclosed low non-specific binding carrier surfaces can be assessed using a standardized procedure for contacting the surface with a labeled protein (e.g., bovine Serum Albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotide, labeled oligonucleotide, etc., under a standard set of incubation and rinsing conditions, followed by detecting the amount of label remaining on the surface, and comparing the signal resulting therefrom to an appropriate calibration standard. In some cases, the label may comprise a fluorescent label. In some cases, the label may comprise a radioisotope. In some cases, the label may comprise any other detectable label known to those of skill in the art. In some cases, the degree of non-specific binding exhibited by a given carrier surface preparation can thus be assessed in terms of the number of non-specifically bound protein molecules (or other molecules) per unit area. In some cases, the low non-specific binding carriers of the present disclosure may exhibit non-specific protein binding (or non-specific binding of other designated molecules such as cyanine dye 3 (Cy 3)) of less than 0.001 molecules/μm ² Less than 0.01 molecules/μm ² Less than 0.1 molecules/μm ² Less than 0.25 molecules/μm ² Less than 0.5 molecules/μm ² Less than 1 molecule/μm ² Less than 10 molecules/μm ² Less than 100 molecules/μm ² Or less than 1,000 molecules/μm ² . Those skilled in the art will recognize that a given support surface of the present disclosure may exhibit non-specific binding anywhere within this range, e.g., less than 86 molecules/μm ² . For example, 1uM of Cy 3-labeled streptavidin (GE Amersham) solution is contacted for 15 minutes in Phosphate Buffer (PBS) buffer and then usedAfter 3 ion water rinses, certain modified surfaces disclosed herein exhibit non-specific protein binding below 0.5 molecules/μm ² . Certain modified surfaces disclosed herein exhibit non-specific binding of Cy3 dye molecules below 0.25 molecules/μm ² . In a separate non-specific binding assay, 1. Mu.M of labeled Cy3 SA (ThermoFisher), 1uM Cy5 SA dye (ThermoFisher), 10uM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10uM aminoallyl-dUTP-ATTO-Rho 11 (Jena Biosciences), 10uM 7-propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10uM 7-propargylamino-7-deaza-dGTP-Cy 3 (Jena Biosciences) were incubated as 384 well plates on a low non-specific binding substrate for 15 minutes at 37 ℃. Each well was rinsed 2-3 times with 50ul of deionized RNase/DNase free water and 2-3 times with 25mm ACES buffer (pH 7.4). 384 well plates were imaged on a GE Typhoon (GE Healthcare Lifesciences, pittsburgh, PA) instrument using a manufacturer specified Cy3, AF555, or Cy5 filter bank (depending on the dye test performed), with PMT gain set at 800 and resolution of 50-100 μm. For higher resolution imaging, images were collected on an Olympus IX83 microscope (OlympusCorp., centerValley, PA) with a Total Internal Reflection Fluorescence (TIRF) objective (20 x, 0.75NA or 100 x, 1.5NA, olympus), sCMOS Andor camera (zyla4.2. Dichroic mirror from Semrock (IDEX Health) &Science, LLC, rochester, new york), e.g., 405, 488, 532, or 633nm dichroic reflector/beamsplitter), and a bandpass filter is selected as 532LP or 645LP, which coincides with the appropriate excitation wavelength. Some modified surfaces disclosed herein exhibit less than 0.25 molecules/μm ² Non-specific binding of dye molecules of (a).

In some cases, the coated flow cell device surfaces disclosed herein can exhibit a ratio of specific to non-specific binding of a fluorophore, such as Cy3, of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the scope herein.

In some cases, one or more surface modifications and/or polymer layers may be applied to the inner flow cell device surface using techniques such as Chemical Vapor Deposition (CVD). In some cases, one or more surface modification and/or polymer layers may be applied to the inner flow cell device surface by flowing one or more suitable chemical coupling agents or coating agents through the capillary or fluid channel prior to use for the intended purpose. In some cases, one or more coating agents may be added to the buffers used, such as nucleic acid hybridization, amplification reactions, and/or sequencing reaction buffers, to dynamically coat the internal flow cell device surfaces.

In some cases, the chemically modified layer may be uniformly applied to the surface of the substrate or carrier structure. Alternatively, the surface of the substrate or support structure may be unevenly distributed or patterned such that the chemically modified layer is confined to one or more discrete regions of the substrate. For example, the substrate surface may be patterned using photolithographic techniques to form an ordered array or random pattern of chemically modified areas on the surface. Alternatively or in combination, the substrate surface may be patterned using, for example, contact printing and/or inkjet printing techniques. In some cases, the ordered array or random pattern of chemically modified discrete regions may comprise at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions, or any intermediate number of discrete regions within the scope herein.

In some cases, when nucleic acid molecules are labeled with Cy3 and images are acquired using an Olympus IX83 inverted fluorescence microscope equipped with a 20 x 0.75NA objective lens, 532nm light source, bandpass and dichroic mirror set adjusted or optimized for 532nm long-pass excitation and Cy3 fluorescence emission filter, semrock 532nm dichroic mirror and camera (andoscmos, zyla 4.2) when used in, for example, nucleic acid hybridization or amplification applications to produce clusters of hybridized or clonally amplified nucleic acid molecules (e.g., a "discrete region" that has been labeled directly or indirectly with a fluorophore), the disclosed low non-specific binding surfaces exhibit contrast to noise ratios (CNR) of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250 or greater than 250, wherein the excitation light intensity is adjusted to avoid saturation of the es surface when the excitation signal is acquired, e.g., in a ph buffer solution (25 mm buffer). As used herein, the contrast to noise ratio (CNR) is calculated as:

Cnr= (S-B)/noise

Where S = foreground signal (e.g., is a fluorescent signal measured from an image generated from a labeled nucleic acid clone or cluster on the surface of the sample carrier), B = background signal (where B = B _{Interstitial substance} +B _{Intracellular} )，B _{Interstitial substance} =background signal measured at positions on the surface of sample carrier between labeled nucleic acid clones or clusters, B _{Intracellular} Background signal measured at the location of a nucleic acid clone or cluster (e.g., determined by contacting the sample carrier surface with labeled non-complementary oligonucleotides and measuring the resulting fluorescence) and noise = signal noise. For example, the contrast to noise ratio (CNR) of images of sequencing surfaces provides a key indicator for assessing nucleic acid amplification specificity and non-specific binding on a carrier. While signal-to-noise ratio (SNR) is generally considered a benchmark for overall signal quality, it can be shown that improved CNR has significant advantages over SNR as a benchmark for signal quality in imaging applications requiring rapid image capture (e.g., nucleic acid sequencing applications where cycle time must be minimized).

In some cases, polymer coated sample carrier structures, e.g., internal flow cell device surfaces comprising the disclosed hydrophilic polymer coatings, can exhibit improved stability to repeated exposure to solvents, temperature changes, pH changes, or long term storage.

Fluid system and fluid flow control module: in some embodiments, the disclosed imaging and/or analysis systems may provide fluid flow control capabilities to deliver a sample or reagent to one or more flow cell devices or flow cell cartridges (e.g., a single capillary flow cell device or a microfluidic channel flow cell device) connected to the system. The reagents and buffers may be stored in bottles, reagent and buffer cartridges, or other suitable containers that are connected to the flow cell inlet by tubing and valve manifolds. The disclosed system may also include a treated sample and waste reservoir in the form of a bottle, cartridge or other suitable container for collecting fluid downstream of the capillary flow cell device or capillary flow cell cartridge. In some embodiments, the fluid flow (or "fluidics") control module may provide programmable switching of flow between different sources, e.g., a sample or reagent reservoir or bottle located in the instrument, and one or more inlets to a central region (e.g., a capillary flow cell or microfluidic device, or a large fluid chamber (e.g., a large fluid chamber within a microfluidic device)). In some cases, the fluid flow control module may provide programmable switching of flow between one or more outlets from a central region (e.g., capillary flow cell or microfluidic device) and different collection points (e.g., processed sample reservoirs, waste reservoirs, etc.) connected to the system. In some cases, the sample, reagents, and/or buffer may be stored in a reservoir integrated with the flow cell cartridge or the microfluidic cartridge itself. In some cases, the treated sample, used reagents, and/or used buffers may be stored in a reservoir integrated with the flow cell cartridge or the microfluidic device cartridge itself.

In some embodiments, the one or more fluid flow control modules may be configured to control delivery of fluid to one or more capillary flow cells, capillary flow cell cartridges, microfluidic devices, microfluidic cartridges, or any combination thereof. In some cases, the one or more fluidic controllers may be configured to control the volumetric flow rate of the one or more fluids or reagents, the linear flow rate of the one or more fluids or reagents, the mixing ratio of the one or more fluids or reagents, or any combination thereof. Control of fluid flow through the disclosed system will typically be performed using pumps (or other fluid actuation mechanisms) and valves (e.g., programmable pumps and valves). Examples of suitable pumps include, but are not limited to, syringe pumps, programmable syringe pumps, peristaltic pumps, diaphragm pumps, and the like. Examples of suitable valves include, but are not limited to, check valves, electromechanical two-way or three-way valves, pneumatic two-way and three-way valves, and the like. In some cases, the flow of fluid through the system may be controlled by applying positive air pressure to one or more inlets of the reagent and buffer containers or to one or more inlets incorporated into a flow cell cartridge (e.g., capillary flow cell or microfluidic cartridge). In some embodiments, the flow of fluid through the system may be controlled by drawing a vacuum at one or more outlets of the waste reservoir or at one or more outlets incorporated into a flow cell cartridge (e.g., capillary flow cell or microfluidic cartridge).

In some cases, different fluid flow control modes are used at different points in the assay or analysis procedure, such as forward flow (with respect to the inlet and outlet of a given capillary flow cell device), reverse flow, oscillatory or pulsatile flow, or a combination thereof. In some applications, for example, during an analytical wash/rinse step, oscillatory or pulsatile flow may be employed to facilitate complete or efficient exchange of fluids within one or more flow cell devices or flow cell cartridges (e.g., capillary flow cell devices or cartridges and microfluidic devices or cartridges).

Similarly, in some cases, different fluid flow rates may be used at different locations within the flow cell device or at different points in the assay or analysis process workflow, for example, in some cases, the volumetric flow rate may vary from-100 ml/s to +100 ml/s. In some embodiments, the absolute value of the volumetric flow rate may be at least 0.001 ml/sec, at least 0.01 ml/sec, at least 0.1 ml/sec, at least 1 ml/sec, at least 10 ml/sec, or at least 100 ml/sec. In some embodiments, the absolute value of the volumetric flow rate may be at most 100 ml/sec, at most 10 ml/sec, at most 1 ml/sec, at most 0.1 ml/sec, at most 0.01 ml/sec, or at most 0.001 ml/sec. The volumetric flow rate of the flow cell device at a given location or point in time may have any value within this range, for example a forward flow rate of 2.5ml/s, a reverse flow rate of-0.05 ml/s, or a value of 0ml/s (i.e., stop flow).

In some embodiments, the fluidic system may be designed to minimize the consumption of critical reagents (e.g., expensive reagents) required to perform, for example, genomic analysis applications. For example, in some embodiments, the disclosed fluidic system may include a first reservoir containing a first reagent or solution, a second reservoir containing a second reagent or solution, and a central region (e.g., a central capillary flow cell or microfluidic device), wherein an outlet of the first reservoir and an outlet of the second reservoir are fluidly coupled to an inlet of the central capillary flow cell or microfluidic device through at least one valve such that a volume of the first reagent or solution flowing per unit time from the outlet of the first reservoir to the inlet of the central capillary flow cell or microfluidic device is less than a volume of the second reagent or solution flowing per unit time from the outlet of the second reservoir to the inlet of the central region. In some embodiments, the first reservoir and the second reservoir may be integrated into a capillary flow cell cartridge or a microfluidic cartridge. In some cases, at least one valve may also be integrated into a capillary flow cell cartridge or microfluidic cartridge.

In some cases, the first reservoir is fluidly coupled to the central capillary flow cell or microfluidic device by a first valve and the second reservoir is fluidly coupled to the central capillary flow cell or microfluidic device by a second valve. In some cases, the first and/or second valve may be, for example, a diaphragm valve, pinch valve, gate valve, or other suitable valve. In some cases, the first reservoir is positioned proximate to the inlet of the central capillary flow cell or the microfluidic device to reduce the dead volume for delivering the first reagent solution. In some cases, the first reservoir is placed closer to the inlet of the central capillary flow cell or microfluidic device than the second reservoir. In some cases, the first reservoir is positioned in close proximity to the second valve so as to reduce dead volume for delivering the first reagent relative to dead volume for delivering the plurality of "second" reagents (e.g., two, three, four, five, or six or more "second" reagents) from the plurality of "second" reservoirs (e.g., two, three, four, five, or six or more "second" reservoirs).

The first reservoir and the second reservoir described above may be used to hold the same or different reagents or solutions. In some cases, the first reagent contained in the first reservoir is different from the second reagent contained in the second reservoir, and the second reagent includes at least one reagent that is commonly used by a plurality of reactions occurring in a central capillary flow cell or microfluidic device. In some cases, for example, in a fluidic system configured for performing nucleic acid sequencing chemistry within a central capillary flow cell or a microfluidic device, the first reagent comprises at least one reagent selected from the group consisting of: polymerases, nucleotides and nucleotide analogs. In some cases, the second reagent comprises a low cost reagent, such as a solvent.

In some cases, the internal volume of the central region (e.g., a central capillary flow cell cartridge or a microfluidic device comprising one or more fluidic channels or fluidic chambers) may be adjusted based on the particular application to be performed (e.g., nucleic acid sequencing). In some embodiments, the central region comprises an internal volume suitable for sequencing a eukaryotic genome. In some embodiments, the central region comprises an internal volume suitable for sequencing a prokaryotic genome. In some embodiments, the central region comprises an internal volume suitable for sequencing a viral genome. In some embodiments, the central region comprises an internal volume suitable for sequencing the transcriptome. For example, in some embodiments, the internal volume of the central region may include a volume of less than 0.05 μl, between 0.05 μl and 0.1 μl, between 0.05 μl and 0.2 μl, between 0.05 μl and 0.5 μl, between 0.05 μl and 0.8 μl, between 0.05 μl and 1 μl, between 0.05 μl and 1.2 μl, between 0.05 μl and 1.5 μl, between 0.1 μl and 1.5 μl, between 0.2 μl and 1.5 μl, between 0.5 μl and 1.5 μl, between 0.8 μl and 1.5 μl, between 1 μl and 1.5 μl, between 1.2 μl and 1.5 μl, or greater than 1.5 μl, or a range defined by any two of the foregoing. In some embodiments, the internal volume of the central region may include a volume of less than 0.5 μl, between 0.5 μl and 1 μl, between 0.5 μl and 2 μl, between 0.5 μl and 5 μl, between 0.5 μl and 8 μl, between 0.5 μl and 10 μl, between 0.5 μl and 12 μl, between 0.5 μl and 15 μl, between 1 μl and 15 μl, between 2 μl and 15 μl, between 5 μl and 15 μl, between 8 μl and 15 μl, between 10 μl and 15 μl, between 12 μl and 15 μl, or greater than 15 μl, or a range defined by any two of the foregoing. In some embodiments, the internal volume of the central region may include a volume of less than 5 μl, between 5 μl and 10 μl, between 5 μl and 20 μl, between 5 μl and 500 μl, between 5 μl and 80 μl, between 5 μl and 100 μl, between 5 μl and 120 μl, between 5 μl and 150 μl, between 10 μl and 150 μl, between 20 μl and 150 μl, between 50 μl and 150 μl, between 80 μl and 150 μl, between 100 μl and 150 μl, between 120 μl and 150 μl, or greater than 150 μl, or a range defined by any two of the foregoing. In some embodiments, the internal volume of the central region may include a volume of less than 50 μl, between 50 μl and 100 μl, between 50 μl and 200 μl, between 50 μl and 500 μl, between 50 μl and 800 μl, between 50 μl and 1000 μl, between 50 μl and 1200 μl, between 50 μl and 1500 μl, between 100 μl and 1500 μl, between 200 μl and 1500 μl, between 500 μl and 1500 μl, between 800 μl and 1500 μl, between 1000 μl and 1500 μl, between 1200 μl and 1500 μl, or greater than 1500 μl, or a range defined by any two of the foregoing. In some embodiments, the internal volume of the central region may include a volume of less than 500 μl, between 500 μl and 1000 μl, between 500 μl and 2000 μl, between 500 μl and 5ml, between 500 μl and 8ml, between 500 μl and 10ml, between 500 μl and 12ml, between 500 μl and 15ml, between 1ml and 15ml, between 2ml and 15ml, between 5ml and 15ml, between 8ml and 15ml, between 10ml and 15ml, between 12ml and 15ml, or greater than 15ml, or any two of the above defined ranges. In some embodiments, the internal volume of the central region may include a volume of less than 5ml, between 5ml and 10ml, between 5ml and 20ml, between 5ml and 50ml, between 5ml and 80ml, between 5ml and 100ml, between 5ml and 120ml, between 5ml and 150ml, between 10ml and 150ml, between 20ml and 150ml, between 50ml and 150ml, between 80ml and 150ml, between 100ml and 150ml, between 120ml and 150ml, or greater than 150ml, or a range defined by any two of the foregoing. In some embodiments, the systems described herein comprise an array or collection of flow cell devices or systems comprising a plurality of discrete capillaries, microfluidic channels, fluidic channels, chambers, or luminal regions, wherein the combined internal volume is, comprises or includes one or more values within the scope disclosed herein.

In some cases, the ratio of the volumetric flow rate for delivering the first reagent to the central capillary flow cell or microfluidic device to the volumetric flow rate for delivering the second reagent to the central capillary flow cell or microfluidic device may be less than 1:20, less than 1:16, less than 1:12, less than 1:10, less than 1:8, less than 1:6, or less than 1:2. In some cases, the ratio of the volumetric flow rate for delivering the first reagent to the central capillary flow cell or microfluidic device to the volumetric flow rate for delivering the second reagent to the central capillary flow cell or microfluidic device may have any value within the range spanned by these values, e.g., less than 1:15.

As noted, the flow cell devices and/or fluidic systems disclosed herein may be configured to enable more efficient use of reagents than is achieved by, for example, other sequencing devices and systems, particularly for various uses of expensive reagents in various sequencing chemistry steps. In some cases, the first reagent comprises a more expensive reagent than the second reagent. In some cases, the first reagent comprises a reaction-specific reagent and the second reagent comprises a non-specific reagent common to all reactions performed in the central capillary flow cell or microfluidic device region, and wherein the reaction-specific reagent is more expensive than the non-specific reagent.

In some cases, advantages may be conveyed in terms of reducing consumption of expensive reagents using the flow cell devices and/or fluid systems disclosed herein. In some cases, for example, utilizing the flow cell devices and/or fluid systems disclosed herein can result in at least a 5%, at least 7.5%, at least 10%, at least 12.5%, at least 15%, at least 17.5%, at least 20%, at least 22.5%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% reduction in reagent consumption compared to reagent consumption encountered when operating, for example, current commercial nucleic acid sequencing systems.

Fig. 31 shows a non-limiting example of a simple fluidic system that includes a single capillary flow cell connected to various fluid flow control assemblies, where the single capillary is light-accessible and compatible in various imaging applications with mounting in a microscope stage or custom imaging instrument for various imaging applications. A plurality of reagent reservoirs are fluidly coupled to the inlet end of a single capillary flow cell device, wherein the flow of reagent through the capillaries at any given point in time is controlled by a programmable rotary valve that allows a user to control the timing and duration of reagent flow. In this non-limiting example, the fluid flow is controlled by means of a programmable syringe pump that provides precise control and timing of the volumetric fluid flow and fluid flow rate.

Fig. 32 shows a non-limiting example of a fluidic system that includes a capillary flow cell cartridge with an integrated diaphragm valve (for reducing or minimizing dead volume and preserving certain critical reagents). The integration of a miniature diaphragm valve into the cartridge allows the valve to be positioned against the inlet of the capillary tube, thereby reducing or minimizing dead volume within the device and reducing the consumption of expensive reagents. Integration of valves and other fluid control components in capillary flow cell cartridges also allows for the integration of larger fluid flow control functions into the cartridge design.

FIG. 33 illustrates a non-limiting example of a capillary flow cell cartridge-based fluidic system for use in combination with a microscope device, the system including a capillary flow cell, a microscope device, and a temperature control mechanism. Wherein the cartridge is bonded or mated with a temperature control component, such as a metal plate, that is in contact with the capillaries within the cartridge and serves as a heat source/sink. The microscope device consists of an illumination system (e.g. comprising a laser, LED or halogen lamp, etc. as light source), an objective lens, an imaging system (e.g. CMOS or CCD camera) and a translation (for moving the cartridge relative to the optical system), which allows for example the acquisition of fluorescence and/or bright field images of different areas of the capillary flow cell as the stage moves.

And a temperature control module: in some embodiments, the disclosed systems will include temperature control functionality to facilitate the accuracy and repeatability of assay or analytical results. Examples of temperature control components that may be incorporated into an instrument system (or capillary flow cell cartridge) design include, but are not limited to, resistive heating elements, infrared light sources, peltier heating or cooling devices, heat sinks, thermistors, thermocouples, and the like. In some cases, a temperature control module (or "temperature controller") may provide programmable temperature changes at specified, adjustable times prior to performing particular assay or analysis steps. In some cases, the temperature controller may provide programmable temperature changes over a specified time interval. In some embodiments, the temperature controller may further provide a temperature cycle between two or more set temperatures having a specified frequency and slope, so that a thermal cycle for the amplification reaction may be performed.

Fig. 34 shows one non-limiting example of controlling the temperature of a flow cell (e.g., a capillary flow cell or a microfluidic device-based flow cell) by using a metal plate placed in contact with the flow cell cartridge. In some cases, the metal plate may be integrated with the cassette base. In some cases, a peltier or resistive heater may be used to control the temperature of the metal plate.

FIG. 35 illustrates one non-limiting method for temperature control of a flow cell (e.g., a capillary or microfluidic channel flow cell) that includes a non-contact thermal control mechanism. In this method, an air temperature control system is used to direct a temperature controlled air stream through a flow cell cartridge (e.g., toward a single capillary flow cell device or a microfluidic channel flow cell device). The air temperature control system includes a heat exchanger (e.g., a resistive heater coil), a heat sink attached to the peltier device, etc., that is capable of heating and/or cooling air and maintaining it at a user-specified constant temperature. The air temperature control system also includes an air delivery device, such as a fan, that directs a heated or cooled air stream to the capillary flow cell box. In some cases, the air temperature control system may be set to a constant temperature T1 such that the air flow and thus the flow cell or cassette (e.g., capillary flow cell or microfluidic channel flow cell) is maintained at a constant temperature T2, depending on the ambient temperature, air flow rate, etc., in some cases T2 may be different from the set temperature T1. In some cases, two or more such air temperature control systems may be installed around a capillary flow cell device or flow cell cartridge to allow the capillary or cartridge to quickly cycle between a plurality of different temperatures by controlling which air temperature control system is activated at a given time. In another approach, the temperature setting of the air temperature control system may be changed, so that the temperature of the capillary flow cell or cartridge may be changed accordingly.

Fluid dispensing robot: in some embodiments, the disclosed systems may include an automated programmable fluid dispensing (or liquid dispensing) system for dispensing reagents or other solutions into, for example, microplates, capillary flow cell devices and cartridges, microfluidic devices and cartridges, and the like. Suitable automated programmable fluid dispensing systems are available from many suppliers, such as Beckman Coulter, perkin Elmer, tecan, velocity 11, and many others. In a preferred aspect of the disclosed system, the fluid dispensing system further comprises a multi-channel dispensing head, such as a 4-channel, 8-channel, 16-channel, 96-channel, or 384-channel dispensing head, for simultaneously delivering a programmable volume of liquid (e.g., ranging from about 1 microliter to several milliliters) to a plurality of wells or locations on a flow cell cartridge or microfluidic cartridge.

Cassette and/or microplate handling (pick and place) robots: in some embodiments, the disclosed systems may include a cassette and/or microplate handling robot system for automatically replacing and positioning a microplate, capillary flow cell box, or microfluidic device cassette relative to an optical imaging system, or for optionally moving a microplate, capillary flow cell box, or microfluidic device cassette between an optical imaging system and a fluid dispensing system. Suitable automated programmable microplate processing robotic systems are available from a number of suppliers including Beckman Coulter, perkin Elmer, tecan, velocity 11 and many others. In a preferred aspect of the disclosed system, an automated microplate handling robotic system is configured to move a collection of microplates containing samples and/or reagents into and out of, for example, a refrigerated storage unit.

Spectrum or imaging module: as described above, in some embodiments, the disclosed analysis systems will include optical imaging capabilities, and may also include other spectroscopic measurement capabilities. For example, the disclosed imaging modules may be configured to operate in any of a variety of imaging modes known to those skilled in the art, including, but not limited to, bright field, dark field, fluorescent, luminescent, or phosphorescent imaging. In some cases, one or more capillary flow cells or microfluidic devices of the fluidic subsystem include a window that allows at least a portion of one or more capillaries or one or more fluidic channels in each flow cell or microfluidic device to be illuminated and imaged.

In some embodiments, single wavelength excitation and emission fluorescence imaging may be performed. In some embodiments, dual wavelength excitation and emission (or multi-wavelength excitation or emission) fluorescence imaging may be performed. In some cases, the imaging module is configured to acquire video images. The choice of imaging mode may affect the design of the flow cell device or cartridge because all or part of the capillary or cartridge must be optically transparent in the spectral range of interest. In some cases, multiple capillaries in a capillary flow cell can be imaged in their entirety within a single image. In some cases, only a single capillary or subset of capillaries, or portions thereof, within a capillary flow cell can be imaged within a single image. In some cases, a series of images may be "tiled" to create a single high resolution image of one, two, several, or all of the multiple capillaries within the cartridge. In some cases, multiple fluidic channels within a microfluidic chip may be imaged in their entirety within a single image. In some cases, only a single fluidic channel or a subset of fluidic channels or portions thereof within a microfluidic chip may be imaged within a single image. In some cases, a series of images may be "tiled" to create a single high resolution image of one, two, several, or all of the multiple fluidic channels within the cartridge.

The spectroscopy or imaging module may comprise a microscope, for example a CMOS equipped with a CCD camera. In some cases, the spectroscopy or imaging module may include, for example, custom instrumentation configured to perform a particular spectroscopy or imaging technique of interest, such as one of the imaging modules described herein. In general, the hardware associated with the spectroscopy or imaging module may include a light source, a detector, and other optical components, as well as a processor or computer.

Light source: any of a variety of light sources may be used to provide imaging or excitation light including, but not limited to, tungsten filament lamps, tungsten halogen lamps, arc lamps, lasers, light Emitting Diodes (LEDs), or laser diodes. In some cases, a combination of one or more light sources with other optical components (e.g., lenses, filters, diaphragms, apertures, mirrors, etc.) may be configured as an illumination system (or subsystem).

A detector: various image sensors may be used for imaging purposes including, but not limited to, photodiode arrays, charge Coupled Device (CCD) cameras, or Complementary Metal Oxide Semiconductor (CMOS) image sensors. As used herein, an "image sensor" may be a one-dimensional (linear) or two-dimensional array sensor. In many cases, a combination of one or more image sensors with other optical components (e.g., lenses, filters, diaphragms, apertures, mirrors, etc.) may be configured as an imaging system (or subsystem). In some cases, for example, where spectroscopic measurements are performed by the system rather than imaging, suitable detectors may include, but are not limited to, photodiodes, avalanche photodiodes, and photomultiplier tubes.

Other optical components: the hardware components of the spectroscopy measurement or imaging module may also include various optical components for manipulating, shaping, filtering, or focusing the light beam passing through the system. Examples of suitable optical components include, but are not limited to, lenses, mirrors, prisms, diaphragms, diffraction gratings, colored glass filters, long pass filters, short pass filters, band pass filters, narrow band interference filters, broadband interference filters, dichroic reflectors, optical fibers, optical waveguides, and the like. In some cases, as described above, the spectroscopy measurement or imaging module may also include one or more translation stages or other motion control mechanisms to move the capillary flow cell device and cartridge relative to the illumination and/or detection/imaging subsystem, or vice versa.

Total internal reflection: in some cases, the optical module or subsystem may be designed to use all or part of the optically transparent walls of the capillaries or microfluidic channels in the flow cell device and cassette as waveguides to transmit excitation light to the capillary or channel lumens by total internal reflection. Total internal reflection occurs at a surface of a capillary or channel lumen when incident excitation light is incident at an angle relative to the surface normal that is greater than the critical angle (determined by the relative refractive indices of the capillary or channel wall material and the aqueous buffer within the capillary or channel), and the light propagates through the capillary or channel wall along the length of the capillary or channel. Total internal reflection creates an evanescent wave at the luminal surface that penetrates a very short distance inside the lumen and can be used to selectively excite fluorophores on the surface, such as labeled nucleotides that have been incorporated into growing oligonucleotides by a polymerase through a solid phase primer extension reaction.

An opaque housing and/or an environmental control chamber: in some embodiments, the disclosed systems may include an opaque housing to prevent stray ambient light from producing intense light and shielding, for example, relatively weak fluorescent signals. In some embodiments, the disclosed systems may include an environmental control chamber that enables the system to operate at tightly controlled temperatures, humidity levels, and the like.

A processor and a computer: in some cases, the disclosed systems may include one or more processors or computers. The processor may be a hardware processor, such as a Central Processing Unit (CPU), a Graphics Processing Unit (GPU), a general purpose processing unit, or a computing platform. The processor may be comprised of any of a variety of suitable integrated circuits, microprocessors, logic devices, field Programmable Gate Arrays (FPGAs), and the like. In some cases, the processor may be a single-core or multi-core processor, or multiple processors may be configured for parallel processing. Although the present disclosure has been described with reference to a processor, other types of integrated circuits and logic devices may also be applied. The processor may have any suitable data manipulation capability. For example, a processor may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations.

The processor or CPU may execute a series of machine readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location. The instructions may be directed to a CPU, which may then be programmed or otherwise configured to implement, for example, the system control methods of the present disclosure. Examples of operations performed by the CPU may include fetch, decode, execute, and write back.

Some processors may include a processing unit of a computer system. The computer system may implement cloud-based data storage and/or computing. In some cases, the computer system may be operably coupled to a computer network ("network") by way of a communication interface. The network may be the internet, an intranet and/or an extranet or a Local Area Network (LAN) in communication with the internet. In some cases, the network is a telecommunications and/or data network. The network may include one or more computer servers that may enable distributed computing, such as cloud-based computing.

The computer system may also include computer memory or memory locations (e.g., random access memory, read only memory, flash memory), electronic storage units (e.g., hard disk), communication interfaces (e.g., network adapters) for communicating with one or more other systems, and peripheral devices (e.g., cache, other storage units, data storage units, and/or electronic display adapters). In some cases, the communication interface may allow the computer to communicate with one or more additional devices. The computer may be capable of receiving input data from a coupled device for analysis. The memory unit, storage unit, communication interface, and peripheral devices may communicate with the processor or CPU through a communication bus (solid line) that may be incorporated into a motherboard, for example. The memory or storage unit may be a data storage unit (or data repository) for storing data. The memory or storage unit may store files, such as drivers, libraries, and saved programs. The memory or storage unit may store user data, such as user preferences and user programs.

The system control, image processing, and/or data analysis methods described herein may be implemented by machine-executable code stored in an electronic storage location (e.g., memory or electronic storage unit) of a computer system. The machine-executable or machine-readable code may be provided in the form of software. During use, code may be executed by a processor. In some cases, the code may be retrieved from a storage unit and stored in memory for ready access by the processor. In some cases, the electronic storage unit may be eliminated and the machine-executable instructions stored in the memory.

In some cases, the code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code. In some cases, code may be compiled during runtime. The code may be provided in a programming language that may be selected to enable execution of the code in a precompiled or just-in-time compiled (as-loaded) manner.

Some aspects of the systems and methods provided herein may be embodied in software. Aspects of the technology may be considered "articles" or "articles of manufacture" in the form of machine (or processor) executable code and/or associated data, typically carried or embodied on some type of machine readable medium. The machine executable code may be stored on an electronic storage unit, such as a memory (e.g., read only memory, random access memory, flash memory) or a hard disk. The "storage" type of medium may include any or all of the tangible memory of a computer, processor, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., that may provide non-transitory storage for software programming at any time. All or part of the software may sometimes communicate over the internet or other various telecommunications networks. Such communication may, for example, enable loading of software from one computer or processor to another computer or processor, such as from a management server or host to a computer platform of an application server. Thus, another type of medium that may carry software elements includes light waves, electric waves, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and through various air links. Physical elements carrying such waves, such as wired or wireless links, optical links, etc., may also be considered as media carrying software. As used herein, unless limited to a non-transitory, tangible "storage" medium, terms, such as computer or machine "readable medium," refer to any medium that participates in providing instructions to a processor for execution.

In some cases, the system control, image processing, and/or data analysis methods of the present disclosure may be implemented by one or more algorithms. The algorithm may be implemented in software when executed by a central processing unit.

System control software: in some cases, the system may include a computer (or processor) and a computer readable medium including code for providing a user interface and manual, semi-automatic, or fully automatic control of all system functions (e.g., controlling fluid flow control modules, temperature control modules, and/or spectroscopy or imaging modules), as well as other data analysis and display options. The system computer or processor may be an integrated component of the system (e.g., a microprocessor or motherboard embedded in an instrument) or may be a stand-alone module, such as a mainframe computer, personal computer, or portable computer. Examples of fluid flow control functions provided by system control software include, but are not limited to, volumetric fluid flow, fluid flow rate, timing and duration of sample and reagent addition, buffer addition, and flushing steps. Examples of temperature control functions provided by system control software include, but are not limited to, specifying a temperature set point, controlling the timing, duration, and rate of temperature rise of temperature changes. Examples of spectroscopic measurement or imaging control functions provided by system control software include, but are not limited to, auto-focusing capabilities, control of illumination or excitation light exposure time and intensity, image acquisition rate, control of exposure time, and data storage options.

Image processing software: in some cases, the system may also include a computer (or processor) and a computer-readable medium including code for providing image processing and analysis capabilities. Examples of image processing and analysis capabilities that may be provided by software include, but are not limited to, manual, semi-automatic, or fully automatic image exposure adjustment (e.g., white balance, contrast adjustment, signal averaging, and other noise reduction capabilities, etc.), automatic edge detection and object recognition (e.g., for identifying clonally amplified clusters of fluorescently labeled oligonucleotides on capillary flow cell device lumen surfaces), automated statistical analysis (e.g., for determining the number of clonally amplified oligonucleotide clusters identified per unit area of capillary lumen surface, or for automatic nucleotide base detection in nucleic acid sequencing applications), and manual measurement capabilities (e.g., for measuring distances between clusters or other objects, etc.). Optionally, the instrument control and image processing/analysis software may be written as separate software modules. In some embodiments, instrument control and image processing/analysis software may be incorporated into the integrated package.

Any of a variety of image processing methods known to those skilled in the art may be used for image processing/preprocessing. Examples include, but are not limited to, canny edge detection methods, canny-Deriche edge detection methods, one-step edge detection methods (e.g., sobel operators), second-order differential edge detection methods, phase-consistency (phase-coherence) edge detection methods, other image segmentation algorithms (e.g., intensity thresholding, intensity clustering methods, intensity histogram-based methods, etc.), feature and pattern recognition algorithms (e.g., generalized hough transforms for detecting arbitrary shapes, circular hough transforms, etc.), and mathematical analysis algorithms (e.g., fourier transforms, fast fourier transforms, wavelet analyses, autocorrelation methods, etc.), or any combination thereof.

Nucleic acid sequencing systems and applications: nucleic acid sequencing provides one non-limiting example of the application of the disclosed flow cell devices (e.g., capillary flow cell devices or cartridges and microfluidic devices and cartridges) and imaging systems. Many "second generation" and "third generation" sequencing techniques utilize a massively parallel cyclic array approach to sequencing by nucleotide incorporation, wherein the accurate decoding of single stranded template oligonucleotide sequences tethered to a solid support depends on successfully classified signals generated by the polymerase adding A, G, C and T nucleotides stepwise to the complementary oligonucleotide strand. These methods typically require modification of an oligonucleotide template with a fixed length of known adaptor sequence, hybridization with a surface tethered capture probe (also referred to herein as an "adaptor" or "primer" tethered to the surface of an internal flow cell) of a known sequence complementary to the adaptor sequence, immobilization on a solid support (e.g., one or more inner cavity surfaces of the disclosed capillary flow cell device or microfluidic chip) in a random or patterned array, and then detection by a series of cyclic single base addition primer extension reactions (using, for example, fluorescently labeled nucleotides) to identify the base sequence in the template oligonucleotide. Thus, these processes require the use of miniaturized fluidic systems that can provide precise, reproducible control of the timing of introduction of reagents into a flow cell performing a sequencing reaction, as well as provide small volumes to reduce or minimize the consumption of expensive reagents.

Existing commercially available NGS flow cells are composed of glass layers that have been etched, ground, and/or treated by other methods to meet the tight dimensional tolerances required for imaging, cooling, and/or other requirements. When the flow cell is used as a consumable, the expensive manufacturing process required for its manufacture results in a cost per sequencing run that is too high to allow scientists and medical professionals in the research and clinical fields to routinely perform sequencing.

The present disclosure provides examples of low cost flow cell architectures that include low cost glass or polymer capillaries or microfluidic channels, fluidic adapters, and cartridge mounts. The use of glass or polymer capillaries extruded in their final cross-sectional geometry can eliminate the need for multiple high precision and expensive glass manufacturing processes. Firmly restricting the orientation of the capillaries or microfluidic channels and providing a convenient fluidic connection using molded plastic and/or elastomeric components further reduces costs. The assembly of laser bonded polymer cartridge bases provides a fast and efficient method of sealing and structurally stabilizing capillaries or channels and flow cell cartridges without the use of fasteners or bonding agents.

The disclosed devices and systems may be configured to perform nucleotide sequencing using any of a variety of "sequencing by nucleotide incorporation", "sequencing by nucleotide binding", "sequencing by nucleotide base pairing", and "sequencing by affinity" biochemistry. Improvements in the flow cell device designs disclosed herein (e.g., including a hydrophilically coated surface that maximizes, for example, the foreground signal of a fluorescent-labeled nucleic acid cluster disposed thereon while minimizing background signal) in combination with improvements in the optical imaging system design for rapid dual-surface flow cell imaging (including simultaneous or near-simultaneous imaging of flow cell surfaces) through improved objective and/or sleeve lens designs (providing greater depth of field and greater field of view) can result in improved CNR of images for base recognition purposes, and reduced reagent consumption (achieved through improved flow cell designs) can result in significantly improved base recognition accuracy, shortened imaging cycle time, shortened overall sequencing reaction cycle time, and improved throughput nucleic acid sequencing at reduced cost per base.

The systems disclosed herein may be configured to implement any of a variety of different sequencing methods using a variety of different sequencing chemistries. For example, FIG. 40 provides a non-limiting example of a flow chart for implementing a method by affinity sequencing. The polymer-nucleotide conjugates can be used to form multivalent binding complexes in which multiple primed target nucleic acid sequences are tethered to a support surface, such as one or more interior surfaces of a flow cell, such that the multivalent binding complex exhibits a duration that is significantly longer than the duration provided by the binding interaction between a single nucleotide and a single primed target nucleic acid sequence. Typically, such a method by affinity sequencing will comprise one or more of the following steps: hybridization of the target nucleic acid sequence to an adapter/primer sequence tethered to the surface of the carrier; clonally amplifying to produce amplified clusters of target sequences on the surface of the carrier; contacting the support surface with a polymer-nucleotide conjugate comprising a plurality of nucleotide moieties conjugated to a polymer core, wherein the polymer-nucleotide conjugate may further comprise one or more detectable labels, such as fluorophores, to produce a stable multivalent binding complex; washing away any excess unbound polymer-nucleotide conjugate; for example, detection of multivalent binding complexes by fluorescence imaging of the support surface; identifying nucleotides in the target nucleic acid sequence (base recognition); for example, the stability of the multivalent binding complex is destroyed by altering the ionic strength, ionic composition, and/or pH of the buffer; flushing the flow cell; and performing a primer extension reaction to add nucleotides comprising complementary bases of the identified nucleotides. This cycle can be repeated to identify additional nucleotide bases in the sequence, and then the sequence data processed and assembled. In some cases, data processing may include calculating a sequencing performance index, such as a Q score, in real-time as the sequencing run is performed or as part of the post-run data processing steps.

In some cases, the disclosed hydrophilic polymer coated flow cell devices used in combination with the optical imaging systems disclosed herein can bring one or more of the following additional advantages to a nucleic acid sequencing system: (i) reduced fluid wash time (faster sequencing cycle time due to reduced non-specific binding), (ii) reduced imaging time (faster turnaround time of assay reading and sequencing cycle), (iii) reduced overall workflow time requirements (due to reduced cycle time), (iv) reduced assay instrument costs (due to improved CNR), (v) improved accuracy of reading (base detection) (due to improved CNR), (vi) improved stability of reagents and reduced reagent usage requirements (thereby reduced reagent costs), and (vii) fewer operational failures due to nucleic acid amplification failures.

The methods, devices, and systems for performing nucleic acid sequencing disclosed herein are suitable for use in a variety of sequencing applications, and for sequencing nucleic acid molecules derived from any of a variety of samples and sources. In some cases, nucleic acids may be extracted from various biological samples, e.g., blood samples, saliva samples, urine samples, cell samples, tissue samples, and the like. For example, the disclosed devices and systems may be used to analyze nucleic acid molecules derived from any of a variety of different cell, tissue or sample types known to those of skill in the art. For example, nucleic acids may be extracted from cells derived from eukaryotes (e.g., animals, plants, fungi, protozoa), archaebacteria, or eubacteria, or tissue samples comprising one or more types of cells. In some cases, nucleic acids may be extracted from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. Nucleic acids are extracted from a wide variety of, for example, primary or immortalized rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. Nucleic acids may be extracted from a variety of different cell, organ or tissue types (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lung, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). Nucleic acids may be extracted from normal or healthy cells. Alternatively or in combination, the acid is extracted from diseased cells (e.g., cancer cells) or pathogenic cells of the infected host. Certain nucleic acids may be extracted from different subsets of cell types, such as immune cells (e.g., T cells, cytotoxic T cells, helper T cells, αβ T cells, γδ T cells, T cell progenitors, B cells, B cell progenitors, lymphocytes, myeloid progenitor cells, lymphocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and/or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells that have been induced to differentiate, rare cells (e.g., circulating Tumor Cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, myeloid cells, progenitor cells, foam cells, mesenchymal cells, or trophoblasts). Other cells are contemplated and are consistent with the disclosure herein.

The nucleic acid may optionally be linked to one or more non-nucleotide moieties, such as labels and other small molecules, macromolecules (e.g., proteins, lipids, sugars, etc.), and solid or semi-solid carriers, such as by covalent bonding or non-covalent bonding at the 5 'or 3' end of the nucleic acid. Labels include any moiety that is detectable using any of a variety of detection methods known to those of skill in the art, and thus allow similar detectability of the attached oligonucleotides or nucleic acids. Some labels, such as fluorophores, emit optically detectable or visible electromagnetic radiation. Alternatively or in combination, some labels include a mass tag that makes the labeled oligonucleotide or nucleic acid visible in mass spectrometry data, or a redox tag that makes the labeled oligonucleotide or nucleic acid detectable by amperometry or voltammetry. Some labels include magnetic labels that facilitate the separation and/or purification of the labeled oligonucleotides or nucleic acids. The nucleotide or polynucleotide is typically not attached to a label and the presence of the oligonucleotide or nucleic acid is detected directly.

A flow cell apparatus configured for sequencing: in some cases, one or more flow cell devices according to the present disclosure may be configured for nucleic acid sequencing applications, for example, wherein two or more inner flow cell device surfaces comprise a hydrophilic polymer coating that further comprises one or more capture oligonucleotides, such as the adaptor/primer oligonucleotides disclosed herein or any other oligonucleotides. In some cases, the hydrophilic polymer coated surface of the disclosed flow cell devices can comprise a plurality of oligonucleotides tethered thereto, which have been selected for sequencing eukaryotic genomes. In some cases, the hydrophilic polymer coated surface of the disclosed flow cell devices can comprise a plurality of oligonucleotides tethered thereto, which have been selected for sequencing a prokaryote genome or portion thereof. In some cases, the hydrophilic polymer coated surface of the disclosed flow cell devices can comprise a plurality of oligonucleotides tethered thereto, which have been selected for sequencing a viral genome or portion thereof. In some cases, the hydrophilic polymer coated surface of the disclosed flow cell devices can comprise a plurality of oligonucleotides tethered thereto, which have been selected for sequencing a transcriptome.

In some cases, the flow cell device of the present disclosure may include a first surface oriented generally toward the interior of the flow channel, a second surface oriented generally toward the interior of the flow channel and also generally toward or parallel to the first surface, a third surface generally toward the interior of the second flow channel, and a fourth surface generally toward the interior of the second flow channel and opposite or parallel to the third surface; wherein the second and third surfaces may be located on opposite sides of or attached to a substantially planar substrate (which may be a reflective, transparent or translucent substrate). In some cases, one or more imaging surfaces within a flow cell may be located within the center of the flow cell, or within or as part of a partition between two subunits or sub-partitions of the flow cell, where the flow cell may include a top surface and a bottom surface, one or both of which may be transparent to the detection modes that may be used; and wherein the surface comprising the oligonucleotide adaptors/primers tethered to the one or more polymer coatings can be placed or inserted into the lumen of a flow cell. In some cases, the top surface and/or the bottom surface does not include attached oligonucleotide adaptors/primers. In some cases, the top surface and/or bottom surface does comprise attached oligonucleotide adaptors/primers. In some cases, the top surface or the bottom surface may comprise attached oligonucleotide adaptors/primers. One or more surfaces placed or inserted into the flow cell lumen may be located on or attached to one side, the opposite side, or both sides of a substantially planar substrate (which may be a reflective, transparent, or translucent substrate).

Typically, at least one of the one or more low non-specific binding coatings on the surface of the flow cell device may comprise functional groups for covalent or non-covalent attachment of oligonucleotide molecules, such as adaptors or primer sequences, or at least one layer may already comprise covalently or non-covalently attached oligonucleotide adaptors or primer sequences when deposited on the surface of the support. In some cases, the oligonucleotides tethered to the polymer molecules of at least one third layer may be distributed at multiple depths throughout the layer.

In some cases, the oligonucleotide adaptors or primer molecules are covalently coupled to the polymer in solution prior to coupling or depositing the polymer on the surface. In some cases, the oligonucleotide adaptors or primer molecules are covalently coupled to the polymer after they have been coupled or deposited on the surface. In some cases, at least one hydrophilic polymer layer comprises a plurality of covalently attached oligonucleotide adaptors or primer molecules. In some cases, at least two, at least three, at least four, or at least five layers of hydrophilic polymer comprise a plurality of covalently attached adaptors or primer molecules.

In some cases, oligonucleotide adaptors or primer molecules may be coupled to one or more layers of hydrophilic polymer using any of a variety of suitable conjugation chemistries known to those skilled in the art. For example, an oligonucleotide adaptor or primer sequence may comprise a moiety that reacts with an amine group, a carboxyl group, a thiol group, or the like. Examples of suitable amine reactive conjugation chemistry that may be used include, but are not limited to, reactions involving isothiocyanate groups, isocyanate groups, acyl azide groups, NHS ester groups, sulfonyl chloride groups, aldehyde groups, glyoxal groups, epoxide groups, oxirane groups, carbonate groups, aryl halide groups, imide ester groups, carbodiimide groups, anhydride groups, and fluorophenyl ester groups. Examples of suitable carboxyl reactive conjugation chemistry include, but are not limited to, reactions involving carbodiimide compounds, such as water-soluble EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide HCL). Examples of suitable thiol-reactive conjugation chemistries include maleimides, haloacetyl groups, and pyridyl disulfides.

One or more types of oligonucleotide molecules may be attached or tethered to the carrier surface. In some cases, one or more types of oligonucleotide adaptors or primers can comprise a spacer sequence, an adaptor sequence for hybridization to a template library nucleic acid sequence to which the adaptors are ligated, a forward amplification primer, a reverse amplification primer, a sequencing primer, and/or a molecular barcoding sequence, or any combination thereof. In some cases, 1 primer or adapter sequence may be tethered to at least one layer of the surface. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adapter sequences may be tethered to at least one layer of the surface.

In some cases, the length of the tethered oligonucleotide adaptors and/or primer sequences can range from about 10 nucleotides to about 100 nucleotides. In some cases, the length of the tethered oligonucleotide adaptors and/or primer sequences can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some cases, the length of the tethered oligonucleotide adaptors and/or primer sequences can be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. Any of the lower and upper values described in this paragraph can be combined to form the scope encompassed by the present disclosure, e.g., in some cases, the tethered oligonucleotide adaptors and/or primer sequences can range in length from about 20 nucleotides to about 80 nucleotides. One skilled in the art will recognize that the length of the tethered oligonucleotide adaptors and/or primer sequences can have any number within this range, for example, about 24 nucleotides.

In some cases, the number of coatings and/or the material composition of each layer is selected so as to adjust the resulting surface density of oligonucleotide adaptors/primers (or other attached molecules) on the surface of the coated flow cell. In some cases, the surface density of oligonucleotide adaptors/primers may be about 1,000 primer molecules/μm ² To about 1,000,000 primer molecules/μm ² Within a range of (2). In some cases, the surface density of the oligonucleotide primer may be at least 1,000, at least 10,000, at least 100,000 molecules/μm ² Or at least 1,000,000 molecules/μm ² . In some cases, the surface density of the oligonucleotide primer may be at most 1,000,000, at most 100,000, at most 10,000 molecules/μm ² Or up to 1,000 molecules/μm ² . Description in this paragraphAny of the lower and upper values described may be combined to form the ranges encompassed within the disclosure, e.g., in some cases, the surface density of the primer may be in the range of about 10,000 molecules/μm ² To about 100,000 molecules/μm ² Within a range of (2). Those skilled in the art will recognize that the surface density of the primer molecules can have any value within this range, for example, about 455,000 molecules/μm ² . In some cases, the surface properties of the capillary or channel lumen coating, including the surface density of tethered oligonucleotide primers, can be adjusted to improve or optimize, for example, solid phase nucleic acid hybridization specificity and efficiency, and/or solid phase nucleic acid amplification rate, specificity, and efficiency.

In some cases, tethered adapter or primer sequences can contain modifications designed to promote the specificity and efficiency of nucleic acid amplification on low-binding vectors. For example, in some cases, the primer may comprise a polymerase termination point such that extension of the primer sequence between the surface ligation point and the modification site is always in single stranded form and serves as a loading site for the 5 'to 3' helicase in some helicase-dependent isothermal amplification methods. Other examples of primer modifications that may be used to create a polymerase termination point include, but are not limited to, inserting a PEG chain between two nucleotides of the primer backbone toward the 5' end, inserting abasic nucleotides (i.e., nucleotides that have neither a purine nor pyrimidine base), or lesions that may be bypassed by helicases.

Nucleic acid hybridization: in some cases, hydrophilic polymer coated flow cell device surfaces disclosed herein can provide advantages when used alone or in combination with improved buffer formulations to perform solid phase nucleic acid hybridization and/or solid phase nucleic acid amplification reactions as part of genotyping or nucleic acid sequencing applications. In some cases, the polymer coated flow cell devices disclosed herein may provide advantages in terms of improved nucleic acid hybridization rates and specificity, as well as improved nucleic acid amplification rates and specificity, which may be achieved by one or more other aspects of the disclosure: (i) primer design (e.g., sequence and modification), (ii) control of tethered primer density on solid support, (iii) surface composition of solid support, (iv) surface polymer density of solid support, (v) use of improved hybridization conditions prior to and during amplification, and/or (vi) use of improved amplification formulation that reduces non-specific primer amplification or increases template amplification efficiency.

In some cases, it may be desirable to alter the surface density of oligonucleotide adaptors or primers tethered to the surface of the coated flow cell and/or the spacing of the tethered adaptors or primers from the surface of the coated flow cell (e.g., by altering the length of the adaptor molecules used to tether the adaptors or primers to the surface) in order to "tune" the carrier for optimal performance, for example, when using a given amplification method. In some cases, adjusting the surface density of tethered oligonucleotide adaptors or primers may affect the level of specific and/or non-specific amplification observed on the surface in a manner that varies depending on the amplification method selected. In some cases, the surface density of tethered oligonucleotide adaptors or primers can be varied by adjusting the proportion of molecular components used to generate the surface of the carrier. For example, where the use of an oligonucleotide primer-PEG conjugate results in a final layer of low-binding carrier, the ratio of oligonucleotide primer-PEG conjugate to unconjugated PEG molecule can be varied. The surface density of the tethered primer molecules can then be estimated or measured using any of a variety of techniques known to those of skill in the art. Examples include, but are not limited to, covalent coupling using radioisotope labeling and counting methods, cleavable molecules comprising an optically detectable label (e.g., a fluorescent label) cleavable from a support surface of a defined region, collected in a fixed volume of an appropriate solvent, and then compared to the fluorescent signal of a calibration solution of known optical label concentration by comparing the fluorescent signal to the fluorescent signal or using fluorescent imaging techniques (provided labeling reaction conditions and image acquisition settings have been noted) to ensure that the fluorescent signal is linearly related to the number of fluorophores on the surface (e.g., no apparent self-quenching of fluorophores on the surface).

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized buffer formulations, can produce a relative hybridization rate that is about 2-fold to about 20-fold faster than conventional hybridization protocols. In some cases, the relative hybridization rate may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold that of a conventional hybridization protocol.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized buffer formulations, can result in less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes of total hybridization reaction time (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the hybridization reaction) for any of these completion indicators.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized buffer formulations, can result in improved hybridization specificity compared to conventional hybridization protocols. In some cases, hybridization specificity may be achieved over 1 base mismatch in 10 hybridization events, 1 base mismatch in 20 hybridization events, 1 base mismatch in 30 hybridization events, 1 base mismatch in 40 hybridization events, 1 base mismatch in 50 hybridization events, 1 base mismatch in 75 hybridization events, 1 base mismatch in 100 hybridization events, 1 base mismatch in 200 hybridization events, 1 base mismatch in 300 hybridization events, 1 base mismatch in 400 hybridization events, 1 base mismatch in 500 hybridization events, 1 base mismatch in 600 hybridization events, 1 base mismatch in 700 hybridization events, 1 base mismatch in 800 hybridization events, 1 base mismatch in 900 hybridization events, 1 base mismatch in 1,000 hybridization events, 1 base mismatch in 2,000 hybridization events, 1 base mismatch in 3,000 base hybridization events, 1 base mismatch in 4,000 base events, 1 base mismatch in 1,000 hybridization events, 1 mismatch in 1,000 hybridization events, 1,000 mismatch in 1,000 hybridization events, 1,9 mismatch in 1,000 hybridization events, 1,000 mismatch in 8 hybridization events, or 1 base mismatch in 10,000 hybridization events.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized buffer formulations, can result in improved hybridization efficiency (e.g., fraction of available oligonucleotide primers on the surface of a carrier that successfully hybridizes to a target oligonucleotide sequence) as compared to conventional hybridization protocols. In some cases, the hybridization efficiency obtainable for any of the input target oligonucleotide concentrations specified below, and within any of the hybridization reaction times specified above, is better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99%. In some cases, for example, where the hybridization efficiency is less than 100%, the resulting surface density of target nucleic acid sequences hybridized to the surface of the carrier may be less than the surface density of oligonucleotide adaptors or primer sequences on the surface.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices in nucleic acid hybridization (or nucleic acid amplification) applications using conventional hybridization (or amplification) protocols or modified or optimized hybridization (or amplification) protocols can result in reduced demands on the input concentration of target (or sample) nucleic acid molecules in contact with the carrier surface. For example, in some cases, the target (or sample) nucleic acid molecule may be contacted with the support surface at a concentration of about 10pM to about 1 μm (i.e., prior to annealing or amplification). In some cases, the target (or sample) nucleic acid molecule may be administered at the following concentrations: at least 10pM, at least 20pM, at least 30pM, at least 40pM, at least 50pM, at least 100pM, at least 200pM, at least 300pM, at least 400pM, at least 500pM, at least 600pM, at least 700pM, at least 800pM, at least 900pM, at least 1nM, at least 10nM, at least 20nM, at least 30nM, at least 40nM, at least 50nM, at least 60nM, at least 70nM, at least 80nM, at least 90nM, at least 100nM, at least 200nM, at least 300nM, at least 400nM, at least 500nM, at least 600nM, at least 700nM, at least 800nM, at least 900nM, or at least 1 μM. In some cases, the target (or sample) nucleic acid molecule may be administered at the following concentrations: at most 1 μM, at most 900nM, at most 800nM, at most 700nM, at most 600nM, at most 500nM, at most 400nM, at most 300nM, at most 200nM, at most 100nM, at most 90nM, at most 80nM, at most 70nM, at most 60nM, at most 50nM, at most 40nM, at most 30nM, at most 20nM, at most 10nM, at most 1nM, at most 900pM, at most 800pM, at most 700pM, at most 600pM, at most 500pM, at most 400pM, at most 300pM, at most 200pM, at most 100pM, at most 90pM, at most 80pM, at most 70pM, at most 60pM, at most 50pM, at most 40pM, at most 30pM, at most 20pM, or at most 10pM. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, e.g., in some cases, the target (or sample) nucleic acid molecule can be administered at a concentration range of about 90pM to about 200 nM. One of skill in the art will recognize that the target (or sample) nucleic acid molecule can be administered at a concentration having any value within this range, for example, about 855 nM.

In some cases, use of the disclosed hydrophilic polymer-coated flow cell devices, alone or in combination with improved or optimized hybridization buffer formulations, can result in a surface density of hybridized target (or sample) oligonucleotide molecules (i.e., prior to any subsequent solid phase or clonal amplification reaction) in the range of about 0.0001 target oligonucleotide molecules/μm ² Up to about 1,000,000 target oligonucleotide molecules per μm ² . In some cases, the surface density of the hybridized target oligonucleotide molecules may be at least 0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000,500. at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000 or at least 1,000,000 molecules/μm ² . In some of the cases where the number of the cases, the surface density of the hybridized target oligonucleotide molecules can be at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000 at most 9,500, at most 9,000, at most 8500, at most 8000, at most 7500, at most 7000, at most 6500, at most 6000, at most 5500, at most 5,000, at most 4,500, at most 4,000, at most 3500, at most 3,000, at most 2500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 5, at most 1, at most 0.5, at most 0.1, at most 0.05, at most 0.01, at most 0.005, at most 0.001, at most 0.0005, or at most 0.0001 molecules/μm ² . Any of the lower and upper values described in this paragraph can be combined to form a range encompassed by the present disclosure, e.g., in some cases, hybridized target oligonucleotidesThe surface density of the molecules may be about 3,000 molecules/μm ² To about 20,000 molecules/μm ² Within the range. Those skilled in the art will recognize that the surface density of hybridized target oligonucleotide molecules can have any number within this range, for example, about 2,700 molecules/μm ² 。

In other words, in some cases, use of the disclosed low-binding vectors, alone or in combination with improved optimized hybridization buffer formulations, may result in a surface density of hybridized target (or sample) oligonucleotide molecules (i.e., prior to any subsequent solid phase or clonal amplification reactions) of about 100 hybridized target oligonucleotide molecules/mm ² Up to about 1X 10 ¹² Target oligonucleotide molecules/mm hybridized ² . In some cases, the surface density of the hybridized target oligonucleotide molecules can be at least 100, at least 500, at least 1,000, at least 4,000, at least 5,000, at least 6,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000, at least 5,000, at least 1×10 ⁷ At least 5X 10 ⁷ At least 1X 10 ⁸ At least 5X 10 ⁸ At least 1X 10 ⁹ At least 5X 10 ⁹ At least 1X 10 ¹⁰ At least 5X 10 ¹⁰ At least 1X 10 ¹¹ At least 5X 10 ¹¹ Or at least 1X 10 ¹² Individual molecules/mm ² . In some cases, the surface density of hybridized target oligonucleotide molecules may be up to 1X 10 ¹² At most 5X 10 ¹¹ At most 1X 10 ¹¹ At most 5X 10 ¹⁰ At most 1X 10 ¹⁰ At most 5X 10 ⁹ At most 1X 10 ⁹ At most 5X 10 ⁸ At most 1X 10 ⁸ At most 5X 10 ⁷ At most 1X 10 ⁷ At most 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most 1,000, at most 500 or at most 100 molecules/mm ² . Any of the lower and upper values described in this paragraph can be combined to form the scope encompassed by the present disclosure, e.g., in some cases the surface density of hybridized target oligonucleotide molecules can be in the range of about 5,000 molecules/mm ² Up to about 50,000 molecules/mm ² Within the range. Those skilled in the art will recognize that the surface density of the hybridized target oligonucleotide molecules can have any number within this range, for example, about 50,700 molecules/mm ² 。

In some cases, the target (or sample) oligonucleotide molecules (or nucleic acid molecules) that hybridize to the oligonucleotide adaptors or primer molecules attached to the low-binding carrier surface can range in length from about 0.02 kilobases (kb) to about 20kb or from about 0.1 kilobases (kb) to about 20kb. In some cases, the target oligonucleotide molecule may be at least 0.001kb, at least 0.005kb, at least 0.01kb, at least 0.02kb, at least 0.05kb, at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 0.6kb, at least 0.7kb, at least 0.8kb, at least 0.9kb, at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb at least 8kb, at least 9kb, at least 10kb, at least 15kb, at least 20kb, at least 30kb, or at least 40kb, or any intermediate value within the ranges described herein, e.g., at least 0.85 in length.

In some cases, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a single-stranded or double-stranded polynucleic acid molecule (e.g., concatemer) that also comprises repeating regularly-occurring monomer units. In some cases, the single-or double-stranded polynucleic acid molecule may be at least 0.001kb, at least 0.005kb, at least 0.01kb, at least 0.02kb, at least 0.05kb, at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb, at least 20kb, at least 30kb, at least 40kb, or any intermediate value within the ranges described herein, e.g., about 2.45kb in length.

In some cases, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a single-stranded or double-stranded polynucleic acid molecule (e.g., concatemer) comprising copies of about 2 to about 100 regularly repeating monomer units. In some cases, the copy number of regularly repeating monomer units may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100. In some cases, the copy number of regularly repeating monomer units may be at most 100, at most 95, at most 90, at most 85, at most 80, at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, or at most 2. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, for example, in some cases, the copy number of regularly repeating monomer units can be in the range of about 4 to about 60. Those skilled in the art will recognize that the copy number of regularly repeating monomer units can have any number within this range, for example about 17. Thus, in some cases, the surface density of hybridized target sequences may exceed the surface density of oligonucleotide primers in terms of the number of copies of target sequences per unit area of the carrier surface, even though the hybridization efficiency is less than 100%.

Nucleic Acid Surface Amplification (NASA): as used herein, the phrase "nucleic acid surface amplification" (NASA) is used interchangeably with the phrase "solid phase nucleic acid amplification" (or simply "solid phase amplification"). In some aspects of the disclosure, nucleic acid amplification formulations are described that provide for increased amplification rates, amplification specificity, and amplification efficiency in combination with the disclosed hydrophilic polymer coated flow cell devices. As used herein, specific amplification refers to amplification of a template library oligonucleotide strand that has been covalently or non-covalently tethered to a solid support. As used herein, non-specific amplification refers to amplification of primer dimers or other non-template nucleic acids. As used herein, amplification efficiency is a measure of the percentage of tethered oligonucleotides on the surface of a carrier that are successfully amplified during a given amplification cycle or amplification reaction. Nucleic acid amplification performed on the surfaces disclosed herein can achieve an amplification efficiency of at least 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95% (e.g., 98% or 99%).

Any of a variety of thermocycling or isothermal nucleic acid amplification protocols can be used with the disclosed low binding vectors. Examples of nucleic acid amplification methods that may be used with the disclosed low-binding vectors include, but are not limited to, polymerase Chain Reaction (PCR), multiple Displacement Amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand Displacement Amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, inter-loop amplification, helicase-dependent amplification, recombinase-dependent amplification, or Single Strand Binding (SSB) protein-dependent amplification.

In some cases, improvements in amplification rate, amplification specificity, and amplification efficiency can be achieved using the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with formulations of amplification reaction components. In addition to comprising nucleotides, one or more polymerases, helicases, single stranded binding proteins, and the like (or any combination thereof), the amplification reaction mixture may be adjusted in a variety of ways to achieve improved performance, including, but not limited to, choice of buffer type, buffer pH, organic solvent mixture, buffer viscosity, detergent and zwitterionic components, ionic strength (including adjustment of monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaines, other additives, and the like.

The use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized amplification reaction formulations, can result in increased amplification rates compared to those obtained using conventional carriers and amplification protocols. In some cases, for any of the amplification methods described above, the relative amplification rates that can be achieved can be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, or at least 20-fold using conventional vectors and amplification protocols.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized buffer formulations, can yield less than 180 minutes, 120 minutes, 90 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, or 10 seconds of total amplification reaction time (i.e., time required to reach 90%, 95%, 98%, or 99% completion of the amplification reaction) for any of these completion indicators.

In some cases, use of the disclosed low-binding carriers, alone or in combination with improved or optimized amplification buffer formulations, can achieve faster amplification reaction times (i.e., the time required to achieve 90%, 95%, 98%, or 99% completion of the amplification reaction) of no more than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes. Similarly, use of the disclosed low-binding carriers alone or in combination with modified or optimized buffer formulations may allow the amplification reaction to be completed in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 cycles, or no more than 30 cycles in some cases.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized amplification reaction formulations, can result in increased specific amplification and/or decreased non-specific amplification as compared to the use of conventional carriers and amplification protocols. In some cases, the resulting ratio of specific amplification to non-specific amplification that can be achieved is at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, or 1,000:1.

In some cases, the use of a hydrophilic polymer coated flow cell device alone or in combination with improved or optimized amplification reaction formulations can result in increased amplification efficiency as compared to the use of conventional carriers and amplification protocols. In some cases, the achievable amplification efficiency is better than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% for any of the amplification reaction times specified above.

In some cases, the length of the clonally amplified target (or sample) oligonucleotide molecules (or nucleic acid molecules) hybridized to the oligonucleotide adaptors or primer molecules attached to the surface of the hydrophilic polymer coated flow cell device can be from about 0.02 kilobases (kb) to about 2kb or from about 0.1 kilobases (kb) to about 20kb. In some cases, the clonally amplified target oligonucleotide molecule may be at least 0.001kb, at least 0.005kb, at least 0.01kb, at least 0.02kb, at least 0.05kb, at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb, or at least 20kb in length, or any intermediate value within the ranges described herein, e.g., at least 0.85kb in length.

In some cases, the clonally amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a single-stranded or double-stranded polynucleic acid molecule (e.g., a concatemer) that also comprises repeating regularly-occurring monomer units. In some cases, the clonally amplified single-or double-stranded polynucleic acid molecule may be at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb or at least 20kb in length, or any intermediate value within the ranges described herein, for example, about 2.45kb in length.

In some cases, the clonally amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a single-stranded or double-stranded polynucleic acid molecule (e.g., concatemer) comprising copies of about 2 to about 100 regularly repeating monomer units. In some cases, the number of copies of regularly repeating monomer units can be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100. In some cases, the copy number of regularly repeating monomer units may be at most 100, at most 95, at most 90, at most 85, at most 80, at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, or at most 2. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, for example, in some cases, the copy number of regularly repeating monomer units can be in the range of about 4 to about 60. Those skilled in the art will recognize that the copy number of regularly repeating monomer units can have any number within this range, for example about 12. Thus, in some cases, the surface density of clonally amplified target sequences may exceed the surface density of oligonucleotide primers even though the hybridization and/or amplification efficiency is less than 100% in terms of the number of copies of target sequences per unit area of the surface of the carrier.

In some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized amplification reaction formulations, can result in increased clone copy numbers as compared to use of conventional carriers and amplification protocols. In some cases, for example, where the clonally amplified target (or sample) oligonucleotide molecule comprises a tandem multimeric repeat sequence of monomeric target sequences, the clone copy number may be much less than that obtained using conventional vectors and amplification protocols. Thus, in some cases, the clone copy number may be from about 1 molecule to about 100,000 molecules (e.g., target sequence molecules) per amplified population. In some cases, the clone copy number may be at least 1, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, or at least 100,000 molecules per amplified population. In some cases, the clone copy number may be at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, at most 1,000, at most 500, at most 100, at most 50, at most 10, at most 5, or at most 1 molecule per amplified colony. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, for example, in some cases, the clone copy number can be in the range of about 2,000 molecules to about 9,000 molecules. Those skilled in the art will recognize that clone copy numbers can have any number within this range, for example, about 2,220 molecules in some cases, and about 2 molecules in other cases.

As described above, in some cases, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a tandem multimeric repeat sequence of a monomeric target sequence. In some cases, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may comprise a plurality of fractionsA seed, each molecule comprising a single monomer target sequence. Thus, use of the disclosed hydrophilic polymer-coated flow cell devices, alone or in combination with improved or optimized amplification reaction formulations, can result in a surface density of about 100 copies of target sequence per mm ² Up to about 1X 10 ¹² Individual target sequence copies/mm ² . In some cases, the surface density of the copy of the target sequence may be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000, at least 5,000, at least 1,000, at least 10 x 10 ⁷ At least 5X 10 ⁷ At least 1X 10 ⁸ At least 5X 10 ⁸ At least 1X 10 ⁹ At least 5X 10 ⁹ At least 1X 10 ¹⁰ At least 5X 10 ¹⁰ At least 1X 10 ¹¹ At least 5X 10 ¹¹ Or at least 1X 10 ¹² Target sequence molecule/mm amplified by individual clones ² . In some cases, the surface density of copies of the target sequence may be up to 1X 10 ¹² At most 5X 10 ¹¹ At most 1X 10 ¹¹ At most 5X 10 ¹⁰ At most 1X 10 ¹⁰ At most 5X 10 ⁹ At most 1X 10 ⁹ At most 5X 10 ⁸ At most 1X 10 ⁸ At most 5X 10 ⁷ At most 1X 10 ⁷ Up to 5,000,000, up to 1,000,000, up to 950,000, up to 90 ten thousand, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, up toUp to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 copies of target sequence per mm ² . Any of the lower and upper values described in this paragraph can be combined to form the scope encompassed by the present disclosure, e.g., in some cases the surface density of copies of a target sequence can be in the range of about 1,000 copies of a target sequence per mm ² Up to about 65,000 copies of the target sequence per mm ² Within a range of (2). Those skilled in the art will recognize that the surface density of copies of the target sequence can have any number within this range, for example about 49,600 copies of the target sequence per mm ² 。

In some cases, use of the disclosed low-binding vectors, alone or in combination with improved or optimized amplification buffer formulations, can result in a surface density of clonally amplified target (or sample) oligonucleotide molecules (or clusters) ranging from about 100 molecules/mm ² Up to about 1X 10 ¹² Individual colonies/mm ² . In some cases, the surface density of the cloned amplified molecules may be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000, at least 5,000, at least 1,000, at least 1×10,000 ⁷ At least 5X 10 ⁷ At least 1X 10 ⁸ At least 5X 10 ⁸ At least 1X 10 ⁹ At least 5X 10 ⁹ At least 1X 10 ¹⁰ At least 5X 10 ¹⁰ At least 1X 10 ¹¹ At least 5X 10 ¹¹ Or at least 1X 10 ¹² Individual molecules/mm ² . In some cases, the surface density of cloned amplified molecules may be up to 1×10 ¹² At most 5X 10 ¹¹ At most 1X 10 ¹¹ At most 5X 10 ¹⁰ At most 1X 10 ¹⁰ At most 5X 10 ⁹ At most 1X 10 ⁹ At most 5X 10 ⁸ At most 1X 10 ⁸ At most 5X 10 ⁷ At most 1X 10 ⁷ At most 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most 1,000, at most 500 or at most 100 molecules/mm ² . Any of the lower and upper values described in this paragraph can be combined to form the scope encompassed by the present disclosure, e.g., in some cases, the surface density of clonally amplified molecules can be about 5,000 molecules/mm ² Up to about 50,000 molecules/mm ² . Those skilled in the art will recognize that the surface density of the clonally amplified colonies may have any number within this range, for example, about 48,800 molecules/mm ² 。

In some cases, use of the disclosed hydrophilic polymer-coated flow cell devices, alone or in combination with improved or optimized amplification reaction formulations, can generate a signal (e.g., a fluorescent signal) from the amplified and labeled nucleic acid population with a coefficient of variation of no greater than 50%, such as 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less than 5%.

Similarly, in some cases, use of the disclosed hydrophilic polymer coated flow cell devices, alone or in combination with improved or optimized amplification reaction formulations, can generate a signal from an amplified and unlabeled nucleic acid population that has a coefficient of variation of no greater than 50%, e.g., 50%, 40%, 30%, 20%, 10%, 5%, or less than 5%.

Fluorescence imaging of the surface of a hydrophilic polymer coated flow cell device: the disclosed hydrophilic polymer coated flow cell devices include, for example, clonal clusters of labeled target nucleic acid molecules disposed thereon, and are useful in any of a variety of nucleic acid analysis applications, such as nucleic acid base identification, nucleic acid base classification, nucleic acid base detection, nucleic acid detection applications, nucleic acid sequencing applications, and nucleic acid-based (genetic and genomic) diagnostic applications. In many of these applications, fluorescence imaging techniques can be used to monitor hybridization, amplification, and/or sequencing reactions performed on low-binding carriers. Fluorescence imaging can be performed using any of the optical imaging modules disclosed herein, as well as using various fluorophores, fluorescence imaging techniques, and other fluorescence imaging instruments known to those of skill in the art.

In some cases, fluorescence imaging techniques can be used to evaluate the performance of nucleic acid hybridization and/or amplification reactions using the disclosed hydrophilic polymer coated flow cell devices and reaction buffer formulations, wherein the contrast to noise ratio (CNR) of the image provides a key indicator for evaluating amplification-specific and non-specific binding on the support. CNR is generally defined as: cnr= (signal-background)/noise. The background term is generally considered to be a signal measured in a specified region of interest (ROI) around a gap region of a specific feature (diffraction limited spot, DLS). As described above, while signal-to-noise ratio (SNR) is generally considered a benchmark for overall signal quality, it can be shown that in applications requiring fast image capture (e.g., sequencing applications where cycle time should be reduced or minimized), improved CNR can provide significant advantages over SNR as a signal quality benchmark. In the case of high CNR, even with moderate improvement in CNR, the imaging time required to reach accurate signal discrimination (and hence accurate base detection in the case of sequencing applications) can be significantly reduced.

Based on most ofIn the overall sequencing approach, background items are typically measured as signals associated with "interstitial" regions. Except for the "interstitial" background (B _{Interstitial substance} ) In addition, an "intracellular" background (B _{Intracellular} ) But also within discrete regions occupied by amplified DNA clones. The combination of these two background signal terms determines the achievable CNR in the image, which then directly affects the requirements of the optical instrument, architecture costs, reagent costs, runtime, costs/genome, ultimately affecting the accuracy and data quality of the sequencing application based on the circular array. B (B) _{Interstitial substance} Background signals come from various sources; some examples include autofluorescence from a sacrificial flow cell, non-specific adsorption of the detection molecules (which may produce spurious fluorescent signals that may mask the foreground signal from the ROI), and the presence of non-specific DNA amplification products (e.g., amplification products produced by primer dimers). In a typical Next Generation Sequencing (NGS) application, the background signal in the current field of view (FOV) will average and subtract over time. Signals from individual DNA clones (i.e., (S) -B in the FOV) _{Interstitial substance} ) A sortable, identifiable feature is produced. In some cases, the intracellular background (B _{Intracellular} ) A mixed fluorescence signal may be contributed, which is not target specific but is present in the same ROI and thus difficult to average and subtract.

Performing nucleic acid amplification on hydrophilic polymer coated substrate surfaces of the present disclosure can reduce B by reducing non-specific binding _{Interstitial substance} Background signal may result in improved amplification of specific nucleic acids and may result in reduced non-specific amplification that affects background signals generated by interstitial and intracellular regions. In some cases, the disclosed low non-specific binding carrier surfaces, optionally in combination with improved hybridization and/or amplification reaction buffer formulations, can increase CNR 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 500-fold, or 1000-fold over those obtained using conventional carriers and hybridization, amplification, and/or sequencing protocols. Although described herein in the context of using fluorescence imaging as a readout or detection mode, the same principles apply to the low levels to be disclosedNonspecific binding carriers and nucleic acid hybridization and amplification reagents are used in other detection modes, including optical detection modes and non-optical detection modes.

Alternative sequencing biochemistry: in addition to the above-described methods of sequencing by nucleotide incorporation, the flow cell devices and optical imaging systems of the present disclosure are also compatible with other emerging biochemical methods of nucleic acid sequencing. Examples include the "sequencing by nucleotide binding" method described in U.S. patent No. 10,655,176B2 and the "sequencing by affinity" method described in U.S. patent No. 10,768,173B2.

Currently, the method of "sequencing by nucleotide binding" developed by Omniome, inc. (San Diego, CA) is based on performing repeated cycles, detecting the formation of stable complexes (e.g., ternary complexes comprising a primed template (tethered to a sample carrier structure), a polymerase and homologous nucleotides at that position) at each position along the template under conditions that prevent covalent incorporation of homologous nucleotides into the primer, and then extending the primer to allow detection of the next position along the template. In sequencing by binding, the nucleotides at each position of the template are detected before the primer is extended to the next position. Typically, the method is used to distinguish between four different nucleotide types that may be present at positions along a nucleic acid template by uniquely labeling each type of ternary complex (i.e., different types of ternary complexes that contain different nucleotide types) or by separately delivering the reagents required to form each type of ternary complex. In some cases, the label may include a fluorescent label, such as a cognate nucleotide or polymerase, that participates in the ternary complex. Thus, the method is compatible with the disclosed flow cell apparatus and imaging system.

Currently, the "through affinity sequencing" method developed by Element Biosciences, inc (San Diego, CA) relies on the increased affinity (or "functional affinity") obtained by forming complexes comprising multiple separate non-covalent binding interactions. The Element method is based on the detection of multivalent binding complexes formed between a fluorescently labeled polymer-nucleotide conjugate, a polymerase and a plurality of primer-engineered target nucleic acid molecules tethered to a sample carrier structure, which allows the detection/base detection step to be separated from the nucleotide incorporation step. Fluorescence imaging is used to detect the bound complex, thereby determining the identity of the n+1 nucleotide in the target nucleic acid sequence (where the primer extension strand is N nucleotides in length). After the imaging step, the multivalent binding complex is destroyed and washed away, the correct blocked nucleotide is incorporated into the primer extension strand, and the cycle is repeated.

In some cases, the polymer-nucleotide conjugates of the present disclosure may comprise multiple nucleotide moieties or nucleotide analog moieties conjugated to the polymer core directly or via a linker, e.g., through the 5' end of the nucleotide. As non-limiting examples, the nucleotide moiety may include a ribonucleotide moiety, a ribonucleotide analog moiety, a deoxyribonucleotide analog moiety, or any combination thereof. In some cases, the nucleotide or nucleotide analog may include deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, adenosine, guanosine, 5-methyl-uridine, and/or cytidine. In some cases, the nucleotide or nucleotide analog moiety may comprise a nucleotide that has been modified to inhibit elongation during a polymerase reaction or sequencing reaction, for example, wherein at least one nucleotide or nucleotide analog is a nucleotide that lacks a 3' hydroxyl group; nucleotides that have been modified to contain a blocking group at the 3' position; and/or nucleotides that have been modified with 3 '-0-azido, 3' -0-azidomethyl, 3 '-0-alkylhydroxyamino, 3' -phosphorothioate, 3 '-malonyl or 3' -0-benzyl.

In some cases, the polymer core may include a linear or branched polymer, for example, a linear or branched polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyglycine, polyvinyl acetate, dextran, protein, or other such polymer, or a copolymer incorporating any two or more of the foregoing, or other polymers known in the art. In some cases, the polymer is PEG. In some cases, the polymer is branched PEG. In some cases, the branched polymer may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more branches or arms, or 2, 4, 8, 16, 32, 64 or more branches or arms. In some cases, the branches or arms may radiate from the central portion.

In some cases, the polymer-nucleotide conjugates may also comprise one or more detectable labels, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than 20 detectable labels. In some cases, the one or more detectable labels may comprise one or more fluorophores (e.g., cyanine dye 3 (Cy 3), cyanine dye 5 (Cy 5), etc.), one or more quantum dots, a Fluorescence Resonance Energy Transfer (FRET) donor, and/or a FRET acceptor.

In some cases, the polymer-nucleotide conjugate may further comprise a binding moiety that binds to each branch or subset of branches of the polymer core. Examples of suitable binding moieties include, but are not limited to, biotin, avidin, streptavidin or analogs, polyhistidine domains, complementary paired nucleic acid domains, G-tetranectin forming nucleic acid domains, calmodulin, maltose binding protein, cellulase, maltose, sucrose, glutathione-S-transferase, glutathione, 0-6-methylguanine-DNA methyltransferase, benzylguanine and derivatives thereof, benzylcysteine and derivatives thereof, antibodies, epitopes, protein a, or protein G. The binding moiety may be any interacting molecule or fragment thereof known in the art that binds to or facilitates interactions between proteins, between proteins and ligands, between proteins and nucleic acids, between nucleic acids, or between small molecule interacting domains or moieties.

As described above, in a method of sequencing by affinity, when the nucleotide moiety of a polymer-nucleotide conjugate is complementary to a nucleotide residue of a target sequence, a multivalent binding complex is formed between, for example, a fluorescently labeled polymer-nucleotide conjugate, a polymerase, and a plurality of primer-functionalized target nucleic acid molecules tethered to a sample carrier structure (e.g., a flow cell surface). The stability of the multivalent binding complex thus formed allows the detection/base detection step to be separated from the nucleotide incorporation step in the sequencing reaction cycle.

The stability of multivalent binding complexes (ternary complexes formed between two or more nucleotide moieties, two or more polymerase molecules, and two or more primer-directed target nucleic acid sequences of a polymer-nucleotide conjugate) can be demonstrated by the extended duration of the complex. For example, in some cases, the multivalent binding complex (ternary complex) may have a duration of less than 0.5 seconds, less than 1 second, greater than 2 seconds, greater than 3 seconds, greater than 4 seconds, greater than 5 seconds, greater than 10 seconds, greater than 15 seconds, greater than 20 seconds, greater than 30 seconds, greater than 60 seconds, greater than 120 seconds, greater than 360 seconds, greater than 720 seconds, greater than 1,440 seconds, greater than 3600 seconds, or longer, or a time within a range defined by any two or more of these values.

The use of polymer-nucleotide conjugates to form multivalent binding complexes with polymerase and primer-specific target nucleic acids can result in an increase in the effective local concentration of nucleotides that is many times higher than the average nucleotide concentration obtained with single unconjugated or unbuckled nucleotides, which in turn increases both the stability of the complex and the signal intensity after the washing step. High signal intensities persist throughout the binding, washing and imaging steps and help shorten image acquisition times. After the imaging step, the multivalent binding complex can be destabilized, for example by changing the ionic composition, ionic strength, and/or pH of the buffer, and washed away. A primer extension reaction can then be performed to extend the complementary strand by one base.

Nucleic acid sequencing System Performance: in some cases, the disclosed nucleic acid sequencing system (including one or more disclosed flow cell devices used in combination with one or more disclosed optical imaging systems, and optionally utilizing one of the emerging sequencing biochemical methods, such as the "through capture sequencing" (or "through affinity sequencing") method described above) can provide improved nucleic acid sequencing performance in: for example, reducing sample input requirements, reducing image acquisition cycle time, reducing sequencing reaction cycle time, reducing sequencing run time, improving accuracy of base detection, reducing reagent consumption and cost, improving sequencing throughput, and reducing sequencing costs.

Nucleic acid sample input (pM): in some cases, because improved hybridization and amplification efficiency can be obtained and high CNR images for base detection can be acquired using the disclosed hydrophilic polymer coated flow cell devices and imaging systems, sample input requirements of the disclosed systems can be significantly reduced. In some cases, the nucleic acid sample input requirements of the disclosed systems can be in the range of about 1pM to about 10,000pM. In some cases, the nucleic acid sample input requirement can be at least 1pM, at least 2pM, at least 5pM, at least 10pM, at least 20pM, at least 50pM, at least 100pM, at least 200pM, at least 500pM, at least 1,000pM, at least 2,000pM, at least 5,000pM, at least 10,000pM. In some cases, the nucleic acid sample input requirements of the disclosed systems can be at most 10,000pM, at most 5,000pM, at most 2,000pM, at most 1,000pM, at most 500pM, at most 200pM, at most 100pM, at most 50pM, at most 20pM, at most 10pM, at most 5pM, at most 2pM, or at most 1pM. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, e.g., in some cases, the nucleic acid sample input requirements of the disclosed system can be in the range of about 5pM to about 500 pM. One skilled in the art will recognize that the nucleic acid sample input requirement can have any value within this range, for example, about 132pM. In an exemplary case, a nucleic acid sample input of about 100pM is sufficient to generate a signal for reliable base detection.

Nucleic acid sample input (nanograms): in some cases, the nucleic acid sample input requirements of the disclosed systems may be in the range of about 0.05 nanograms to about 1,000 nanograms. In some cases, the nucleic acid sample input requirement is at least 0.05 nanogram, at least 0.1 nanogram, at least 0.2 nanogram, at least 0.4 nanogram, at least 0.6 nanogram, at least 0.8 nanogram, at least 1.0 nanogram, at least 2 nanogram, at least 4 nanogram, at least 6 nanogram, at least 8 nanogram, at least 10 nanogram, at least 20 nanogram, at least 40 nanogram, at least 60 nanogram, at least 80 nanogram, at least 100 nanogram, at least 200 nanogram, at least 400 nanogram, at least 600 nanogram, at least 800 nanogram, or at least 1,000 nanogram. In some cases, the nucleic acid sample input requirement may be at most 1,000 nanograms, at most 800 nanograms, at most 600 nanograms, at most 400 nanograms, at most 200 nanograms, at most 100 nanograms, at most 80 nanograms, at most 60 nanograms, at most 40 nanograms, at most 20 nanograms, at most 10 nanograms, at most 8 nanograms, at most 6 nanograms, at most 4 nanograms, at most 2 nanograms, at most 1 nanograms, at most 0.8 nanograms, at most 0.6 nanograms, at most 0.4 nanograms, at most 0.2 nanograms, at most 0.1 nanograms, or at most 0.05 nanograms. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, e.g., in some cases, the nucleic acid sample input requirements of the disclosed system can range from about 0.6 nanograms to about 400 nanograms. One skilled in the art will recognize that the nucleic acid sample input requirement may have any value within this range, for example, about 2.65 nanograms.

FOV image required for # tiling flow cell: in some cases, the field of view (FOV) of the disclosed optical imaging module is large enough that the multichannel (or multi-lane) flow cell of the present disclosure (i.e., the fluidic channel portion thereof) can be imaged by tiling about 10 FOV images (or "frames") to about 1,000 FOV images (or "frames"). In some cases, an image of an entire multichannel flow cell may require tiling at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1,000 FOV images (or "frames"). In some cases, images of an entire multichannel flow cell may need to be tiled up to 1,000, up to 950, up to 900, up to 850, up to 800, up to 750, up to 700, up to 650, up to 600, up to 550, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 FOV images (or "frames"). Any of the lower and upper values described in this paragraph may be combined to form the scope encompassed by the present disclosure, for example, in some cases, the image of the entire multichannel flow cell may require tiling about 30 to about 100 FOV images. Those skilled in the art will recognize that in some cases the number of FOV images required may have any value within this range, for example, about 54 FOV images.

Imaging cycle time: in some cases, the combination of a large FOV, image sensor response sensitivity, and/or fast FOV transition time enables shortening the imaging cycle time (i.e., the time required to acquire a sufficient number of FOV images to tile the entire multichannel flow cell (or fluid channel portion thereof). In some cases, the imaging cycle time may be in the range of about 10 seconds to about 10 minutes. In some cases, the imaging cycle time may be at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes. At least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes. In some cases, the imaging cycle time may be at most 10 minutes, at most 9 minutes, at most 8 minutes, at most 7 minutes, at most 6 minutes, at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes, at most 1 minute, at most 50 seconds, at most 40 seconds, at most 30 seconds, at most 20 seconds, or at most 10 seconds. Any of the lower and upper values described in this paragraph may be combined to form the ranges included in the present disclosure, for example, in some cases, the imaging cycle time may be in the range of about 20 seconds to about 1 minute. Those skilled in the art will recognize that in some cases, the imaging cycle time may have any value within this range, for example, about 57 seconds.

Sequencing cycle time: in some cases, a shortened sequencing reaction step (e.g., due to reduced washing time requirements of the disclosed hydrophilic polymer coated flow cell) can result in a shortened overall sequencing cycle time. In some cases, the sequencing cycle time of the disclosed system can be in the range of about 1 minute to about 60 minutes. In some cases, the sequencing cycle time may be at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, or at least 60 minutes. In some cases, the sequencing reaction cycle time may be at most 60 minutes, at most 55 minutes, at most 50 minutes, at most 45 minutes, at most 40 minutes, at most 35 minutes, at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, at most 10 minutes, at most 9 minutes, at most 8 minutes, at most 7 minutes, at most 6 minutes, at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes, or at most 1 minute. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, for example, in some cases, the sequencing cycle time can be in the range of about 2 minutes to about 15 minutes. Those skilled in the art will recognize that in some cases, the sequencing cycle time may have any value within this range, e.g., about 1 minute, 12 seconds.

Sequencing read length: in some cases, enhanced CNR images can be achieved by using the disclosed hydrophilic polymer coated flow cell devices in combination with the disclosed imaging systems, and in some cases, using milder sequencing biochemistry methods can enable longer sequencing read lengths for the disclosed systems. In some cases, the maximum (single read) read length may be in the range of about 50bp to about 500bp. In some cases, the maximum (single read) read length may be at least 50bp, at least 100bp, at least 150bp, at least 200bp, at least 250bp, at least 300bp, at least 350bp, at least 400bp, at least 450bp, or at least 500bp. In some cases, the maximum (single read) read length may be at most 500bp, at most 450bp, at most 400bp, at most 350bp, at most 300bp, at most 250bp, at most 200bp, at most 150bp, at most 100bp, or at most 50bp. Any of the lower and upper values described in this paragraph may be combined to form the ranges encompassed by the present disclosure, for example, in some cases the maximum (single read) read length may be in the range of about 100bp to about 450 bp. Those skilled in the art will recognize that in some cases the maximum (single read) read length may have any value within this range, for example, about 380bp.

Sequencing run time: in some cases, the sequencing run time of the disclosed nucleic acid sequencing system can be in the range of about 8 hours to about 20 hours. In some cases, the sequencing run time is at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, or at least 20 hours. In some cases, the sequencing run time is at most 20 hours, at most 18 hours, at most 16 hours, at most 14 hours, at most 12 hours, at most 10 hours, at most 9 hours, or at most 8 hours. Any of the lower and upper values described in this paragraph may be combined to form the ranges encompassed by the present disclosure, for example, in some cases, the sequencing run time may be in the range of about 10 hours to about 16 hours. Those skilled in the art will recognize that in some cases, the sequencing run time may have any value within this range, for example, about 7 hours 35 minutes.

Average base detection accuracy: in some cases, the disclosed nucleic acid sequencing systems can provide an average base detection accuracy of at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% during a sequencing run. In some cases, the disclosed nucleic acid sequencing systems can provide at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% correct average base detection accuracy per 1,000 bases, 10,0000 bases, 25,000 bases, 50,000 bases, 75,000 bases, or 100,000 bases detected.

Average Q score: in some cases, the quality or accuracy of a sequencing run can be assessed by calculating a Phred quality score (also referred to as a quality score or "Q score") that indicates the probability that a given base was erroneously detected by the sequencing system. For example, in some cases, base detection accuracy of a particular sequencing chemistry and/or sequencing system may be assessed against a large empirical data set derived from sequencing runs on a library of known nucleic acid sequences. The Q score may then be calculated according to the following equation:

Q＝-10log ₁₀ P

where P is the base detection error probability. For example, a Q score of 30 indicates that the probability of a base detection error is 1 (or base detection accuracy is 99.9%) for every 1000 base detections.

In some cases, the disclosed nucleic acid sequencing systems can provide more accurate base reads. For example, in some cases, the disclosed nucleic acid sequencing systems can provide a Q score for base detection accuracy in a sequencing run, ranging from about 20 to about 50. In some cases, the running average Q score may be at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q score may have any value within this range, for example, about 32.

Q score relative to% nucleotide identified: in some cases, the disclosed nucleic acid sequencing systems can provide a Q score of greater than 20 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides. In some cases, the disclosed nucleic acid sequencing systems can provide a Q score of greater than 25 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides. In some cases, the disclosed nucleic acid sequencing systems can provide a Q score of greater than 30 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides. In some cases, the disclosed nucleic acid sequencing systems can provide a Q score of greater than 35 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides. In some cases, the disclosed nucleic acid sequencing systems can provide a Q score of greater than 40 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides. In some cases, the disclosed nucleic acid sequencing systems can provide a Q score of greater than 45 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides. In some cases, the disclosed compositions and methods for nucleic acid sequencing can provide a Q score of greater than 50 for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the identified terminal (or n+1) nucleotides.

Reagent consumption: in some cases, the disclosed nucleic acid sequencing systems can have lower reagent consumption rates and costs due to, for example, the use of the disclosed flow cell devices and fluid systems that minimize fluid channel volumes and dead volumes. Thus, in some cases, the reagents that may be required per Gbase sequencing by the disclosed nucleic acid sequencing system are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% on a volumetric basis compared to the reagents consumed by the Illumina MiSeq sequencer.

Sequencing flux: in some cases, the disclosed nucleic acid sequencing systems can provide a sequencing throughput in the range of about 50 Gbase/run to about 200 Gbase/run. In some cases, the sequencing throughput may be at least 50 Gbase/run, at least 75 Gbase/run, at least 100 Gbase/run, at least 125 Gbase/run, at least 150 Gbase/run, at least 175 Gbase/run, or at least 200 Gbase/run. In some cases, the sequencing throughput may be at most 200 Gbase/run, at most 175 Gbase/run, at most 150 Gbase/run, at most 125 Gbase/run, at most 100 Gbase/run, at most 75 Gbase/run, or at most 50 Gbase/run. Any of the lower and upper values described in this paragraph can be combined to form the ranges encompassed by the present disclosure, e.g., in some cases, the sequencing throughput can range from about 75 Gbase/run to about 150 Gbase/run. Those skilled in the art will recognize that in some cases, the sequencing throughput may have any value within this range, for example, about 119 Gbase/run.

Sequencing cost: in some cases, the disclosed nucleic acid sequencing system can provide nucleic acid sequencing at a cost of about $5 per Gbase to about $30 per Gbase. In some cases, the sequencing cost may be at least $ 5 per Gbase, at least $ 10 per Gbase, at least $ 15 per Gbase, at least $ 20 per Gbase, at least $ 25 per Gbase, or at least $ 30 per Gbase. In some cases, the sequencing cost may be $ 30 per Gbase, $ 25 per Gbase, $ 20 per Gbase, $ 15 per Gbase, $ 10 per Gbase, or $ 30 per Gbase. Any of the lower and upper values described in this paragraph may be combined to form the scope encompassed by the present disclosure, e.g., in some cases, the sequencing cost may be in the range of about $10 per Gbase to about $15 per Gbase. Those skilled in the art will recognize that in some cases, the sequencing cost may have any value within this range, for example, about $7.25 per Gbase.

Examples

These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

Example 1-design specifications for fluorescence imaging modules for genomics

A non-limiting example of the design specifications of the fluorescence imaging module of the present disclosure is provided in table 1.

Table 1. Design specification examples of fluorescence imaging modules for genomics.

Example 2-fabrication of glass microfluidic flow cell devices

Wafer level fabrication of microfluidic devices for use as flow cells may be constructed from, for example, one, two or three layers of glass, such as borosilicate glass, fused silica glass or quartz, using one of the processing methods shown in fig. 36A-36C and processing techniques such as focused femtosecond laser ablation and/or laser glass bonding.

In fig. 36A, a first wafer is processed with a laser (e.g., a laser producing femtosecond laser radiation) to ablate wafer material and provide a patterned surface. The patterned wafer surface may include a plurality of microfluidic devices (e.g., 12 devices per 210mm diameter wafer), each of which may include a plurality of fluidic channels. The processed wafer may then be diced into individual microfluidic chips comprising open fluidic channels, which may optionally be subsequently sealed, for example, by sealing with a membrane or clamping the device to another carrier surface.

In fig. 36B, a first wafer is processed to form a patterned surface, which can then be placed in contact with a second wafer and bonded thereto to seal the fluid channel. Depending on the materials used, e.g., glass wafers, silicon wafers, etc., bonding may be performed using, for example, a thermal bonding process, an anodic bonding process, a laser glass bonding process, etc. The second wafer covers and/or seals grooves, notches, and/or holes on the wafer having the patterned surface to form fluid channels and/or fluid chambers (e.g., interior portions) of the device at the junction of the two wafer assemblies. The bonded structure may then be diced into individual microfluidic chips, e.g. 12 microfluidic chips per 210mm diameter wafer.

In fig. 36C, a first wafer is processed to form a pattern of fluid channels, which are cut or etched through the entire thickness of the wafer (i.e., open on either surface of the wafer). The first wafer is then sandwiched between and bonded to the second wafer on one side and the third wafer on the other side. Depending on the materials used, e.g., glass wafers, silicon wafers, etc., bonding may be performed using, for example, a thermal bonding process, an anodic bonding process, a laser glass bonding process, etc. The second wafer and the third wafer cover and/or seal grooves, recesses, and/or holes in the first wafer to form fluid channels and/or fluid chambers (e.g., interior portions) of the device. The bonded structure may then be diced into individual microfluidic chips, e.g. 12 microfluidic chips per 210mm diameter wafer.

EXAMPLE 3 coating of flow cell surfaces with hydrophilic Polymer coatings

The glass flow cell device was coated by: the prepared glass channels were washed with KOH, then rinsed with ethanol, and then silanized at 65℃for 30 minutes. The fluidic channel surface was activated with EDC-NHS for 30 min, then the oligonucleotide primers were grafted by incubating the activated surface with 5 μm primer for 20 min, then blunted with 30 μm amino-capped polyethylene glycol (PEG-NH 2).

A multi-layered surface was made according to the above method, wherein after the PEG-NH2 passivation step, the multi-arm PEG-NHs was flowed through the fluidic channel, followed by the addition of PEG-NH2, optionally followed by incubation with PEG-NHs, and optionally followed by incubation with multi-arm PEG-NH 2. For these surfaces, the primers can be grafted at any step, especially after the final addition of the multi-arm PEG-NH 2.

Example 4-flow cell apparatus for nucleic acid sequencing

Figures 37 A1-37A-4 illustrate non-limiting examples of one-piece glass microfluidic chip/flow cell designs. In this design, the fluid channels and inlet/outlet holes may be fabricated using, for example, focused femtosecond laser radiation. There are two flow channels ("lanes") in the flow cell device, each flow channel comprising, for example, two rows of 26 frames each (i.e., where one "frame" is an image area equivalent to the field of view of the corresponding imaging module), such that tiling 2×26=52 images is sufficient to image the entire flow channel. The fluid channel may have a depth of, for example, about 100 μm. The fluid passage 1 has an inlet hole A1 and an outlet hole A2, and the fluid passage 2 has an inlet hole B1 and an outlet hole B2. The flow cell device may also include a 1D linear, human readable and/or machine readable bar code, and optionally a 2D matrix bar code.

Figures 37B-1 through 37B-3 illustrate non-limiting examples of two-piece glass microfluidic chip/flow cell designs. In this design, the fluid channels and inlet/outlet holes may be fabricated using, for example, focused femtosecond laser ablation or photolithographic and chemical etching processes. The 2 pieces may be joined together using any of the various techniques described above. The inlet and outlet apertures may be positioned on the top layer of the structure and oriented in such a way that they are in fluid communication with at least one of the fluid channels and/or fluid chambers formed in the interior of the device. In a flow cell device there are two flow channels, such as the device shown in fig. 37 A1-37A-4, each flow channel comprising, for example, 2 rows of 26 frames each. The fluid channel may have a depth of, for example, about 100 μm. The fluid passage 1 has an inlet hole A1 and an outlet hole A2, and the fluid passage 2 has an inlet hole B1 and an outlet hole B2. The flow cell device may also include a 1D linear, human readable and/or machine readable bar code, and optionally a 2D matrix bar code.

FIGS. 37C-1 through 37C-4 illustrate non-limiting examples of three-piece glass microfluidic chip/flow cell designs. In this design, the fluid channels and inlet/outlet holes may be fabricated using, for example, focused femtosecond laser ablation or photolithographic and chemical etching processes. The 3 pieces may be joined together using any of the various techniques described above. A first wafer (including a through pattern of fluid channels or fluid chambers) may be sandwiched between and bonded to a second wafer on one side and a third wafer on the other side. The inlet and outlet apertures may be positioned on the top layer of the structure and oriented in such a way that they are in fluid communication with at least one of the fluid channels and/or fluid chambers formed inside the device. There are two flow channels in the flow cell device, as shown in FIGS. 37A 1-37A-4 and 37B-1-37B-3, with 2 rows of each flow channel, with 26 frames per row. The depth of the fluid channel may be, for example, about 100 μm. The fluid passage 1 has an inlet hole A1 and an outlet hole A2, and the fluid passage 2 has an inlet hole B1 and an outlet hole B2. The flow cell device may also include a 1D linear, human readable and/or machine readable bar code, and optionally a 2D matrix bar code.

EXAMPLE 5 imaging of nucleic acid clusters in capillary flow cells

Nucleic acid clusters are established within capillaries and fluorescence imaging is performed. The test was performed using a flow device with capillary tubes. An example of the generated cluster image is given in fig. 38. The figure shows that nucleic acid clusters formed by amplification in the lumen of the capillary flow cell device disclosed herein can be reliably formed and visualized.

Example 6 Plastic sample Carrier Structure

In some cases, the disclosed sample carrier structures can be fabricated from polymers. Examples of materials that may be used to fabricate the sample carrier structure, e.g., capillary flow cell device, include, but are not limited to, polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HOPE), cyclic Olefin Polymer (COP), cyclic Olefin Copolymer (COC), polyethylene terephthalate (PET), or any combination thereof. Various compositions constituting glass and plastic substrates are also contemplated.

Modification of a polymer surface for the purposes of the surface coating disclosed herein involves rendering the surface reactive to other chemical groups (-R) including amines. In some cases, these reactive surfaces may be stored at room temperature for extended periods of time, for example, for at least 3 months or longer when prepared on a suitable substrate. Such surfaces may be further grafted with R-PEG and R-primer oligomers for surface amplification of nucleic acids, as described elsewhere herein. Any of a number of methods known in the art may be used to modify the plastic surface, such as Cyclic Olefin Polymer (COP). For example, ti: sapphire laser ablation, UV-mediated ethylene glycol methacrylate photo-grafting, plasma treatment or mechanical agitation (e.g., sandblasting or polishing, etc.) to create hydrophilic surfaces that can remain reactive for many chemical groups, such as amine groups, for months. These groups can then bind to inactivating polymers (e.g., PEG) or biomolecules (e.g., DNA or proteins) without losing biochemical activity. For example, ligation of DNA primer oligomers allows for amplification of DNA on a passivated plastic surface while reducing or minimizing non-specific adsorption of proteins, fluorophore molecules, or other hydrophobic molecules.

In addition, in some cases, surface modifications may be combined with, for example, laser printing or UV masking to create a patterned surface. This allows for patterned attachment of DNA oligomers, proteins, or other moieties, providing surface-based enzymatic activity, binding, detection, or processing. For example, DNA oligomers may be used only to amplify DNA within patterned features, or to capture amplified long DNA concatemers in a patterned manner. In some embodiments, enzyme islands may be created in patterned areas capable of reacting with a solution-based matrix. Because plastic surfaces are particularly suited for these modes of processing, plastic sample carrier surfaces or flow cell devices may be considered particularly advantageous in some embodiments contemplated herein.

Furthermore, plastics may be more easily injection molded, embossed, stamped or 3D printed than glass substrates to form any shape, including microfluidic devices, and thus may be used to create surfaces for binding and analyzing biological samples in a variety of configurations (e.g., sample-result microfluidic chips for biomarker detection or DNA sequencing).

Specific and localized DNA amplification can be performed on modified plastic surfaces, resulting in nucleic acid spots with ultra-high contrast-to-noise ratios and very low background when probed with fluorescent markers.

Hydrophilized amine-reactive cyclic olefin polymer surfaces with amine-primers and amine-PEG can be prepared and demonstrated to support rolling circle amplification. When probed with a fluorophore-labeled primer, or when labeled dntps are added to the hybridized primer by a polymerase, bright spots of DNA amplicon are observed to exhibit a signal-to-noise ratio greater than 100, very low background, indicating highly specific amplification, and very low levels of non-specific protein binding to hydrophobic fluorophores, which is a marker for high precision detection required by systems (e.g., fluorescence-based DNA sequencers).

Example 7-prophetic example of sequencing using a structured illumination imaging system

A structured illumination imaging system 4100, such as the non-limiting example shown in fig. 41, can be used in combination with a flow cell 4187 comprising a low non-specific binding surface to perform nucleic acid sequencing. The target nucleic acid sequence hybridizes to an adapter/primer sequence attached to a low non-specific binding surface 4188 inside a flow cell 4187 at a high surface density and is clonally amplified using hybridization and amplification buffers specifically formulated for the surface to enhance specific hybridization and amplification rates.

The flow cell 4187 is installed in the structured illumination imaging system 4100 and a sequencing reaction cycle is initiated that includes the use of, for example, the polymer-nucleotide conjugate chemistry described above and the workflow shown in fig. 40. If the nucleotide portion of the polymer-nucleotide conjugate is complementary to a nucleotide of the target sequence, the fluorescently labeled polymer nucleotide conjugate is introduced into a flow cell 4187 and contacted with its surface 4188 to form a multivalent binding complex. Excess unbound polymer-nucleotide conjugate is then rinsed away.

For each detection step, a series of images of the surface 4188 are captured by using different orientations of the diffraction grating (e.g., 4130A) in at least one branch of the illumination light path and at a plurality of different locations of the optical phase modulator (e.g., 4140A) to project a pattern of illumination light stripes onto the surface 4188. After image acquisition, the series of images are processed using an image reconstruction algorithm to generate a higher resolution image than can be obtained using only the diffraction limited optics. This process may be repeated for multiple locations on the surface 4188 to create a tiled image of the inner flow cell surface. Optionally, the focal plane may be adjusted and the process may be repeated to generate a high resolution image of the second inner flow cell surface 4189.

The combination of high contrast-to-noise ratio images (achieved using the disclosed low binding surfaces and multi-labeled polymer-nucleotide conjugate sequencing chemistry) with efficient processing of relatively small numbers of images acquired using a structured illumination imaging system to image the flowcell surface at super resolution (thus enabling the use of higher target sequence cluster surface densities) may help to increase overall sequencing throughput.

Example 8-prophetic example of double-surface imaging Using a multiplexed read head

A multiplexed read head such as schematically shown in fig. 44A and 44B is designed to perform duplex imaging. The read head includes a plurality of microfluorometers assembled so that they remain in a fixed position relative to each other and can be scanned in a direction horizontal to a pair of opposed internal flow cell surfaces to acquire an image of an elongate column of each surface. As in fig. 44A, a first subset of the plurality of microfluorometers is configured to acquire an image of a first inner flow cell surface and a second subset of the plurality of microfluorometers is configured to acquire an image of a second inner flow cell surface facing the first inner surface and separated therefrom by a thickness of the intermediate fluid channel.

Flow cells comprising low non-specific binding surface coatings are used for nucleic acid sequencing. The target nucleic acid sequence is hybridized to an adapter/primer sequence attached to a low non-specific binding surface inside the flow cell and clonally amplified using hybridization and amplification buffers specifically formulated for the surface to enhance specific hybridization and amplification rates.

The flow cell is installed in an imaging system that includes a multiplexed read head and initiates a sequencing reaction cycle that includes the use of, for example, the polymer-nucleotide conjugate chemistry described above and the workflow shown in fig. 40. If the nucleotide portion of the polymer-nucleotide conjugate is complementary to a nucleotide of the target sequence, the fluorescently labeled polymer nucleotide conjugate is introduced into a flow cell and contacted with an inner surface to form a multivalent binding complex. Excess unbound polymer-nucleotide conjugate is then rinsed away.

For each detection step, as shown in fig. 44B, the multiplexed read head is scanned in at least one direction parallel to the flow cell inner surface (or the flow cell may be scanned relative to the multiplexed read head) and images of the first inner flow cell surface and the second inner flow cell surface are simultaneously acquired, while the autofocus mechanism may maintain an appropriate working distance between the objective lens of the multiplexed read head and at least one of the inner flow cell surfaces.

The ability to image both flow cell surfaces simultaneously using a single pass scan of the flow cell (depending on the design of the read head) can greatly increase sequencing throughput.

Further numbered embodiments

The present disclosure also provides the following exemplary embodiments.

1. A nucleic acid molecule sequencing system comprising:

a) A flow cell having an inner surface comprising a plurality of primed target nucleic acid sequences coupled thereto, wherein a primed target nucleic acid sequence of the plurality of primed target nucleic acid sequences has a polymerase bound thereto;

b) A fluid flow controller configured to control sequential and iterative delivery of reagents to the inner surface of the flow cell;

c) An imaging module, the imaging module comprising:

i) A structured illumination system; and

ii) an image acquisition system configured to acquire an image of the inner surface of the flow cell; and

d) A processor, wherein the processor is programmed to instruct the system to perform an iterative method comprising:

i) Contacting the plurality of primer-formulated target nucleic acid sequences coupled to the inner surface of the flow cell with a nucleotide composition to form a transient binding complex between the plurality of primer-formulated target nucleic acid sequences and a plurality of nucleotide moieties when the nucleotide moieties of the nucleotide composition are complementary to the nucleotides of the primer-formulated target nucleic acid sequences; and

ii) imaging the interior surface of the flow cell to detect the transient binding complex, thereby determining the identity of the nucleotide of the primer-materialized target nucleic acid sequence.

2. The system of embodiment 1, wherein the structured illumination system comprises an optical system designed to project a periodic light pattern on the inner surface of the flow cell, and wherein the relative orientation or phase shift of a plurality of the periodic light patterns can be changed in a known manner.

3. The system of embodiment 1, wherein the structured illumination system comprises a first optical arm comprising: a first light emitter for emitting light; and a first beam splitter for splitting light emitted by the first light emitter to project a first plurality of fringes on the inner surface of the flow cell.

4. The system of embodiment 3, wherein the structured illumination system further comprises a second optical arm comprising: a second light emitter for emitting light; and a second beam splitter for splitting light emitted by the second light emitter to project a second plurality of fringes on the inner surface of the flow cell.

5. The system of embodiment 4 wherein the structured illumination system further comprises an optical element for combining the optical paths of the first and second arms.

6. The system of embodiment 4 or embodiment 5, wherein the first beam splitter comprises a first transmissive diffraction grating and the second beam splitter comprises a second transmissive diffraction grating.

7. The system of embodiment 4 or embodiment 5, wherein the first light emitter and the second light emitter emit unpolarized light, and wherein the first transmissive diffraction grating and the second transmissive diffraction grating are to diffract unpolarized light emitted by a respective one of the first light emitter and the second light emitter.

8. The system of embodiment 6 or embodiment 7, wherein the optical element for combining the optical paths of the first plurality of fringes and the second plurality of fringes comprises a mirror having an aperture, wherein the mirror is arranged to reflect light diffracted by the first transmissive diffraction grating, and the aperture is arranged to pass at least first order light diffracted by the second transmissive diffraction grating.

9. The system of embodiment 8, further comprising: one or more optical elements for phase shifting the first plurality of fringes and the second plurality of fringes.

10. The system of embodiment 9, wherein the one or more optical elements for phase shifting the first plurality of fringes and the second plurality of fringes comprise a first rotating optical window phase shifting the first plurality of fringes and a second rotating optical window phase shifting the second plurality of optical fringes.

11. The system of embodiment 9 or embodiment 10, wherein the one or more optical elements for phase shifting the first plurality of fringes and the second plurality of fringes comprises a first linear motion stage for translating the first diffraction grating and a second linear motion stage for translating the second diffraction grating.

12. The system of any one of embodiments 9-11, wherein the one or more optical elements for phase shifting the first plurality of fringes and the second plurality of fringes comprises a single rotating optical window, wherein the single rotating optical window is located behind the mirror having an aperture in an optical path to the sample.

13. The system of embodiment 12, wherein the axis of rotation of the single rotating optical window is offset from the optical axis of each grating by about 45 degrees.

14. The system of any one of embodiments 9-13, wherein the first plurality of fringes and the second plurality of fringes are angularly offset from about 90 degrees on the sample plane.

15. The system of embodiment 14, wherein the sample comprises a plurality of features regularly patterned in a rectangular array or a hexagonal array.

16. The system of any of embodiments 9-15, further comprising an objective lens for projecting each of the first plurality of fringes and the second plurality of fringes onto the sample.

17. The system of any of embodiments 9 through 16, further comprising one or more beam blockers for blocking zero order light emitted by each of the first diffraction grating and the second diffraction grating.

18. The system of embodiment 17, wherein the one or more beam blockers comprise a bragg grating.

19. The system of any one of embodiments 6-18, wherein the optical element for combining the optical paths of the first and second arms comprises a polarizing beam splitter, wherein the first diffraction grating diffracts vertically polarized light, and wherein the second diffraction grating diffracts horizontally polarized light.

20. The system of any one of embodiments 4-19, wherein the first beam splitter and the second beam splitter each comprise a beam splitter cube or plate.

21. The system of any of embodiments 3-20, wherein the first beam splitter comprises a first reflective diffraction grating and the second beam splitter comprises a second reflective diffraction grating.

22. The system of any of embodiments 1-21, wherein the structured illumination system comprises a plurality of beam splitter slides comprising a plurality of beam splitters mounted on a linear translation stage such that the plurality of beam splitters have a fixed orientation relative to an optical axis of the system.

23. The system of embodiment 22, wherein the plurality of beam splitters comprises a plurality of diffraction gratings.

24. The system of embodiment 23, wherein the plurality of diffraction gratings comprises two diffraction gratings.

25. The system of any of embodiments 1-24, wherein the structured illumination system comprises a fixed two-dimensional diffraction grating used in combination with a spatial filter wheel to project a one-dimensional diffraction pattern onto the inner surface of the flow cell.

26. The system of any one of embodiments 1 through 25, wherein the image acquisition system comprises a customized tube lens that, in combination with an objective lens, is capable of imaging the first inner flow cell surface and the second inner flow cell surface with substantially the same image resolution.

27. The system of any one of embodiments 1 to 26, wherein the nucleotide composition comprises a conjugated polymer-nucleotide composition.

28. The system of embodiment 27, wherein the conjugated polymer-nucleotide composition comprises a plurality of nucleotide moieties conjugated to a polymer core.

29. The system of embodiment 28, wherein the plurality of nucleotide moieties comprises nucleotides, nucleotide analogs, or any combination thereof.

30. The system of embodiment 28 or embodiment 29, wherein the plurality of nucleotide portions comprises a plurality of identical nucleotide portions.

31. The system of any one of embodiments 1 to 30, wherein the nucleotide composition lacks a polymerase prior to formation of the transient binding complex.

32. A method of sequencing a nucleic acid molecule, comprising:

a) Providing a plurality of primed target nucleic acid sequences tethered to a surface, wherein a primed target nucleic acid sequence of the plurality of primed target nucleic acid sequences has a polymerase bound thereto;

b) Contacting the plurality of primed target nucleic acid sequences with a nucleotide composition to form a transient binding complex between the plurality of primed target nucleic acid sequences and a plurality of nucleotide portions when the nucleotide portions of the nucleotide composition are complementary to the nucleotides of the primed target nucleic acid sequences; and

c) Detecting the transient binding complex to determine the identity of the nucleotide of the primer-materialized target nucleic acid sequence, wherein the detecting comprises:

i) Illuminating the surface with light provided by a structured illumination system under a first set of illumination conditions to project a first plurality of fringes oriented in a particular direction on the surface;

ii) capturing a first plurality of phase images of the surface, wherein the position of the first plurality of fringes moves across the surface during capturing of the first plurality of images;

iii) Illuminating the surface with light provided by the structured illumination system under a second set of illumination conditions to project a second plurality of fringes on the surface, wherein the second plurality of fringes are angularly offset from the first plurality of fringes on the surface; and

iv) capturing a second plurality of phase images of the surface illuminated with the second plurality of fringes, wherein the position of the second plurality of fringes moves across the surface during capturing of the second plurality of fringes.

33. The method of embodiment 32, wherein the structured illumination system comprises a first optical arm comprising: a first light emitter for emitting light; and a first diffraction grating for diffracting light emitted by the first light emitter to project the first plurality of fringes oriented in a particular direction onto the surface.

34. The method of embodiment 33, wherein the structured illumination system comprises a second optical arm comprising: a second light emitter for emitting light; and a second diffraction grating for diffracting light emitted by the second light emitter to project the second plurality of fringes angularly offset from the first plurality of fringes onto the surface.

35. The method of any of embodiments 32-34, wherein the structured illumination system comprises a plurality of beam splitter slides comprising a plurality of beam splitters mounted on a linear translation stage such that the plurality of beam splitters have a fixed orientation relative to an optical axis of the system, and wherein the first set of illumination conditions corresponds to a first position of the linear translation stage and the second set of illumination conditions corresponds to a second position of the linear translation stage.

36. The method of embodiment 35, wherein the plurality of beam splitters comprises a plurality of diffraction gratings.

37. The method of embodiment 36, wherein the plurality of diffraction gratings comprises two diffraction gratings.

38. The method of any of embodiments 32-37, wherein the structured illumination system comprises a fixed two-dimensional diffraction grating used in combination with a spatial filter wheel to project a one-dimensional diffraction pattern on the surface, and wherein the first set of illumination conditions corresponds to a first location of the spatial filter wheel and the second set of illumination conditions corresponds to a second location of the spatial filter wheel.

39. The method of any of embodiments 34-38, wherein the first diffraction grating and the second diffraction grating are transmissive diffraction gratings, wherein the structured illumination system comprises a mirror having an aperture to reflect light diffracted by the first diffraction grating and pass at least first order light diffracted by the second diffraction grating.

40. The method of any of embodiments 32 to 39, further comprising: one or more images having a higher resolution than each of the first and second plurality of captured phase images are computationally reconstructed using at least the first and second plurality of captured phase images.

41. The method of embodiment 40, wherein the first plurality of stripes are angularly offset from the second plurality of stripes on the surface by about 90 degrees.

42. The method of any one of embodiments 32 to 41, wherein the surface comprises a plurality of features regularly patterned in a rectangular array or a hexagonal array.

43. The method of any of embodiments 32-42, wherein the first plurality of fringes and the second plurality of fringes are phase shifted by rotating a single optical window located in an optical path between the surface and each of the first diffraction grating and the second diffraction grating, wherein a rotational axis of the single rotating optical window is offset from an optical axis of each of the diffraction gratings.

44. The method of any of embodiments 34-43 wherein after capturing the first plurality of phase images, the first optical arm is turned off and the second optical arm of the structured illumination system is turned on.

45. The method of any of embodiments 34-44 wherein the first diffraction grating and the second diffraction grating are mechanically fixed during image capture.

46. The method of any one of embodiments 32 to 45, wherein the nucleotide composition comprises a conjugated polymer-nucleotide composition.

47. The method of embodiment 46, wherein the conjugated polymer-nucleotide composition comprises a plurality of nucleotide moieties conjugated to a polymer core.

48. The method of embodiment 47, wherein the plurality of nucleotide moieties comprises nucleotides, nucleotide analogs, or any combination thereof.

49. The method of embodiment 47 or embodiment 48, wherein the plurality of nucleotide portions comprises a plurality of identical nucleotide portions.

50. The method of any one of embodiments 32 to 49, wherein the method is used to determine the identity of the n+1 or terminal nucleotide of the primer strand of the primer-materialized target nucleic acid sequence.

51. The method of any one of embodiments 32 to 50, wherein the nucleotide composition lacks a polymerase prior to forming the transient binding complex.

52. A detection apparatus, comprising:

a) A readhead assembly comprising a plurality of microfluorometers,

wherein the plurality of microfluorometers are maintained in a fixed position relative to each other to form a multiplexed read head,

wherein at least one of the first subset of the plurality of microfluorometers is configured to acquire wide field images of different areas of the first sample plane, and

wherein at least one of the second subset of the plurality of microfluorometers is configured to acquire wide field images of different regions of the second sample plane.

53. The detection device of embodiment 52, further comprising a translation stage configured to move the read head assembly in at least one direction parallel to the first and second sample planes.

54. The test device of embodiments 52 or 53, further comprising a sample stage configured to hold a flow cell comprising a first inner surface and a second inner surface such that the first inner surface is held at the first sample plane and the second inner surface is held at the second sample plane.

55. The detection device of any one of embodiments 52 to 54, wherein at least one of the plurality of microfluorometers is configured to acquire a wide field image having a field of view of at least 1 mm.

56. The detection device of any one of embodiments 52 to 55, wherein at least one of the plurality of microfluorometers is configured to acquire a wide field image having a field of view of at least 1.5 mm.

57. The detection device of any one of embodiments 52 to 56, wherein at least one of the microfluorometers further comprises a dedicated autofocus mechanism.

58. The detection device of embodiment 57, wherein the autofocus mechanism for the first microfluorometer is configured to integrate data from the autofocus mechanism for the second microfluorometer, whereby the autofocus mechanism for the first microfluorometer changes the focus of the first microfluorometer based on the focus position of the first microfluorometer and the focus position of the second microfluorometer.

59. The detection device of any one of embodiments 52 to 58, wherein the individual microfluorometers further comprise an objective lens, a beam splitter, and a detector, wherein the beam splitter is positioned to direct excitation radiation from an excitation radiation source to the objective lens and to direct emission radiation from the objective lens to the detector.

60. The detection device of embodiment 59, wherein the at least one separate microfluorometer further comprises a separate excitation radiation source.

61. The detection device of embodiment 59 or 60, wherein the excitation radiation source directs the excitation radiation to an objective lens of two or more individual microfluorometers of the plurality of microfluorometers such that the two or more individual microfluorometers share the excitation radiation source.

62. The detection device of any one of embodiments 59 to 61, wherein two or more individual micro-fluorometers of the plurality of micro-fluorometers further comprise or share at least two excitation radiation sources.

63. The test device of any one of embodiments 59-62, wherein the numerical aperture of the objective lens of an individual one of the plurality of microfluorometers is 0.2-0.5.

64. The detection device of any one of embodiments 52 to 63, wherein a micrometer of the plurality of micrometers is configured to acquire images at a resolution sufficient to distinguish features less than 50 micrometers apart.

65. The detection device of any one of embodiments 52 to 64, wherein a micrometer of the plurality of micrometers is configured to have a depth of field that is less than a separation distance between the first interior surface and the second interior surface of the flow cell.

66. The detection device of any one of embodiments 52 to 65, wherein the first subset of the plurality of microfluorometers is configured to acquire a wide field image at a first fluorescence emission wavelength and the second subset of the plurality of microfluorometers is configured to acquire a wide field image at a second fluorescence emission wavelength.

67. A method for determining the identity of a nucleotide in a target nucleic acid sequence, comprising:

a) Providing a plurality of primed target nucleic acid sequences, wherein a primed target nucleic acid sequence in the plurality of primed target nucleic acid sequences has a polymerase bound thereto;

translating the multiplexed read head in at least one direction parallel to a surface tethered by the plurality of primer-containing target nucleic acid sequences,

Wherein the multiplexed read head comprises a plurality of microfluorometers held in a fixed position relative to each other, an

Wherein at least one of the plurality of microfluorometers is configured to acquire a wide field image of a different surface area than other of the plurality of microfluorometers.

68. The method of embodiment 67, wherein the nucleotide composition comprises a conjugated polymer-nucleotide composition.

69. The method of embodiment 68, wherein the conjugated polymer-nucleotide composition comprises a plurality of nucleotide moieties conjugated to a polymer core.

70. The method of embodiment 69, wherein the plurality of nucleotide moieties comprises nucleotides, nucleotide analogs, or any combination thereof.

71. The method of embodiment 69 or embodiment 70, wherein the plurality of nucleotide portions comprises a plurality of identical nucleotide portions.

72. The method of any one of embodiments 67 to 71, wherein the method is used to determine the identity of the n+1 or terminal nucleotide of the primer strand of the primer-materialized target nucleic acid sequence.

73. The method of any one of embodiments 67 to 72, wherein the nucleotide composition lacks a polymerase prior to forming the transient binding complex.

74. The method of any one of embodiments 67 to 73, wherein the plurality of primer-directed target nucleic acid sequences are tethered to a first interior surface and a second interior surface of a flow cell, and wherein a first subset of the plurality of microfluorometers is configured to acquire wide field images of different regions of the first interior surface of the flow cell and a second subset of the plurality of microfluorometers is configured to acquire wide field images of different regions of the second interior surface of the flow cell.

75. A nucleic acid molecule sequencing system comprising:

a) A flow cell having at least one interior surface comprising a plurality of primed target nucleic acid sequences coupled thereto, wherein a primed target nucleic acid sequence of the plurality of primed target nucleic acid sequences has a polymerase bound thereto;

b) A fluid flow controller configured to control sequential and iterative delivery of reagents to the at least one interior surface of the flow cell;

c) An imaging module configured to image the at least one interior surface of the flow cell, wherein the imaging module comprises:

a multiplexed read head assembly comprising a plurality of microfluorometers held in a fixed position relative to each other;

Wherein at least one of the plurality of microfluorometers is configured to acquire a wide field image of at least one surface region that is different from other of the plurality of microfluorometers; and

i) Contacting the plurality of primer-formulated target nucleic acid sequences coupled to the at least one interior surface of the flow cell with a nucleotide composition to form a transient binding complex between the plurality of primer-formulated target nucleic acid sequences and a plurality of nucleotide moieties when the nucleotide moieties of the nucleotide composition are complementary to the nucleotides of the primer-formulated target nucleic acid sequences; and

ii) imaging the at least one interior surface of the flow cell using the multiplexed read head to detect the transient binding complex, thereby determining the identity of the nucleotides of the primed target nucleic acid sequence.

76. The system of embodiment 75, wherein the nucleotide composition comprises a conjugated polymer-nucleotide composition.

77. The system of embodiment 76, wherein the conjugated polymer-nucleotide composition comprises a plurality of nucleotide moieties conjugated to a polymer core.

78. The system of embodiment 77, wherein the plurality of nucleotide moieties comprises nucleotides, nucleotide analogs, or any combination thereof.

79. The system of embodiment 77 or embodiment 78, wherein the plurality of nucleotide portions comprises a plurality of identical nucleotide portions.

80. The system of any one of embodiments 75 to 79, wherein the method is used to determine the identity of the n+1 or terminal nucleotide of the primer strand of the primer-materialized target nucleic acid sequence.

81. The system of any one of embodiments 75 to 80, wherein the nucleotide composition lacks a polymerase prior to formation of the transient binding complex.

82. The method of any one of embodiments 75 to 81, wherein the plurality of primer-directed target nucleic acid sequences are tethered to a first interior surface and a second interior surface of the flow cell, and wherein a first subset of the plurality of microfluorometers is configured to acquire wide field images of different regions of the first interior surface of the flow cell and a second subset of the plurality of microfluorometers is configured to acquire wide field images of different regions of the second interior surface of the flow cell.

83. The system of any one of embodiments 75 to 82, further comprising a translation stage configured to move the multiplexed read head assembly in at least one direction parallel to the first and second sample planes.

84. The system of any one of embodiments 75 to 83, wherein at least one of the plurality of microfluorometers is configured to acquire a wide field image having a field of view of at least 1 mm.

85. The system of any one of embodiments 75 to 84, wherein at least one of the plurality of microfluorometers is configured to acquire a wide field image having a field of view of at least 1.5 mm.

86. The system of any of embodiments 74-85, wherein at least one of the microfluorometers further comprises a dedicated autofocus mechanism.

87. The system of embodiment 86, wherein the autofocus mechanism for the first microfluorometer is configured to integrate data from the autofocus mechanism for the second microfluorometer such that the autofocus mechanism for the first microfluorometer changes the focus of the first microfluorometer based on the focus position of the first microfluorometer and the focus position of the second microfluorometer.

88. The system of any one of embodiments 75-87, wherein individual ones of the plurality of microfluorometers further comprise an objective lens, a beam splitter, and a detector, wherein the beam splitter is positioned to direct excitation radiation from an excitation radiation source to the objective lens and to direct emission radiation from the objective lens to the detector.

89. The system of embodiment 88, wherein at least one individual microfluorometer further comprises an individual excitation radiation source.

90. The system of embodiment 89, wherein the excitation radiation source directs the excitation radiation to an objective lens of two or more individual microfluorometers of the plurality of microfluorometers such that the two or more individual microfluorometers share the excitation radiation source.

91. The system of any of embodiments 88 to 90, wherein two or more individual microfluorometers of the plurality of microfluorometers further comprise or share at least two excitation radiation sources.

92. The system of any of embodiments 88 to 91, wherein the numerical aperture of the objective lens of an individual micro-fluorometer of the plurality of micro-fluorometers is 0.2 to 0.5.

93. The system of any one of embodiments 75 to 92, wherein a microfluorometer of the plurality of microfluorometers is configured to acquire images at a resolution sufficient to distinguish features less than 50 microns apart.

94. The system of any one of embodiments 82-93, wherein a microfluorometer of the plurality of microfluorometers is configured to have a depth of field that is less than a separation distance between the first and second inner surfaces of the flow cell.

95. The system of any one of embodiments 82 to 94, wherein the first subset of the plurality of microfluorometers is configured to acquire a wide field image at a first fluorescence emission wavelength and the second subset of the plurality of microfluorometers is configured to acquire a wide field image at a second fluorescence emission wavelength.

96. A method of sequencing a nucleic acid molecule, the method comprising:

a) Providing a surface; wherein the surface comprises:

i) A substrate;

ii) at least one hydrophilic polymer coating;

iii) A plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating; and

iv) at least one discrete region of the surface comprising a plurality of clonally amplified sample nucleic acid molecules immobilized onto the plurality of attached oligonucleotide molecules, wherein the plurality of immobilized clonally amplified sample nucleic acid molecules are present at a distance less than λ/(2 x NA), wherein λ is the central wavelength of the excitation energy source and NA is the numerical aperture of the imaging system.

b) Simultaneously applying a random light switching chemistry to the plurality of clonally amplified sample nucleic acid molecules to cause the plurality of clonally amplified sample nucleic acid molecules to fluoresce in up to four different colors in an on and off event by random light switching; and

c) Detecting the on and off events in the color channel of each color in real time as they occur for the plurality of clonally amplified sample nucleic acid molecules to determine the identity of the nucleotides of the clonally amplified sample nucleic acid molecules.

97. The method of embodiment 96, wherein the concentration of the reagent for random light switching is sufficiently high such that the probability of an on event for a given nucleotide of a given clonally amplified sample nucleic acid molecule from the plurality of clonally amplified sample nucleic acid molecules occurring simultaneously with an on event for a given nucleotide of a clonally amplified sample nucleic acid molecule adjacent to the given clonally amplified sample nucleic acid molecule is less than about 0.5%.

98. The method of embodiment 96, further comprising controlling the rate at which the on and off events occur to control the probability that an on event for a given nucleotide of a given clonally amplified sample nucleic acid molecule occurs simultaneously with an on event for a nucleotide of a clonally amplified sample nucleic acid molecule adjacent to the given clonally amplified sample nucleic acid molecule.

99. The method of embodiment 98, wherein controlling the rate at which the on and off events occur comprises adjusting the concentration of nucleotides and enzymes in the random photoswitching chemistry.

100. The method of embodiment 96, further comprising determining whether an illumination intensity of the detected event in the color channel is greater than a predetermined threshold.

101. The method of embodiment 96, further comprising determining whether a spot size of the detected event in the color channel is greater than a predetermined threshold.

102. The method of embodiment 96, wherein the at least one hydrophilic polymer coating comprises PEG.

103. The method of embodiment 96, wherein detecting comprises acquiring an image of the surface, wherein the image exhibits a contrast to noise ratio (CNR) of at least 40.

104. The method of embodiment 96, wherein detecting comprises acquiring an image of the surface, wherein the image exhibits a contrast to noise ratio (CNR) of at least 60.

105. The method of embodiment 96, wherein the substrate comprises glass.

106. The method of embodiment 96, wherein the substrate comprises plastic.

107. The method of embodiment 96, wherein the surface is located inside the flow channel.

108. The method of embodiment 96, wherein the at least one hydrophilic polymer layer comprises a branched hydrophilic polymer having at least 8 branches.

109. The method of embodiment 96, wherein the background fluorescence intensity measured at a region of the surface laterally displaced from the at least one discrete region is no greater than 2 times the intensity measured at the at least one discrete region prior to the clonal amplification.

110. The method of embodiment 96, wherein the sample nucleic acid molecule comprises a single-stranded polynucleic acid molecule comprising a repeat of a regularly occurring monomeric unit.

111. The method of embodiment 110, wherein the single stranded polynucleic acid molecule is at least 10kb in length.

112. The method of embodiment 110, further comprising double stranded monomer copies of the regularly occurring monomer units.

113. The method of embodiment 96, wherein the surface comprises a first layer comprising a monolayer of polymer molecules tethered to the surface of the substrate; a second layer comprising polymer molecules tethered to the polymer molecules of the first layer; a third layer comprising polymer molecules tethered to the polymer molecules of the second layer, wherein at least one layer comprises branched polymer molecules.

114. The method of embodiment 113, wherein the third layer further comprises an oligonucleotide tethered to the polymer molecule of the third layer.

115. The method of embodiment 114, wherein the oligonucleotides tethered to the polymer molecules of the third layer are distributed at multiple depths of the third layer.

116. The method of embodiment 113, further comprising a fourth layer comprising branched polymer molecules tethered to the polymer molecules of the third layer; and a fifth layer comprising polymer molecules tethered to the branched polymer molecules of the fourth layer.

117. The method of embodiment 116, wherein the polymer molecule of the fifth layer further comprises an oligonucleotide tethered to the polymer molecule of the fifth layer.

118. The method of embodiment 117, wherein the oligonucleotides tethered to the polymer molecules of the fifth layer are distributed at multiple depths of the fifth layer.

119. The method of embodiment 96, wherein the at least one hydrophilic polymer coating comprises a molecule selected from the group consisting of: polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), poly (vinylpyridine), poly (vinylpyrrolidone) (PVP), poly (acrylic acid) (PAA), polyacrylamide, poly (N-isopropylacrylamide) (PNIPAM), poly (methyl methacrylate) (PMA), poly (2-hydroxyethyl methacrylate) (PHEMA), poly (oligo (ethylene glycol) monomethyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that the various alternatives to the embodiments of the invention described herein may be employed in any combination in practicing the invention. The following claims are intended to define the scope of the inventive concepts and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. An imaging system, comprising:

a) A sample container comprising a surface having a plurality of attachment elements, wherein a single oligonucleotide molecule is attached to each of the attachment elements, and wherein the average distance between adjacent ones of the attachment elements is less than an abbe limit; and

b) An imager positioned to image light switching occurring at the plurality of attachment elements.

2. The imaging system of claim 1, wherein the sample container comprises a flow cell comprising the plurality of attachment elements at a plurality of sample locations.

3. The imaging system of claim 1, wherein each of the plurality of attachment elements on the sample container is within a field of view of the imager for imaging the light switch.

4. The imaging system of claim 1, wherein an average distance between the adjacent elements is less than 20nm.

5. The imaging system of claim 1, wherein the surface comprises at least one hydrophilic polymer coating.

6. The imaging system of claim 1, wherein the plurality of attachment elements comprises a branched hydrophilic polymer having at least 8 branches.

7. The imaging system of claim 1, wherein the imager is positioned to exhibit a contrast to noise ratio CNR of at least 40 for the optically switched imaging of the surface.

8. The imaging system of claim 1, wherein the imager is positioned to exhibit a contrast to noise ratio CNR of at least 60 for the optically switched imaging of the surface.

9. A super-resolution imaging system, comprising:

(a) An excitation light source;

(b) Depleting the light source;

(c) An objective lens;

(d) An optical path comprising an optical assembly that produces an array of regions, wherein each region comprises an active region comprising light from the excitation light source, the active region surrounded by a depletion region comprising light from the depletion light source;

(e) At least one image sensor that receives and integrates signals from said areas over time and generates integrated signals for individual points illuminated by a single annular area; and

(f) A processor that determines fluorescence of the respective spots from the integrated signal.

10. The super resolution imaging system according to claim 9, further comprising at least one tube lens disposed in an optical path between the objective lens and the at least one image sensor.

11. The super resolution imaging system as claimed in claim 9, wherein the at least one tube lens includes an asymmetric convex-convex lens, a convex-flat lens, an asymmetric concave-concave lens, and an asymmetric convex-concave lens in that order.

12. The super resolution imaging system according to claim 9, wherein the spots correspond to fluorescent nucleic acid molecules on a solid support.

13. The super resolution imaging system as claimed in claim 9, wherein each region is a circular ring region.

14. The super resolution imaging system according to claim 9, wherein the excitation light source includes an excitation laser for each region in the array and the depletion light source includes a depletion laser for each region in the array, and wherein for each region in the array of regions the optical path is used to direct light from the respective excitation and depletion lasers to the annular region produced.

15. The super resolution imaging system as claimed in claim 9, wherein the at least one image sensor includes an image sensor for each respective region in the array of regions.

16. The super resolution imaging system according to claim 9, wherein the optical path includes a deflector that directs light from the excitation light source and directs light from the depletion light source in a time dependent manner to generate the array of regions.

17. The super resolution imaging system according to claim 9, wherein the optical path includes a phase mask to split light from the excitation light source into a plurality of excitation light beams and to split light from the depletion light source into a plurality of depletion light beams to produce the array of regions.

18. The super resolution imaging system according to claim 9, wherein the optical path includes a waveguide to create a standing wave within the waveguide with the light from the depletion light source.

19. The super resolution imaging system according to claim 9, wherein the at least one image sensor includes a single image sensor to detect light from the sample.

20. The super resolution imaging system as claimed in claim 19, wherein the single image sensor comprises a multi-channel photon sensor.

21. The super resolution imaging system as claimed in claim 20, wherein the multi-channel photon sensor comprises a CCD image sensor.

22. The super resolution imaging system as claimed in claim 9, wherein the regions in the array of regions are distributed in a grid comprising a plurality of rows and a plurality of columns.

23. The super resolution imaging system as claimed in claim 9, further comprising a scanning system that moves the sample to move the array of regions relative to the sample.

24. A super-resolution imaging system, comprising:

(a) An excitation light source;

(b) Depleting the light source;

(c) An objective lens;

(d) An optical path comprising an optical assembly that produces a plurality of pattern areas, wherein each pattern area comprises excitation light from the excitation light source and depletion light from the depletion light source; and

(e) At least one image sensor that receives and integrates signals from fluorophores illuminated by the pattern region and generates fluorescence of the fluorophores based on the integrated signals.

25. The super resolution imaging system as claimed in claim 24, further comprising at least one tube lens disposed in the optical path between the objective lens and the at least one image sensor.

26. The super resolution imaging system according to claim 24, wherein the fluorophore has a dark state with a lifetime of greater than or equal to 100ms.

27. The super resolution imaging system as claimed in claim 24, wherein the pattern region includes a first activated light region surrounded by a second depleted light region.

28. An imaging system, comprising:

a) A flow cell comprising a first inner surface and a second inner surface, wherein the first inner surface or the second inner surface comprises at least 5 x 10 ⁸ A nucleic acid population coupled thereto;

b) An objective lens;

c) At least one image sensor for acquiring an image of a nucleic acid colony; and

d) An optical element disposed in an optical path between the objective lens and the at least one image sensor,

wherein the imaging system has a numerical aperture NA of greater than 0.2 and greater than 1.0mm ² And (2) the field of view FOV of

Wherein the optical element corrects imaging performance such that images of the first inner surface of the flow cell and the second inner surface of the flow cell have the same optical resolution.

29. The imaging system of claim 28, wherein the flow cell has a wall thickness of at least 700 μιη and a fluid-filled gap between the first inner surface and the second inner surface is at least 50 μιη.

30. The imaging system of claim 28, wherein the optical element is an optical compensator.

31. The imaging system of claim 28, further comprising a field programmable gate array FPGA.

32. The imaging system of claim 28, wherein the FOV is greater than 2.0mm ² 。

33. The imaging system of claim 32, wherein the optical resolution of the images of the first interior surface and the second interior surface is diffraction limited over the entire FOV.

34. The imaging system of claim 28, wherein the optical element is a motion actuated compensator.

35. The imaging system of claim 28, further comprising a focusing mechanism that refocuses the imaging system between acquiring images of the first inner surface and the second inner surface.

36. The imaging system of claim 28, wherein the imaging system images two or more fields of view on at least one of the first inner surface or the second inner surface.

37. The imaging system of claim 28, wherein the imaging system comprises a dichroic mirror and a bandpass filter set optimized for Cy3 emission, and wherein the image is acquired under non-signal saturation conditions while immersing the first inner surface or the second inner surface in 25mm maces pH 7.4 buffer.

38. The imaging system of claim 28, further comprising 2 imaging channels.

39. The imaging system of claim 28, wherein the imaging system further comprises means for extracting a plurality of nucleic acid molecules from the sample for sequencing.

40. The imaging system of claim 28, wherein the at least one image sensor comprises pixels having a pixel size selected such that a spatial sampling frequency of the imaging system is at least twice an optical resolution of the imaging system.

41. The imaging system of claim 28, wherein the first inner surface of the flow cell is disposed in the optical path between the objective lens and the second inner surface of the flow cell.

42. The imaging system of claim 28, further comprising a liquid between the first inner surface and the second inner surface.

43. The imaging system of claim 28, wherein the flow cell is a capillary flow cell.

44. The imaging system of claim 43, wherein the capillary flow cell is a multi-lumen capillary flow cell.

45. The imaging system of claim 28, wherein the flow cell comprises two or more flow channels.

46. The imaging system of claim 28, wherein at least one optical element is located in a permanent position between the objective lens and the at least one image sensor.

47. The imaging system of claim 28, further comprising a fluorescence filter.

48. The imaging system of claim 28, further comprising a fluorescence excitation filter that selectively transmits fluorescence emitted by a fluorescent label coupled to the covalently coupled nucleic acid population.

49. The imaging system of claim 28A system wherein at least one of said first interior surface and said second interior surface comprises at least 1 x 10 covalently coupled thereto ⁹ And (3) each of the nucleic acid communities.

50. The imaging system of claim 28, wherein at least one of the first inner surface and the second inner surface comprises at least 5 x 10 covalently coupled thereto ⁹ And (3) individual said nucleic acid colonies.