HK40014693B - Apparatuses, systems and methods for imaging micro-objects - Google Patents

Description

Devices, systems and methods for imaging micro-objects

Related applications

This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/429,066, filed December 1, 2016, the disclosure of which is incorporated herein by reference.

All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Background Art

As the field of microfluidics continues to develop, microfluidic devices have become a convenient platform for processing and manipulating micro-objects such as biological cells. For example, light-activated microfluidic devices provide some desired capabilities, including the ability to select and manipulate individual micro-objects. Typically, light-activated microfluidic devices (e.g., optoelectronic tweezers (OET) devices) utilize optically induced dielectrophoresis (DEP) to manipulate micro-objects. For example, micro-objects can be moved around a microfluidic device and incorporated within the microfluidic device. The simultaneous manipulation, analysis, and selection of micro-objects such as single cells can be valuable in biological discovery and development, as well as single-cell annotation and genomics.

However, conventional microscopes are not designed to observe micro-objects in microfluidic devices, especially light-actuated microfluidic devices. Therefore, images of micro-objects obtained using conventional microscopes may have large aberrations, which reduces the quality of the image. In addition, the optical equipment design in conventional microscopes may have a certain amount of out-of-focus light in the image, which may cause high-intensity noise in the image and reduce the contrast and resolution of the image. In addition, due to the limited compact space available for the optical equipment of microfluidic devices, there are generally limitations on the optical equipment. Therefore, there is a need to develop devices, systems and related methods for imaging and manipulating micro-objects to overcome the above-mentioned problems and challenges.

Summary of the Invention

The present disclosure relates to optical devices, systems, and methods for imaging and manipulating micro-objects. In particular, the present disclosure relates to optical devices, systems, and methods for imaging and manipulating micro-objects in light-actuated microfluidic devices.

Disclosed herein is an optical device for imaging and/or manipulating micro-objects in a microfluidic device, such as a light-actuated microfluidic (LAMF) device. The optical device may include a first light source, a structured light modulator, a first tube lens, an objective lens, a dichroic beam splitter, a second tube lens, and an image sensor. The structured light modulator may be configured to receive an unstructured light beam from the first light source and transmit a structured light beam to the first tube lens. The structured light beam may be adapted to selectively activate one or more of a plurality of dielectrophoresis (DEP) electrodes on a surface of a substrate of the LAMF device. The first tube lens may be configured to capture the structured light beam from the structured light modulator. The objective lens may be configured to image at least a portion of a housing of the microfluidic device within a field of view. The housing may include a flow region and/or a plurality of isolation docks, each of the plurality of isolation docks being fluidically connected to the flow region. The dichroic beam splitter may be configured to reflect (or transmit) the structured light beam from the first tube lens to the objective lens and to transmit (or reflect) an image beam received from the objective lens to the second tube lens. The second tube lens can be configured to receive the image beam from the dichroic beam splitter and transmit the beam to the image sensor. The image sensor can be configured to receive the image beam from the second tube lens and thereby generate an image of at least a portion of the housing of the microfluidic device. The optical apparatus can be configured to perform imaging, analysis, and manipulation of one or more micro-objects within the housing of the microfluidic device.

In some embodiments, the first tube lens has a clear aperture greater than 45 mm and is configured to capture all light beams from the structured light modulator. In some embodiments, the structured light modulator comprises an effective area of at least 15 mm (e.g., at least 15.5 mm, 16.0 mm, 16.5 mm, 17.0 mm, or greater). In some embodiments, the first tube lens has an effective focal length of approximately 162 mm or less (e.g., approximately 161 mm, approximately 160 mm, approximately 159 mm, approximately 158 mm, approximately 157 mm, approximately 156 mm, approximately 155 mm, or less). In some embodiments, the first tube lens has an effective focal length of approximately 155 mm.

In some embodiments, the optical device may further include a second light source configured to provide unstructured bright field illumination. In some embodiments, the optical device may further include a third light source. The second (or third) light source may be, for example, an LED or a laser light source. The laser light source may be configured to heat a surface within the housing of the microfluidic device and/or a fluid medium within the housing. The heating of the surface or medium may result in the generation of gas (e.g., bubbles).

In some embodiments, the optical apparatus can further include a nest configured to hold the microfluidic device during imaging. The nest can also be configured to provide at least one electrical connection to the microfluidic device and/or fluidic connection.

In some embodiments, the structured light modulator transmits (or transmits) multiple illumination beams. In some embodiments, the optical device is configured to illuminate multiple isolation docks using multiple illumination points. For example, each of the multiple isolation docks can be illuminated with a single illumination point, and the size of each illumination point can be determined to illuminate all or a portion of the isolation dock it is illuminating. In some embodiments, each of the multiple illumination points has a size of approximately 60 microns by 120 microns. In some embodiments, each of the multiple illumination points has an area of approximately 7,000 to approximately 20,000 square microns (e.g., any range defined by two of the aforementioned endpoints of approximately 7,000 square microns to approximately 10,000 square microns, approximately 10,000 square microns to approximately 15,000 square microns, or approximately 15,000 square microns to approximately 20,000 square microns).

In some embodiments, the optical device is further configured to focus a portion of the housing within the field of view simultaneously on the image sensor and the structured light modulator. In some embodiments, the optical device is further configured to image only a portion of the housing (e.g., an interior area of the flow region and/or each of the plurality of isolation docks) onto the image sensor to reduce overall noise for high image quality. In some embodiments, the structured light modulator is located at a conjugate plane of the image sensor. In some embodiments, the optical device is further configured to perform confocal imaging. In other embodiments, the optical device includes a sliding lens slidably positioned between the structured light modulator and the first barrel lens, wherein the sliding lens is configured to support stacked microscopy techniques.

In some embodiments, the objective lens is configured to minimize aberrations in an image of at least a portion of the plurality of isolation docks. In some embodiments, the second tube lens is configured to correct for residual aberrations of the objective lens. In some embodiments, the optical device may further include a correction lens configured to correct for residual aberrations of the objective lens. The correction lens may be located in front of the objective lens (i.e., between the objective lens and the microfluidic device) or behind the objective lens (i.e., between the objective lens and the dichroic beam splitter).

In some embodiments, the optical device may further include a control unit configured to adjust the illumination pattern of the structured light modulator to selectively activate one or more of the plurality of DEP electrodes and generate a DEP force to move the one or more micro-objects within the plurality of isolated docks. In some embodiments, the optical device may further include a control unit configured to adjust the illumination pattern of the structured light modulator to illuminate a selected area within the microfluidic device (e.g., a portion of the flow area and/or a portion of one or more isolated docks) and, optionally, one or more micro-objects located within the selected area.

Disclosed herein is a system for imaging and manipulating micro-objects. The system may include a microfluidic device, such as a light-actuated microfluidic (LAMF) device, an optical device, and a nest. The microfluidic device may include a housing and a substrate, the substrate including a surface and a plurality of dielectrophoresis (DEP) electrodes on the surface. In some embodiments, the housing of the microfluidic device includes a flow region and optionally a plurality of isolation docks, each of the plurality of isolation docks being fluidically connected to the flow region. The flow region and the plurality of isolation docks may be disposed on the substrate surface. The optical device, which may be any of the optical devices described herein, may be configured to perform imaging, analysis, and/or manipulation of one or more micro-objects within the housing.

In some embodiments, the system further comprises a control unit configured to adjust the illumination pattern of the structured light modulator to selectively activate one or more of the plurality of DEP electrodes of the substrate of the microfluidic device, thereby generating sufficient DEP force to move the one or more units within the housing. In some embodiments, the system further comprises a control unit configured to adjust the illumination pattern of the structured light modulator to illuminate a selected area (e.g., a portion of the flow area and/or a portion of one or more isolation docks) within the microfluidic device and, optionally, one or more micro-objects located within the selected area.

In some embodiments, the system is configured to illuminate at least a portion of the housing with a plurality of illumination points, including any portion of the flow area and/or the plurality of isolation docks within the field of view. For example, each isolation dock in the field of view can be illuminated with one or more illumination points, and the size of each illumination point can be determined to illuminate all or a portion of the isolation dock it is illuminating. In some embodiments, each of the plurality of illumination points has a size of approximately 60 microns by 120 microns. In some embodiments, each of the plurality of illumination points has an area of approximately 7,000 to approximately 20,000 square microns (e.g., any range defined by two of the aforementioned endpoints of approximately 7,000 square microns to approximately 10,000 square microns, approximately 10,000 square microns to approximately 15,000 square microns, or approximately 15,000 square microns to approximately 20,000 square microns).

Disclosed herein is a method for manipulating one or more micro-objects of a sample. The method may include the step of loading a sample containing the one or more micro-objects into a microfluidic device, such as a light-actuated microfluidic (LAMF) device. The microfluidic device may have a housing comprising a substrate having a surface and a plurality of dielectrophoresis (DEP) electrodes on the surface. The microfluidic device may also include a flow region and optionally a plurality of isolation docks, each of the plurality of isolation docks being fluidically connected to the flow region. The method may include the step of applying a voltage potential across the microfluidic device.

The method may further include the step of selectively activating a DEP force adjacent to at least one micro-object located within the microfluidic device by projecting structured light onto a first location on a surface of a substrate of the microfluidic device using an optical device, wherein the first location is located adjacent to a second location on the surface of the substrate, the second location being located below the at least one micro-object. The optical device may be any optical device described herein.

The method may further include the step of shifting the position of the generated DEP force by moving the structured light from the first position on the surface of the substrate of the microfluidic device to a third position on the surface of the substrate using the optical apparatus.

In some embodiments, the method can further include the step of capturing the image of at least a portion of the housing of the microfluidic device using the image sensor. In some embodiments, the imaged portion of the housing of the microfluidic device includes a flow area and/or at least one isolation dock and at least one micro-object.

In some embodiments, the structured light projected onto the first location on the substrate surface comprises a plurality of illumination points. In some embodiments, the first location on the substrate surface is located in a flow region of the microfluidic device, and the third location on the substrate surface is located within one of a plurality of isolation bays. In some embodiments, the structured light projected onto the first location on the substrate surface comprises a shape resembling a line segment or a symbol. In some embodiments, the structured light projected onto the first location on the substrate surface has a shape resembling the outline of a polygon (e.g., a square, rectangle, diamond, pentagon, etc.), a circle, etc.

In some embodiments, the method may further include a step of selectively activating DEP forces adjacent to a plurality of micro-objects located within the microfluidic device by projecting structured light onto a plurality of first locations on the surface of a substrate of the microfluidic device using an optical device, wherein each of the plurality of first locations is located near a corresponding second location on the surface of the substrate, and the corresponding second location is located below a corresponding micro-object in the plurality of first locations.

In some embodiments, the method may further include the step of shifting positions of DEP forces generated adjacent to the plurality of micro-objects by moving the imaged structured light from a plurality of first positions on the substrate surface to a plurality of corresponding third positions on the substrate surface using an optical device.

In some embodiments, the method may further include the step of capturing an image of at least a portion of the housing, the step including imaging only the flow region within the imaged portion of the housing and/or the interior region of each isolator dock, thereby reducing overall noise to achieve high image quality. In some embodiments, the method may further include the step of analyzing the image to provide feedback and adjustments to the first position.

Disclosed herein is a method for imaging one or more micro-objects of a sample. The method may include loading a sample containing the one or more micro-objects into a microfluidic device having a housing containing a flow area, capturing multiple images of at least a portion of the housing containing the one or more micro-objects using multiple corresponding illumination patterns projected into the at least a portion of the housing, and combining the multiple images to generate a single image of the one or more micro-objects located in the portion of the housing. In some embodiments, each of the multiple illumination patterns is generated using structured light and is different from other illumination patterns in the multiple illumination patterns. In some embodiments, the multiple images are captured using an optical system, which may be any optical system disclosed herein. In some embodiments, combining the multiple images includes processing each of the multiple images to remove out-of-focus background light.

In some embodiments, the illumination pattern projected into at least a portion of the housing and the corresponding image captured at the image sensor are simultaneously in focus. In some embodiments, the plurality of corresponding illumination patterns are configured to scan across the field of view (e.g., the entire field of view) within the housing.

Disclosed herein is a tube lens for an optical device of a microfluidic device, such as a light-actuated microfluidic (LAMF) device. The tube lens may include a first surface having a convex shape and a first positive radius of curvature, a second surface having a second radius of curvature, a third surface having a concave shape and a third negative radius of curvature, a fourth surface having a concave shape and a fourth negative radius of curvature, and a clear aperture having a diameter greater than 45 mm, wherein a front focal point and a back focal point of the tube lens are not equidistantly spaced from a midpoint and are asymmetrical.

In some embodiments, the back focal length (BFL) is minimized. In some embodiments, the tube lens has an effective focal length (EFL) of approximately 155 mm and a back focal length (BFL) of approximately 135 mm. In some embodiments, the tube lens has an effective focal length (EFL) of approximately 162 mm and a back focal length (BFL) of approximately 146 mm. In some embodiments, the tube lens has an effective focal length (EFL) of approximately 180 mm and a back focal length (BFL) of approximately 164 mm.

In some embodiments, the tube lens has an effective focal length (EFL) of approximately 155 mm, wherein the first radius of curvature is approximately 91 mm, the second radius of curvature is approximately 42 mm, the third negative radius of curvature is approximately -62 mm, and the fourth negative radius of curvature is approximately -116 mm.

In some embodiments, the tube lens has an effective focal length (EFL) of approximately 162 mm, wherein the first radius of curvature is approximately 95 mm, the second radius of curvature is approximately 54 mm, the third negative radius of curvature is approximately -56 mm, and the fourth negative radius of curvature is approximately -105 mm.

In some embodiments, the tube lens has an effective focal length (EFL) of approximately 180 mm, wherein the first radius of curvature is approximately 95 mm, the second radius of curvature is approximately 64 mm, the third negative radius of curvature is approximately -60 mm, and the fourth negative radius of curvature is approximately -126 mm.

In some embodiments, the tube lens has an effective focal length (EFL) of approximately 200 mm, wherein the first radius of curvature is approximately 160 mm, the second radius of curvature is approximately -62 mm, the third negative radius of curvature is approximately -80 mm, and the fourth negative radius of curvature is approximately -109 mm.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the present invention are set forth with particularity in the claims below. The features and advantages of the present disclosure may be better understood by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings, wherein:

FIG. 1A illustrates an example of a microfluidic device including associated control equipment and a system for use with the microfluidic device according to some embodiments of the present disclosure.

1B and 1C respectively illustrate vertical and horizontal cross-sectional views of a microfluidic device according to some embodiments of the present disclosure.

2A and 2B illustrate vertical and horizontal cross-sectional views, respectively, of a microfluidic device having an isolation dock according to some embodiments of the present disclosure.

2C shows a detailed horizontal cross-sectional view of an isolation dock according to some embodiments of the present disclosure.

2D illustrates a partial horizontal cross-sectional view of a microfluidic device having an isolation dock according to some embodiments of the present disclosure.

2E and 2F illustrate detailed horizontal cross-sectional views of an isolation dock according to some embodiments of the present disclosure.

FIG2G illustrates a microfluidic device with an isolation dock according to some embodiments of the present disclosure.

FIG2H illustrates a microfluidic device according to some embodiments of the present disclosure.

FIG. 3A illustrates a system that may be used to manipulate and observe a microfluidic device according to some embodiments of the present disclosure.

FIG. 3B illustrates an optical device for a microfluidic device according to some embodiments of the present disclosure.

4A is a schematic diagram of a system including an optical device and a microfluidic device according to some embodiments of the present disclosure.

FIG. 4B illustrates an example of multiple isolation docks in the microfluidic device of FIG. 4A .

4C shows a first tube lens of an optical device configured to capture all light beams from the structured light modulator in FIG. 4A .

5A is a schematic diagram of multiple light sources used in optical devices and microfluidic devices according to some other embodiments of the present disclosure.

5B illustrates an example dichroic beam splitter for use with multiple light sources of the optical device of FIG. 5A .

5C is a schematic diagram of another embodiment of a system including an optical apparatus and a microfluidic device.

6A is a schematic diagram of a system including an optical device having an excitation filter and an emission filter according to some other embodiments of the present disclosure.

6B is a schematic diagram of a system including an optical device according to some other embodiments of the present disclosure, wherein a beam splitter is configured to reflect a light beam from a first light source.

6C is a schematic diagram of a system including an optical device having a correction lens to compensate for aberrations according to yet other embodiments of the present disclosure.

7A is an optical schematic of an example tube lens for an optical device of a microfluidic device.

7B is an optical schematic diagram of another example tube lens for an optical apparatus for a microfluidic device.

7C is an optical schematic diagram of yet another example tube lens for an optical apparatus for a microfluidic device.

7D is an optical schematic diagram of another example tube lens for an optical apparatus for a microfluidic device.

8A-8D illustrate various embodiments of optical configurations that may be used by the optical system.

Figure 9A shows a schematic diagram of a simplified portion of the optical train of some embodiments.

Figure 9B shows a schematic diagram of a simplified portion of an optical train of some embodiments that has been modified to include a sliding lens for stacked microscopy techniques.

DETAILED DESCRIPTION

This specification describes exemplary embodiments and applications of the present invention. However, the present invention is not limited to these exemplary embodiments and applications, nor to the manner in which the exemplary embodiments and applications operate or are described herein. In addition, the accompanying drawings may show simplified or partial views, and the sizes of the elements in the accompanying drawings may be exaggerated or disproportionate. In addition, since the terms "on...", "attached to," "connected to," "coupled to," or similar words are used herein, an element (e.g., a material, layer, substrate, etc.) may be "on another element," "attached to," "connected to," or "coupled to" another element, regardless of whether the element is directly on, attached to, connected to, or coupled to the other element, or whether there are one or more spacer elements between the element and the other element. In addition, unless the context dictates otherwise, directions (e.g., on, under, top, bottom, side, up, down, directly below, directly above, above, below, horizontal, vertical, "x," "y," "z," etc.), if provided, are provided only by way of example and for ease of illustration and discussion rather than in a limiting sense. Where a list of elements is referred to (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or any combination of all of the listed elements. The section divisions in the specification are for ease of review only and do not limit any combination of the described elements.

As used herein, "substantially" means a device that is adequate for its intended purpose. Thus, the term "substantially" allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, etc., such as would be expected by one of ordinary skill in the art but which do not significantly affect overall performance. When used in relation to a numerical value or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within ten percent.

The term "plurality" means more than one.

As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

As used herein, the term "positioned" includes within its meaning "located at"

As used herein, a "microfluidic device" or "microfluidic apparatus" is a device comprising one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprising fluidically interconnected circuit elements, including but not limited to one or more regions, one or more flow paths, one or more channels, one or more chambers and/or docks, and at least one port configured to allow fluid (and optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, the microfluidic circuits of a microfluidic device will include a flow region, which may include a microfluidic channel and at least one chamber, and will hold a volume of fluid less than about 1 mL, for example, less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In certain embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. The microfluidic circuit can be configured to have a first end fluidically connected to a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected to a second port (e.g., an outlet) in the microfluidic device.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is a type of microfluidic device having microfluidic circuitry comprising at least one tubing element configured to hold a volume of fluid less than about 1 μL, for example, less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL, or less. The nanofluidic device can include a plurality of tubing elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000 or more). In certain embodiments, one or more (e.g., all) of at least one tubing element are configured to hold a volume of fluid of approximately 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 10 nL, 500 pL to 10 nL, 750 pL to 10 nL, 750 pL to 20 nL, 750 pL to 20 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one tubing element are configured to hold a volume of fluid of approximately 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.

As used herein, " microfluidic channel " or " flow channel " refer to the flow area of the microfluidic device with the length significantly longer than both horizontal and vertical dimensions.For example, flow channel can be at least 5 times of the length of horizontal or vertical dimension, for example at least 10 times of length, at least 25 times of length, at least 100 times of length, at least 200 times of length, at least 500 times of length, at least 1,000 times of length, at least 5,000 times of length or longer.In some embodiments, the length of flow channel is in the range of about 50,000 microns to about 500,000 microns, including any range therebetween.In some embodiments, horizontal dimension is in the range of from about 100 microns to about 1000 microns (for example, about 150 microns to about 500 microns), and vertical dimension is in the range of about 25 microns to about 200 microns, for example, about 40 to about 150 microns.It should be noted that flow channel can have a variety of different spatial configurations in microfluidic device, is therefore not limited to completely linear elements. For example, the flow channel can include one or more portions having any of the following structures: curves, bends, spirals, slopes, dips, bifurcations (e.g., multiple distinct flow paths), and any combination thereof. Additionally, the flow channel can have varying cross-sectional areas along its path, widening and converging to provide a desired fluid flow therein.

As used herein, the term "obstacle" generally refers to a bump or similar structure that is large enough to partially (but not completely) impede the movement of a target micro-object between two distinct regions or conduit elements in a microfluidic device. The two distinct regions/conduit elements can be, for example, a microfluidic isolation dock and a microfluidic channel, or a connection region and an isolation region of a microfluidic isolation dock.

As used herein, the term "constriction" generally refers to a narrowing of the width of a tubing element (or the interface between two tubing elements) in a microfluidic device. This constriction can be located, for example, at the interface between a microfluidic isolation dock and a microfluidic channel, or at the interface between an isolation region and a connection region of the microfluidic isolation dock.

] As used herein, the term "transparent" refers to a material that allows visible light to pass through without substantially altering the light as it passes through.

As used herein, the term "micro-object" generally refers to any microscopic object that can be isolated and/or manipulated according to the present invention. Non-limiting examples of micro-objects include: inanimate micro-objects, such as microparticles; microbeads (e.g., polystyrene beads, LuminexTM beads, etc.); magnetic beads; microrods; microfilaments; quantum dots, etc.; biological micro-objects, such as cells; biological organelles; vesicles or complexes; synthetic vesicles; liposomes (e.g., synthesized or derived from membrane preparations); lipid nanorafts, etc.; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.). Beads can include covalently or non-covalently linked moieties/molecules, such as fluorescent markers, proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species that can be used in assays. Lipid nanorafts have been described in, for example, Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,” Methods Enzymol., 464: 211-231.

As used herein, the term "cell" is used interchangeably with the term "biological cell". Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptile cells, bird cells, fish cells, etc., prokaryotic cells, bacterial cells, fungal cells, protozoan cells, etc., cells dissociated from tissues, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, etc., immune cells, such as T cells, B cells, natural killer cells, macrophages, etc., embryos (e.g., zygotes), oocytes, OVAs, sperm cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, etc. Mammalian cells can be, for example, from humans, mice, rats, horses, goats, sheep, cattle, primates, etc.

A colony of biological cells is a "clone" if all living cells in the colony that are capable of reproduction are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells in a clonal colony are derived from a single parent cell no more than 10 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single parent cell no more than 14 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single parent cell no more than 17 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single parent cell no more than 20 divisions. The term "clonal cells" refers to cells of the same clonal colony.

As used herein, a "colony" of biological cells refers to 2 or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells).

As used herein, the term "culture unit(s)" refers to an environment comprising fluid and gaseous components and optionally a surface, which provides the conditions necessary to keep cells viable and/or proliferating.

A "component" of a fluid medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, etc.

As used herein with respect to a fluid medium, "diffuse" and "diffusion" refer to the thermodynamic movement of components of a fluid medium down a concentration gradient.

The phrase "flow of a medium" means the overall movement of a fluid medium primarily due to any mechanism other than diffusion. For example, the flow of a medium may involve the movement of a fluid medium from one point to another due to a pressure differential between the points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of a liquid, or any combination thereof. When one fluid medium flows into another, turbulence and mixing of the media may result.

The phrase "substantially non-flowing" refers to a flow rate of a fluid medium that, averaged over time, is less than the diffusion rate of components of a material (e.g., a target analyte) into or within the fluid medium. The diffusion rate of components of such a material may depend on, for example, temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein with respect to different regions within a microfluidic device, the phrase "fluidically connected" means that when the different regions are substantially filled with a fluid, such as a fluid medium, the fluid in each region is connected to form a single body of fluid. This does not mean that the fluids (or fluid media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules), which compositions change as the solutes follow their respective concentration gradients and/or fluid flow through the device.

A microfluidic (or nanofluidic) device may include a "swept" region and a "non-swept" region. As used herein, a "swept" region includes one or more fluidically interconnected tubing elements of a microfluidic circuit, each of which experiences a flow of a medium when the fluid flows through the microfluidic circuit. The tubing elements of a swept region may include, for example, regions, channels, and all or part of a chamber. As used herein, an "unswept" region includes one or more fluidically interconnected tubing elements of a microfluidic circuit, each of which does not substantially experience a flow of fluid when the fluid flows through the microfluidic circuit. An unswept region may be fluidically connected to a swept region as long as the fluid connection is configured to diffuse but not substantially flow a medium between the swept region and the unswept region. Thus, a microfluidic device may be configured to substantially isolate an unswept region from a flow of a medium in a swept region, while substantially only achieving diffusional fluid communication between the swept region and the unswept region. For example, a flow channel of a microfluidic device is an example of a swept region, while an isolated region of a microfluidic device (described in further detail below) is an example of an unswept region.

As used herein, a "flow path" refers to one or more fluidically connected tubing elements (e.g., channels, regions, chambers, etc.) that define and experience the flow trajectory of a medium. Thus, a flow path is an example of a swept region of a microfluidic device. Other tubing elements (e.g., unswept regions) may be fluidically connected to the tubing elements comprising the flow path without experiencing the flow of the medium in the flow path.

As used herein, the "clear aperture" of a "lens" (or "lens assembly") is the diameter or size of the portion of the lens (or lens assembly) that can be used for its intended purpose. Due to manufacturing constraints, it is not practically possible to produce a clear aperture that is equal to the actual physical diameter of a lens (or lens assembly).

As used herein, the term "active area" refers to the portion of an image sensor or structured light modulator that can be used to image structured light or provide structured light to a particular optical device, respectively. The active area is limited by the optical device, such as the aperture stop of the optical path within the optical device. Although the active area corresponds to a two-dimensional surface, the measurement of the active area generally corresponds to the length of a diagonal line through opposite corners of a square having the same area.

As used herein, an "image beam" is an electromagnetic wave reflected or emitted from a device surface, micro-object, or fluid medium as viewed by an optical device. The device can be a microfluidic device, such as a light-activated microfluidic (LAMF) device. The micro-object and fluid medium can be located within such a microfluidic device.

As used herein, μm refers to micrometer, μm ³ refers to cubic micrometer, pL refers to picoliter, nL refers to nanoliter, and μL (or uL) refers to microliter.

Loading method. For example, but not limited to the loading of biological micro-objects or micro-objects of beads can involve the use of fluid flow, gravity, dielectrophoresis (DEP) force, electrowetting, magnetic force, etc., or any combination thereof, as described herein. The DEP force can be optically actuated and/or electrically actuated, such as by activating electrodes/electrode areas with a time/space pattern, for example, by photoelectric tweezers (OET) structures. Similarly, the electrowetting force can be optically actuated and/or electrically actuated, such as by activating electrodes/electrode areas with a time/space pattern, for example, by photoelectrowetting (OEW) structures.

The present disclosure relates to optical devices, systems, and methods for observing and manipulating micro-objects. Specifically, the present disclosure relates to an optical device for observing and manipulating micro-objects in a microfluidic device (e.g., a light-actuated microfluidic device), and related systems and methods.

Disclosed herein is an optical device for observing and/or manipulating micro-objects in a microfluidic device. The optical device is configured to perform imaging, analysis, and manipulation of one or more micro-objects within a housing of the microfluidic device. The optical device may include a first light source, a structured light modulator, a first tube lens, an objective lens, a dichroic beam splitter, a second tube lens, and an image sensor. The structured light modulator is configured to receive an unstructured light beam from the first light source and transmit a structured light beam for imaging and/or selectively activating one or more of a plurality of dielectrophoresis (DEP) electrodes on a surface of a substrate of the microfluidic device, including any of the light-actuated microfluidic devices described herein. The first tube lens is configured to capture the structured light beam from the structured light modulator. The objective lens is configured to image a field of view that includes at least a portion of the housing of the microfluidic device. The dichroic beam splitter is configured to reflect (or transmit) a light beam from the first tube lens to the objective lens and to transmit (or reflect) an image light beam received from the objective lens to the second tube lens. The second tube lens is configured to receive the image beam from the dichroic beam splitter and transmit the image beam to the image sensor. The image sensor is configured to receive the image beam from the second tube lens and generate an image of the field of view therefrom.

Disclosed herein is a system for observing and manipulating micro-objects. The system may include a microfluidic device and an optical apparatus for imaging and/or manipulating micro-objects in the microfluidic device. The microfluidic device may include a housing having a substrate. The microfluidic device may also include a flow region and a plurality of isolation docks, each of which is fluidically connected to the flow region. The substrate may include a surface and a plurality of dielectrophoresis (DEP) electrodes on or consisting of the surface. The microfluidic device may also include a cover, which may include a ground electrode that is transparent to visible light. Details of such a microfluidic device are described elsewhere herein and in the art. See, for example, International Patent Application Publication No. WO2016/094507, filed December 9, 2015; U.S. Patent No. 9,403,172, filed October 10, 2013; and International Patent Application Publication No. WO2014/074367, filed October 30, 2013. The optical apparatus may be configured to perform imaging, analysis, and manipulation of one or more micro-objects within the housing. The optical device may include a first light source, a structured light modulator, a first and second tube lenses, an objective lens, a dichroic beam splitter, and an image sensor. The structured light modulator may be configured to receive light from the first light source and transmit a structured light beam to selectively image and/or activate one or more of the plurality of DEP electrodes on the surface of the substrate of the microfluidic device. The first tube lens may be configured to capture light from the structured light modulator. The objective lens may be configured to image a field of view that includes at least a portion of a flow region within the microfluidic device and/or a portion of a plurality of isolation docks. The dichroic beam splitter may be configured to reflect or transmit the structured light beam from the first tube lens to the objective lens and transmit or reflect an image beam received from the objective lens to the second tube lens. The second tube lens is configured to receive an image beam from the dichroic beam splitter and transmit the image beam to the image sensor. The image sensor is configured to receive the image beam and generate an image of the field of view from the image beam.

Disclosed herein are microfluidic devices and systems for operating and observing such devices. Figure 1A illustrates the example of a microfluidic device 100 and a system 150, which can be used for screening and detecting antibody-producing cells that secrete antibodies that bind (e.g., specifically bind) to an antigen of interest. A perspective view of the microfluidic device 100 is represented by a partial excision of its lid 110 to provide a partial view in the microfluidic device 100. The microfluidic device 100 typically includes a microfluidic circuit 120, which includes a flow path 106 through which a fluid medium 180 can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit 120. Although a single microfluidic circuit 120 is illustrated in Figure 1A, suitable microfluidic devices can include multiple (e.g., 2 or 3) such microfluidic circuits. In any case, the microfluidic device 100 can be configured as a nanofluidic device. 1A , the microfluidic circuit 120 includes a plurality of microfluidic isolation docks 124, 126, 128, and 130, each having an opening (e.g., a single opening) in fluid communication with the flow path 106. As described further below, the microfluidic isolation docks include various features and structures that have been optimized to retain micro-objects within a microfluidic device, such as the microfluidic device 100, even when a medium 180 flows through the flow path 106. However, before turning to the foregoing, a brief description of the microfluidic device 100 and the system 150 is provided.

As generally shown in FIG1A , the microfluidic circuit 120 is defined by the housing 102. Although the housing 102 can be physically constructed in different configurations, in the example shown in FIG1A , the housing 102 is depicted as including a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108, and the cover 110 can be attached to each other. For example, the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104, and the cover 110 can be disposed over the microfluidic circuit structure 108. The microfluidic circuit structure 108 can, together with the support structure 104 and the cover 110, define elements of the microfluidic circuit 120.

As shown in FIG1A , the support structure 104 can be located at the bottom, and the cover 110 can be located on top of the microfluidic circuit 120, or the support structure 104 and cover 110 can be configured in other orientations. For example, the support structure 104 can be located at the top of the microfluidic circuit 120, and the cover 110 can be located at the bottom of the microfluidic circuit 120. Regardless, there can be one or more ports 107, each of which includes a passageway for entering or exiting the housing 102. Examples of passageways include valves, doors, through-holes, etc. As shown, the port 107 is a through-hole formed by a gap in the microfluidic circuit structure 108. However, the port 107 can be located in other components of the housing 102, such as the cover 110. Only one port 107 is shown in FIG1A , but the microfluidic circuit 120 can have two or more ports 107. For example, there can be a first port 107 that serves as an inlet for fluid entering the microfluidic circuit 120, and a second port 107 that serves as an outlet for fluid exiting the microfluidic circuit 120. Whether port 107 functions as an inlet or an outlet may depend on the direction of fluid flow through flow path 106 .

The support structure 104 may include one or more electrodes (not shown) and a substrate or multiple interconnected substrates. For example, the support structure 104 may include one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates may be electrically connected to a single electrode). The support structure 104 may also include a printed circuit board assembly ("PCBA"). For example, the semiconductor substrates may be mounted on the PCBA.

The microfluidic tubing structure 108 can define the tubing elements of the microfluidic tubing 120. Such tubing elements can include spaces or regions that can be fluidically interconnected when the microfluidic tubing 120 is filled with fluid, such as flow regions (which can include or can be one or more flow channels), chambers, docks, traps, etc. In the microfluidic tubing 120 shown in Figure 1A, the microfluidic tubing structure 108 includes a frame 114 and a microfluidic tubing material 116. The frame 114 can partially or completely surround the microfluidic tubing material 116. The frame 114 can be, for example, a relatively rigid structure that substantially surrounds the microfluidic tubing material 116. For example, the frame 114 can include a metal material.

The microfluidic tubing material 116 can be patterned with chambers or the like to define the tubing elements and interconnections of the microfluidic tubing 120. The microfluidic tubing material 116 can include a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), etc.), which can be breathable. Other examples of materials that can constitute the microfluidic tubing material 116 include molded glass, etchable materials such as silicone (e.g., photopatternable silicone or “PPS”), photoresist (e.g., SU8), etc. In some embodiments, such material, i.e., the microfluidic tubing material 116, can be rigid and/or substantially impermeable. In any event, the microfluidic tubing material 116 can be disposed on the support structure 104 and within the frame 114.

The cover 110 can be an integral part of the frame 114 and/or the microfluidic tubing material 116. Alternatively, as shown in FIG1A , the cover 110 can be a structurally distinct component. The cover 110 can comprise the same or different material as the frame 114 and/or the microfluidic tubing material 116. Similarly, the support structure 104 can be a separate structure from the frame 114 or the microfluidic tubing material 116 as shown, or an integral part of the frame 114 or the microfluidic tubing material 116. Likewise, the frame 114 and the microfluidic tubing material 116 can be separate structures as shown in FIG1A or integral parts of the same structure.

In some embodiments, the cover 110 can include a rigid material. The rigid material can be glass or a material with similar properties. In some embodiments, the cover 110 can include a deformable material. The deformable material can be a polymer, such as PDMS. In some embodiments, the cover 110 can include both rigid and deformable materials. For example, one or more portions of the cover 110 (e.g., one or more portions located above the isolation docks 124, 126, 128, 130) can include a deformable material that interfaces with the rigid material of the cover 110. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes can include a conductive oxide, such as indium tin oxide (ITO), which can be coated on glass or a similar insulating material. Alternatively, the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices have been described, for example, in US 2012/0325665 (Chiou et al.), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 can be modified (e.g., by modifying all or part of the inwardly facing surface toward the microfluidic conduit 120) to support cell adhesion, viability, and/or growth. The modification can include a coating of a synthetic or natural polymer. In some embodiments, the cover 110 and/or support structure 104 are transparent to light. The cover 110 can also include at least one gas permeable material (e.g., PDMS or PPS).

1A also shows a system 150 for operating and controlling a microfluidic device, such as microfluidic device 100. System 150 includes a power source 192, an imaging device 194 (incorporated within imaging module 164, wherein device 194 itself is not shown in FIG. 1A ), and a tilting device 190 (incorporated within tilting module 166, wherein device 190 itself is not shown in FIG. 1 ).

The power supply 192 can provide power to the microfluidic device 100 and/or the tilting device 190, providing a bias voltage or current as needed. The power supply 192 can, for example, include one or more alternating current (AC) and/or direct current (DC) voltage or current sources. The imaging device 194 (part of the imaging module 164 discussed below) can include a device for capturing images of the interior of the microfluidic circuit 120, such as a digital camera. In some cases, the imaging device 194 also includes a detector with a fast frame rate and/or high sensitivity (e.g., for low-light applications). The imaging device 194 can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beam can be in the visible spectrum and can, for example, include fluorescent emission. The reflected light beam can include reflected emission from an LED or a broad spectrum lamp, such as a mercury lamp (e.g., a high-pressure mercury lamp) or a xenon arc lamp. As discussed with respect to FIG. 3B , imaging device 194 may also include a microscope (or optical apparatus), which may or may not include an eyepiece.

The system 150 also includes a tilting device 190 (part of the tilting module 166 described below) that is configured to rotate the microfluidic device 100 about one or more rotational axes. In some embodiments, the tilting device 190 is configured to support and/or hold the housing 102 including the microfluidic conduit 120 about at least one axis such that the microfluidic device 100 (and therefore the microfluidic conduit 120) can be held in a horizontal orientation (i.e., 0° relative to the x-axis and y-axis), a vertical orientation (i.e., 90 degrees relative to the x-axis and/or y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and the microfluidic conduit 120) relative to an axis is referred to herein as the "tilted" orientation of the microfluidic device 100 (and the microfluidic conduit 120). For example, the tilting device 190 can tilt the microfluidic device 100 relative to the x-axis by 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90°, or any angle therebetween. The horizontal orientation (and therefore the x-axis and y-axis) is defined as being perpendicular to the vertical axis defined by gravity. The tilting device can also tilt the microfluidic device 100 (and the microfluidic circuit 120) by more than 90° relative to the x-axis and/or the y-axis, or by 180° relative to the x-axis or the y-axis, so as to fully intertune the microfluidic device 100 (and the microfluidic circuit 120). Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) about a rotational axis defined by the flow path 106 or some other portion of the microfluidic circuit 120.

In some cases, the microfluidic device 100 is tilted into a vertical orientation such that the flow path 106 is positioned above or below one or more isolation docks. As used herein, the term "above" means that the flow path 106 is positioned higher than one or more isolation docks on a vertical axis defined by gravity (i.e., objects in an isolation dock above the flow path 106 have a higher gravitational potential energy than objects in the flow path). As used herein, the term "below" means that the flow path 106 is positioned lower than one or more isolation docks on a vertical axis defined by gravity (i.e., objects in an isolation dock below the flow path 106 have a lower gravitational potential energy than objects in the flow path).

In some cases, the tilting device 190 tilts the microfluidic device 100 about an axis that is parallel to the flow path 106. In addition, the microfluidic device 100 can be tilted to an angle less than 90° so that the flow path 106 is located above or below one or more isolation docks, rather than directly above or below the isolation docks. In other cases, the tilting device 190 tilts the microfluidic device 100 about an axis that is perpendicular to the flow path 106. In other cases, the tilting device 190 tilts the microfluidic device 100 about an axis that is neither parallel nor perpendicular to the flow path 106.

The system 150 may also include a medium source 178. The medium source 178 (e.g., container, reservoir, etc.) may include multiple parts or containers, each part or container being used to hold a different fluid medium 180. Thus, the medium source 178 may be a device that is external to and separate from the microfluidic device 100, as shown in FIG1A. Alternatively, the medium source 178 may be located entirely or partially within the housing 102 of the microfluidic device 100. For example, the medium source 178 may include a reservoir that is part of the microfluidic device 100.

FIG1A also shows a simplified block diagram depicting an example of a control and monitoring device 152 that forms part of the system 150 and can be used in conjunction with the microfluidic device 100. As shown, an example of such a control and monitoring device 152 includes a main controller 154 that can control other control and monitoring devices, such as: a media module 160 for controlling a media source 178; a motion module 162 for controlling the movement and/or selection of micro-objects (not shown) and/or media (e.g., droplets of media) in the microfluidic circuit 120; an imaging module 164 for controlling an imaging device 194 (e.g., a camera, a microscope, a light source, or any combination thereof for capturing images (e.g., digital images); and a tilt module 166 for controlling a tilt device 190. The control device 152 may also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the device 152 may also include a display device 170 and input/output devices 172.

The main controller 154 may include a control module 156 and a digital memory 158. The control module 156 may include, for example, a digital processor configured to operate according to machine-executable instructions (e.g., software, firmware, source code, etc.) stored as non-transitory data or signals in the memory 158. Alternatively or additionally, the control module 156 may include hard-wired digital circuits and/or analog circuits. The media module 160, motion module 162, imaging module 164, tilt module 166, and/or other modules 168 may be similarly configured. Thus, the functions, process actions, actions, or steps of the processes described herein with respect to the microfluidic device 100 or any other microfluidic device can be performed by any one or more of the main controller 154, media module 160, motion module 162, imaging module 164, tilt module 166, and/or other modules 168 configured as described above. Similarly, the main controller 154, media module 160, motion module 162, imaging module 164, tilt module 166 and/or other modules 168 can be communicatively coupled to send and receive data used in any of the functions, processes, process actions, actions or steps described herein.

The media module 160 controls the media source 178. For example, the media module 160 can control the media source 178 to input a selected fluid medium 180 into the housing 102 (e.g., through the inlet port 107). The media module 160 can also control the removal of the medium from the housing 102 (e.g., through the outlet port (not shown)). Thus, one or more media can be selectively input into and removed from the microfluidic circuit 120. The media module 160 can also control the flow of the fluid medium 180 in the flow path 106 within the microfluidic circuit 120. For example, in some embodiments, the media module 160 prevents the flow of the medium 180 in the flow path 106 and through the housing 102 before the tilt module 166 causes the tilt device 190 to tilt the microfluidic device 100 to a desired tilt angle.

The motion module 162 can be configured to control the selection, capture, and movement of micro-objects (not shown) in the microfluidic circuit 120. As described below with respect to Figures 1B and 1C, the housing 102 can include dielectrophoresis (DEP), optoelectronic tweezers (OET), and/or photoelectrowetting (OEW) structures (not shown in Figure 1A), and the motion module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects (not shown) and/or droplets of medium (not shown) in the flow path 106 and/or the isolation docks 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example, the imaging module 164 can receive and process image data from the imaging device 194. The image data from the imaging device 194 can include any type of information captured by the imaging device 194 (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of labels such as fluorescent markers, etc.). Using the information captured by the imaging device 194, the imaging module 164 can also calculate objects (e.g., micro-objects, droplets of medium) and/or the rate of movement of such objects within the microfluidic device 100.

The tilt module 166 can control the tilting motion of the tilting device 190. Alternatively or additionally, the tilt module 166 can control the tilting rate and timing to optimize the transfer of the micro-objects to one or more isolation docks via gravity. The tilt module 166 is communicatively coupled to the imaging module 164 to receive data describing the movement of the micro-objects and/or media droplets in the microfluidic circuit 120. Using this data, the tilt module 166 can adjust the tilt of the microfluidic circuit 120 to adjust the rate at which the microfluids and/or media droplets move in the microfluidic circuit 120. The tilt module 166 can also use this data to iteratively adjust the position of the micro-objects and/or media droplets in the microfluidic circuit 120.

In the example shown in Figure 1A, microfluidic circuit 120 is shown to include a microfluidic channel 122 and isolation docks 124, 126, 128, 130. Each dock includes a single opening leading to the microfluidic channel 122, and the rest are closed so that the dock can substantially isolate the micro-objects in the dock from the fluid medium 180 and/or the micro-objects in the flow path 106 of the channel 122 or in other docks. The wall of the isolation dock extends from the inner surface 109 of the base to the inner surface of the cover 110 for sealing. The opening of the dock to the channel 122 is oriented to be at an angle to the fluid 106 of the fluid medium 180 so that the fluid 106 is not directed into the dock. The fluid can be tangential or orthogonal to the plane of the opening of the dock. In some cases, docks 124, 126, 128, 130 are configured to physically support one or more micro-objects in the microfluidic circuit 120. As will be described and illustrated in detail below, the isolation docks of the present invention may include a variety of shapes, surfaces, and features that are optimized for use with DEP, OET, flowing fluids, and/or gravity.

Microfluidic circuit 120 can comprise the microfluid isolation dock of any number.Although five isolation docks are shown, microfluidic circuit 120 can have less or more isolation dock.As shown in the figure, the microfluid isolation dock 124,126,128 and 130 of microfluidic circuit 120 each comprise different features and shapes, which can provide one or more benefits useful in screening antibody production cells, for example, an antibody production cell is separated from another antibody production cell.Microfluidic isolation dock 124,126,128 and 130 can provide other benefits, for example, promotes single cell loading and/or the growth of the colony (for example clonal colony) of the cell that produces antibody.In some embodiments, microfluidic circuit 120 comprises a plurality of identical microfluid isolation docks.

In some embodiments, the microfluidic circuit 120 includes a plurality of microfluidic isolation docks, wherein two or more isolation docks include different structures and/or features for different benefits of screening antibody-producing cells. A microfluidic device that can be used to screen antibody-producing cells can include any isolation docks 124, 126, 128, and 130 or variations thereof, and/or can include docks similar to those shown in Figures 2B, 2C, 2D, 2E, and 2F as described below.

In the embodiment shown in FIG1A , a single channel 122 and flow path 106 are shown. However, other embodiments may include multiple channels 122, each configured to include a flow path 106. The microfluidic circuit 120 also includes an inlet valve or port 107 in fluid communication with the flow path 106 and a fluid medium 180, whereby the fluid medium 180 can flow into the channel 122 via the inlet port 107. In some cases, the flow path 106 includes a single path. In some cases, the single path is arranged in a zigzag pattern, whereby the flow path 106 travels two or more times along the microfluidic device 100 in alternating directions.

In some cases, microfluidic circuit 120 includes multiple parallel channels 122 and flow paths 106, wherein the fluid medium 180 within each flow path 106 flows in the same direction. In some cases, the fluid medium within each flow path 106 flows in at least one of a forward or backward direction. In some cases, multiple isolation docks are configured (e.g., relative to channels 122) so that the isolation docks can be loaded with target micro-objects in parallel.

In some embodiments, the microfluidic circuit 120 further includes one or more micro-object traps 132. The traps 132 are typically formed in a wall that forms a boundary of the channel 122 and can be positioned opposite an opening of one or more of the microfluidic isolation docks 124, 126, 128, 130. In some embodiments, the traps 132 are configured to receive or trap a single micro-object from the flow path 106. In some embodiments, the traps 132 are configured to receive or trap a plurality of micro-objects from the flow path 106. In some cases, the traps 132 include a volume that is approximately equal to the volume of a single target micro-object.

The trap 132 may further comprise an opening configured to assist the flow of target micro-objects into the trap 132. In some cases, the trap 132 comprises an opening having a height and width approximately equal to the size of a single target micro-object, thereby preventing larger micro-objects from entering the micro-object trap. The trap 132 may further comprise other features configured to help retain the target micro-objects within the trap 132. In some cases, the trap 132 is aligned with and located on opposite sides of the opening of the microfluidic isolation dock relative to the channel 122, such that when the microfluidic device 100 is tilted about an axis parallel to the channel 122, the captured micro-objects leave the trap 132 with a trajectory that causes the micro-objects to fall into the opening of the isolation dock. In some cases, the trap 132 comprises a side channel 134 that is smaller than the target micro-object to facilitate flow through the trap 132, thereby increasing the likelihood of capturing the micro-objects in the trap 132.

In some embodiments, dielectrophoresis (DEP) forces are applied to the fluid medium 180 (e.g., in the flow path and/or isolation dock) via one or more electrodes (not shown) to manipulate, transport, separate, and classify micro-objects located therein. For example, in some embodiments, DEP forces are applied to one or more portions of the microfluidic circuit 120 to transfer a single micro-object from the flow path 106 to a desired microfluidic isolation dock. In some embodiments, DEP forces are used to prevent micro-objects within an isolation dock (e.g., isolation dock 124, 126, 128, or 130) from shifting therefrom. Additionally, in some embodiments, DEP forces are used to selectively remove micro-objects from an isolation dock that was previously collected in accordance with the teachings of the present invention. In some embodiments, DEP forces comprise optoelectronic tweezers (OET) forces.

In other embodiments, photoelectrowetting (OEW) forces are applied via one or more electrodes (not shown) to one or more locations in the support structure 104 (and/or lid 110) of the microfluidic device 100 (e.g., locations that help define flow paths and/or isolation docks) to manipulate, transport, separate, and sort droplets located in the microfluidic circuit 120. For example, in some embodiments, OEW forces are applied to one or more locations in the support structure 104 (and/or lid 110) to transfer a single droplet from the flow path 106 to a desired microfluidic isolation dock. In some embodiments, the OEW forces are used to prevent droplets within an isolation dock (e.g., isolation dock 124, 126, 128, or 130) from being displaced therefrom. Additionally, in some embodiments, the OEW forces are used to selectively remove droplets from an isolation dock that was previously collected according to the teachings of the present invention.

In some embodiments, DEP forces and/or OEW forces are combined with other forces, such as fluid and/or gravity, to manipulate, transport, separate, and sort micro-objects and/or droplets within the microfluidic circuit 120. For example, the housing 102 can be tilted (e.g., by tilting device 190) to position the flow path 106 and the micro-objects therein above the microfluidic isolation dock, and gravity can transport the micro-objects and/or droplets into the dock. In some embodiments, the DEP force and/or OEW force can be applied before the other forces. In other embodiments, the DEP force and/or OEW force can be applied after the other forces. In other cases, the DEP force and/or OEW force can be applied simultaneously with the other forces or in an alternating manner with the other forces.

Figures 1B, 1C, and 2A to 2H show various embodiments of microfluidic devices that can be used in the practice of the present invention. Figure 1B depicts an embodiment in which the microfluidic device 200 is configured as an optically actuated electric device. A variety of optically actuated electric devices are known in the art, including devices with optoelectronic tweezers (OET) structures and devices with optoelectronic electrowetting (OEW) structures. Examples of suitable OET structures are described in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety: U.S. Patent No. RE44,711 (Wu et al.) (originally published as U.S. Patent No. 7,612,355); U.S. Patent No. 7,956,339 (Ohta et al.); U.S. Patent No. 9,403,172 (Wu et al.); and U.S. Patent Application Publication No. 20160184821 (Hobbs et al.). Examples of OEW structures are shown in U.S. Patent No. 6,958,132 (Chiou et al.); U.S. Patent Application Publication No. 2012/0024708 (Chiou et al.); and U.S. Patent No. 9,815,056 (Wu et al.), each of which is incorporated herein by reference in its entirety. Yet another example of an optically actuated electrokinetic device includes a combined OET/OEW structure, examples of which are shown in U.S. Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599 (Khandros et al.) and their corresponding PCT Publications WO 2015/164846 and WO 2015/164847, all of which are incorporated herein by reference in their entirety.

For example, examples of microfluidic devices having docks in which cells producing antibodies can be placed, cultured, monitored, and/or screened are described in U.S. patent applications Nos. 20140116881 (Chapman et al.), 20150151298 (Hobbs et al.), and 20150165436 (Chapman et al.), each of which is incorporated herein by reference in its entirety. Each of the aforementioned applications further describes a microfluidic device configured to generate dielectrophoresis (DEP) forces, such as optoelectronic tweezers (OET) or configured to provide photoelectrowetting (OEW). For example, the optoelectronic tweezers device shown in FIG. 2 of U.S. Patent Application Publication No. 20140116881 (Chapman et al.) is an example of a device that can be used in embodiments of the present invention to select and move a single biological micro-object or a group of biological micro-objects.

Microfluidic device power configuration. As described above, the control and monitoring equipment of the system may include a power module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of the microfluidic device. The microfluidic device can have a variety of power configurations, depending on the type of object being moved and other considerations. For example, a dielectrophoresis (DEP) structure can be used to select and move micro-objects in the microfluidic circuit. Therefore, the support structure 104 and/or the cover 110 of the microfluidic device 100 may include a DEP structure for selectively inducing DEP forces on micro-objects in the fluid medium 180 in the microfluidic circuit 120, thereby selecting, capturing and/or moving individual micro-objects or groups of micro-objects. Alternatively, the support structure 104 and/or the cover 110 of the microfluidic device 100 may include an electrowetting (EW) structure for selectively inducing EW forces on droplets in the fluid medium 180 in the microfluidic circuit 120, thereby selecting, capturing and/or moving individual droplets or groups of droplets.

An example of a microfluidic device 200 including a DEP structure is shown in Figures 1B and 1C. Although for simplicity, Figures 1B and 1C respectively show a side cross-sectional view and a top cross-sectional view of the housing 102 of the microfluidic device 200 having an open area/chamber 202, it should be understood that the area/chamber 202 can be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, an isolation dock, a flow region, or a flow channel. In addition, the microfluidic device 200 can include other fluidic circuit elements. For example, the microfluidic device 200 can include multiple growth chambers or isolation docks and/or one or more flow regions or flow channels, such as those described herein with respect to the microfluidic device 100. The DEP structure can be incorporated into any such fluidic circuit element of the microfluidic device 200, or selected portions thereof. It should also be understood that any of the microfluidic device components and system components described above or below can be incorporated into and/or used in conjunction with the microfluidic device 200. For example, the system 150 including the control and monitoring apparatus 152 described above may be used with a microfluidic device 200 that includes one or more of the media module 160 , motion module 162 , imaging module 164 , tilt module 166 , and other modules 168 .

As shown in FIG1B , the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode-activated substrate 206 covering the bottom electrode 204, and a lid 110 having a top electrode 210 spaced apart from the bottom electrode 204. The top electrode 210 and the electrode-activated substrate 206 define opposing surfaces of a region/chamber 202. Thus, a dielectric 180 contained within the region/chamber 202 provides a resistive connection between the top electrode 210 and the electrode-activated substrate 206. A power supply 212 is also shown, configured to connect to the bottom electrode 204 and the top electrode 210 and generate a bias voltage between the electrodes as needed for generating a DEP force in the region/chamber 202. The power supply 212 can be, for example, an alternating current (AC) power supply.

In certain embodiments, the microfluidic device 200 shown in Figures 1B and 1C may have an optically actuated DEP structure. Thus, changing the pattern 218 of light from the light source 216, which can be controlled by the power module 162, can selectively activate and deactivate the changing pattern of the DEP electrode at the region 214 of the inner surface 208 of the electrode activation substrate 206. (Hereinafter, the region 214 of the microfluidic device having the DEP structure is referred to as the "DEP electrode region." As shown in Figure 1C, the light pattern 218 directed onto the inner surface 208 of the electrode activation substrate 206 can illuminate the selected DEP electrode region 214a (shown in white) in, for example, a square pattern. The DEP electrode region 214 that is not illuminated (cross-hatched) is referred to as the "dark" DEP electrode region 214 hereinafter. The relative electrical impedance of the DEP electrode activation substrate 206 (i.e., from the bottom electrode 204 up to the electrode activation substrate 206) is determined by the relative electrical impedance of the DEP electrode activation substrate 206. 10) is greater than the relative electrical impedance through the medium 180 in the region/chamber 202 at each dark DEP electrode region 214 (i.e., from the inner surface 208 of the electrode-active substrate 206 to the top electrode 210 of the lid 110). However, the illuminated DEP electrode region 214a exhibits a reduced relative impedance of the entire electrode-active substrate 206 that is less than the relative impedance of the entire medium 180 in the region/chamber 202 at each illuminated DEP electrode region 214a.

When the power source 212 is activated, the aforementioned DEP structure generates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214, which in turn generates a localized DEP force that attracts or repels nearby micro-objects (not shown) in the fluid medium 180. Thus, by varying the light pattern 218 projected from the light source 216 into the microfluidic device 200, DEP electrodes that attract or repel micro-objects in the fluid medium 180 can be selectively activated and deactivated at many different such DEP electrode regions 214 on the inner surface 208 of the region/chamber 202. Whether the DEP force attracts or repels nearby micro-objects can depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or the micro-objects (not shown).

The square pattern 220 of illuminated DEP electrode areas 214a shown in FIG1C is merely an example. Any pattern of DEP electrode areas 214 can be illuminated (and thereby activated) by the light pattern 218 projected into the device 200, and the pattern of illuminated/activated DEP electrode areas 214 can be repeatedly changed by changing or moving the light pattern 218.

In some embodiments, the electrode-activated substrate 206 may include or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode-activated substrate 206 may be featureless. For example, the electrode-activated substrate 206 may include or consist of a hydrogenated amorphous silicon (a-Si:H) layer. The a-Si:H layer may contain, for example, approximately 8% to 40% hydrogen (calculated as 100*number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si:H layer may have a thickness of approximately 500 nm to approximately 2.0 microns. In such embodiments, the DEP electrode regions 214 may be created anywhere and in any pattern on the inner surface 208 of the electrode-activated substrate 206 according to the optical pattern 218. Thus, the number and pattern of the DEP electrode regions 214 need not be fixed, but may correspond to the optical pattern 218. Examples of microfluidic devices having a DEP structure including a light-guiding layer as described above have been described, for example, in US Pat. No. RE44,711 (Wu et al.) (originally issued as US Pat. No. 7,612,355).

In other embodiments, the electrode-activated substrate 206 may include a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and conductive layers forming a semiconductor integrated circuit, such as is known in the semiconductor field. For example, the electrode-activated substrate 206 may include a plurality of phototransistors, including, for example, lateral bipolar phototransistors, each phototransistor corresponding to a DEP electrode region 214. Alternatively, the electrode-activated substrate 206 may include electrodes (e.g., conductive metal electrodes) controlled by a phototransistor switch, wherein each such electrode corresponds to a DEP electrode region 214. The electrode-activated substrate 206 may include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern may, for example, be an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in rows and columns, as shown in FIG. 2B . Alternatively, the pattern may be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes forming a hexagonal lattice. Regardless of the pattern, circuit elements can form electrical connections between the DEP electrode regions 214 at the inner surface 208 of the electrode-activated substrate 206 and the bottom electrode 210, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 218. When not activated, each electrical connection can have a high impedance, such that at the corresponding DEP electrode region 214, the relative impedance of the entire electrode-activated substrate 206 (i.e., from the bottom electrode 204 to the inner surface 208 of the electrode-activated substrate 206 that interfaces with the dielectric 180 in the region/chamber 202) is greater than the relative impedance of the entire dielectric 180 (i.e., from the inner surface 208 of the electrode-activated substrate 206 to the top electrode 210 of the lid 110). However, when activated by light in the light pattern 218, the relative impedance of the entire electrode-activated substrate 206 is less than the relative impedance of the entire dielectric 180 at each illuminated DEP electrode region 214, thereby activating the DEP electrode at the corresponding DEP electrode region 214 as described above. Thus, DEP electrodes that attract or repel micro-objects (not shown) in medium 180 can be selectively activated and deactivated at many different DEP electrode regions 214 at inner surface 208 of electrode-active substrate 206 in region/chamber 202 in a manner determined by light pattern 218 .

Examples of microfluidic devices having electrode-activated substrates that include phototransistors are described, for example, in U.S. Pat. No. 7,956,339 (Ohta et al.) (see, for example, device 300 illustrated in Figures 21 and 22 and the description thereof), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode-activated substrates that include electrodes controlled by phototransistor switches are described, for example, in U.S. Pat. No. 9,403,172 (Short et al.) (see, for example, devices 200, 400, 500, 600, and 900 shown throughout the figures and the description thereof).

In some embodiments of a DEP-structured microfluidic device, the top electrode 210 is part of the first wall (or lid 110) of the housing 102, and the electrode-activated substrate 206 and the bottom electrode 204 are part of the second wall (or support structure 104) of the housing 102. The region/chamber 202 may be between the first and second walls. In other embodiments, the electrode 210 is part of the second wall (or support structure 104), and one or both of the electrode-activated substrate 206 and/or the electrode 210 are part of the first wall (or lid 110). Alternatively, a light source 216 may be used to illuminate the housing 102 from below.

Utilizing the microfluidic device 200 of FIG. 1B-1C having a DEP structure, the motion module 162 can select a micro-target (not shown) in the medium 180 in the region/chamber 202 by activating a first set of one or more DEP electrodes at a DEP electrode region 214A of the inner surface 208 of the substrate 206 by projecting a light pattern 218 into the device 200 to activate the electrodes in a pattern (e.g., a square pattern 220) that surrounds and captures the micro-object. The module 162 can then move the captured micro-object by moving the light pattern 218 relative to the device 200 to activate a second set of one or more DEP electrodes at the DEP electrode region 214. Alternatively, the device 200 can be moved relative to the light pattern 218.

Regardless of the configuration of the microfluidic device 200, a power supply 212 can be used to provide a potential (e.g., an AC voltage potential) to power the circuits of the microfluidic device 200. The power supply 212 can be the same as the power supply 192 referenced in FIG. 1, or a component of the power supply 192 referenced in FIG. 1. The power supply 212 can be configured to provide an AC voltage and/or current to the top electrode 210 and the bottom electrode 204. For the AC voltage, as described above, the power supply 212 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate a sufficiently strong net DEP force (or electrowetting force) to capture and move individual micro-objects (not shown) in the region/chamber 202, and/or, as also described above, to change the wetting characteristics of the inner surface 208 (i.e., the dielectric layer and/or the hydrophobic coating on the dielectric layer) of the support structure 104 in the region/chamber 202. Such frequency ranges and average or peak power converters are known in the art. See, e.g., U.S. Patent No. 6,958,132 (Chiou et al.), U.S. Patent No. RE44,711 (Wu et al.) (originally published as U.S. Patent No. 7,612,355), U.S. Patent Application Publication Nos. US2014/0124370 (Short et al.), US2016/0184821 (Hobbs et al.), US2015/0306598 (Khandros et al.), and US2015/0306599 (Khandros et al.).

Isolation Docks. Non-limiting examples of universal isolation docks 224, 226, and 228 are shown within the microfluidic device 230 shown in Figures 2A to 2C. Each isolation dock 224, 226, and 228 can include an isolation structure 232 that defines an isolation region 240 and a connection region 236 that fluidically connects the isolation region 240 to the channel 122. The connection region 236 can include a proximal opening 234 to the channel 122 and a distal opening 238 to the isolation region 240. The connection region 236 can be configured such that the maximum penetration depth of the flow of a fluid medium (not shown) flowing from the channel 122 into the isolation docks 224, 226, 228 does not extend into the isolation region 240. Therefore, due to the connection region 236, micro-objects (not shown) or other materials (not shown) disposed in the isolation region 240 of the isolation docks 224, 226, 228 can be isolated therefrom and substantially unaffected by the flow of the medium 180 in the channel 122.

Isolation docks 224, 226, and 228 of Figures 2A-2C each have a single opening that leads directly into channel 122. The opening of the isolation dock leads directly into channel 122. Electrode activation substrate 206 covers both channel 122 and isolation docks 224, 226, and 228. The upper surface of electrode activation substrate 206 within the housing of the isolation dock forms the floor of the isolation dock. Electrode activation substrate 206, disposed at or substantially at the same level as the upper surface of electrode activation substrate 206 within channel 122 (or flow region if a channel is not present), forms the floor of the flow channel (or flow region) of the microfluidic device. Electrode activation substrate 206 can be featureless or can have an irregular or patterned surface that varies from its highest height to its lowest depression by less than about 3 microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.8 microns, 0.7 microns, 0.6 microns, 0.5 microns, 0.4 microns, 0.3 microns, 0.2 microns, 0.1 microns, or less. The variation in height of the upper surface of the substrate across the channel 122 (or flow area) and the isolation dock can be less than about 3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3%, or 0.1% of the height of the wall of the isolation dock or the microfluidic device. Although microfluidic device 200 is described in detail, this also applies to any of the microfluidic devices 100, 230, 250, 280, 290 described herein.

In one embodiment, the channel 122 and the isolation dock 224, 226, 228 can be configured to accommodate one or more fluid media 180. In the example shown in Figures 2A to 2B, port 222 is connected to channel 122 and allows fluid media 180 to be introduced into or removed from the microfluidic device 230. Before introducing fluid media 180, the microfluidic device can be primed with a gas such as carbon dioxide gas. Once the microfluidic device 230 includes fluid media 180, the fluid 242 of the fluid media 180 in the channel 122 can be selectively generated and stopped. For example, as shown in the figure, port 222 can be arranged at different positions (for example, opposite ends) of the channel 122, and can lead to the fluid 242 of another port 222 used as an outlet from a port 222 used as an inlet.

2C shows a detailed view of an example of an isolation dock 224 of the present invention. An example of a micro-object 246 is also shown.

As is known, the flow 242 of the fluid medium 180 in the microfluidic channel 122 through the proximal opening 234 of the isolation dock 224 can cause a secondary fluid 244 of the fluid medium 180 to flow into and/or out of the isolation dock 224. In order to isolate the micro-objects 246 in the isolation region 240 of the isolation dock 224 from the secondary fluid 244, the length Lcon of the connection region 236 of the isolation dock 224 (i.e., from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth Dp of the secondary fluid 244 into the connection region 236. The penetration depth Dp of the secondary fluid 244 depends on the velocity of the fluid medium 180 flowing in the channel 122 and various parameters related to the configuration of the channel 122 and the configuration of the proximal opening 234 of the connection region 236 to the channel 122. For a given microfluidic device, the structure of the channel 122 and the opening 234 will be fixed, while the velocity of the flow 242 of the fluid medium 180 in the channel 122 will be variable. Thus, for each isolation dock 224, a maximum velocity Vmax of the fluid medium 180 flowing 242 in the channel 122 can be identified that ensures that the penetration depth Dp of the secondary fluid 244 does not exceed the length Lcon of the connection region 236. As long as the velocity of the fluid medium 180 flowing 242 in the channel 122 does not exceed the maximum velocity Vmax, the resulting secondary fluid 244 can be confined to the channel 122 and the connection region 236 and maintained outside the isolation region 240. Therefore, the fluid medium 180 flowing 242 in the channel 122 will not draw the micro-objects 246 out of the isolation region 240. Conversely, regardless of the fluid medium 180 flowing 242 in the channel 122, the micro-objects 246 located in the isolation region 240 will remain in the isolation region 240.

Furthermore, as long as the velocity of the fluid medium 180 flowing 242 in the channel 122 does not exceed Vmax, the fluid medium 180 flowing 242 in the channel 122 will not displace contaminant particles (e.g., microparticles and/or nanoparticles) from the channel 122 into the isolated region 240 of the isolation dock 224. Having the length Lcon of the connection region 236 greater than the maximum penetration depth Dp of the secondary fluid 244 can prevent contamination of one isolation dock 224 with contaminant particles from the channel 122 or another isolation dock (e.g., isolation docks 226, 228 in FIG. 2D ).

Because the channels 122 and connection regions 236 of the isolation docks 224, 226, 228 can be affected by the flow 242 of the medium 180 in the channels 122, the channels 122 and connection regions 236 can be considered swept (or flowing) regions of the microfluidic device 230. On the other hand, the isolation regions 240 of the isolation docks 224, 226, 228 can be considered unswept (or non-flowing) regions. For example, components (not shown) in the first fluid medium 180 in the channels 122 can mix with the second fluid medium 248 in the isolation regions 240 substantially solely by diffusion of components of the first medium 180 from the channels 122 through the connection regions 236 into the second fluid medium 248 in the isolation regions 240. Similarly, components of the second medium 248 in the isolation region 240 (not shown) can be mixed with the first medium 180 in the channel 122 substantially solely by diffusion of components of the second medium 248 from the isolation region 240 through the connecting region 236 into the first medium 180 in the channel 122. In some embodiments, the degree of fluid medium exchange between the isolation region of the isolation dock and the flow region by diffusion is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than the total fluid exchange. The first medium 180 can be the same medium as the second medium 248 or a different medium. Furthermore, the first medium 180 and the second medium 248 can be the same at the outset and then become different (e.g., by adjusting the passage conditions of the second medium 248 by one or more cells in the isolation region 240 or by changing the flow of the medium 180 through the channel 122).

The maximum penetration depth Dp of the secondary fluid 244 caused by the flow 242 of the fluid medium 180 in the channel 122 can depend on a number of parameters, as described above. Examples of such parameters include: the shape of the channel 122 (e.g., the channel can direct the medium into the connecting region 236, divert the medium away from the connecting region 236, or direct the medium into the channel 122 in a direction substantially perpendicular to the proximal opening 234 of the connecting region 236); the width Wch (or cross-sectional area) of the channel 122 at the proximal opening 234; and the width Wcon (or cross-sectional area) of the connecting region 236 at the proximal opening 234; the velocity V of the flow 242 of the fluid medium 180 in the channel 122; the viscosity of the first medium 180 and/or the second medium 248, etc.

In some embodiments, the dimensions of the channel 122 and the isolation docks 224, 226, 228 can be oriented relative to the vector of the flow 242 of the fluid medium 180 in the channel 122: the channel width Wch (or the cross-sectional area of the channel 122) can be substantially perpendicular to the flow 242 of the medium 180; the width Wcon (or the cross-sectional area) of the connection region 236 at the opening 234 can be substantially parallel to the flow 242 of the medium 180 in the channel 122; and/or the length Lcon of the connection region can be substantially perpendicular to the flow 242 of the medium 180 in the channel 122. The foregoing are examples only, and the relative positions of the channel 122 and the isolation docks 224, 226, 228 can be oriented in other ways relative to each other.

2C , the width Wcon of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238. Thus, the width Wcon of the connection region 236 at the distal opening 238 can be within any of the ranges identified herein for the width Wcon of the connection region 236 at the proximal opening 234. Alternatively, the width Wcon of the connection region 236 at the distal opening 238 can be greater than the width Wcon of the connection region 236 at the proximal opening 234.

As shown in FIG2C , the width Wiso of the isolation region 240 at the distal opening 238 can be substantially the same as the width Wcon of the connection region 236 at the proximal opening 234. Thus, the width Wiso of the isolation region 240 at the distal opening 238 can be within any of the ranges identified herein for the width Wcon of the connection region 236 at the proximal opening 234. Alternatively, the width Wiso of the isolation region 240 at the distal opening 238 can be greater than or less than the width Wcon of the connection region 236 at the proximal opening 234. Furthermore, the distal opening 238 can be smaller than the proximal opening 234, and the width Wcon of the connection region 236 can narrow between the proximal and distal openings 234, 238. For example, the connection region 236 can narrow between the proximal and distal openings using various geometric shapes (e.g., chamfering or beveling the connection region). Furthermore, any portion or subportion of the connection region 236 can be narrowed (e.g., the portion of the connection region adjacent to the proximal opening 234).

2D-2F depict another exemplary embodiment of a microfluidic device 250 comprising microfluidic circuits 262 and flow channels 264, which are variations of the circuits 132 and channels 134 of the corresponding microfluidic device 100 of FIG1 . The microfluidic device 250 also has a plurality of isolation docks 266, which are further variations of the isolation docks 124, 126, 128, 130, 224, 226, or 228 described above. In particular, it should be understood that the isolation docks 266 of the device 250 shown in FIG2D-2F can replace any of the isolation docks 124, 126, 128, 130, 224, 226, or 228 described above in the devices 100, 200, 230, 280, 290, or 320. Likewise, microfluidic device 250 is another variation of microfluidic device 100 and may also have the same or different DEP structures as microfluidic devices 100, 200, 230, 280, 290, 320 described above, as well as any other microfluidic system components described herein.

The microfluidic device 250 of Figures 2D to 2F includes a support structure (not visible in Figures 2D to 2F, but can be the same as or substantially similar to the support structure 104 of the device 100 depicted in Figure 1A), a microfluidic tubing structure 256, and a lid (not visible in Figures 2D to 2F, but can be the same as or substantially similar to the lid 122 of the device 100 depicted in Figure 1A). The microfluidic tubing structure 256 includes a frame 252 and a microfluidic tubing material 260, which can be the same as or substantially similar to the frame 114 and microfluidic tubing material 116 of the device 100 shown in Figure 1A. As shown in Figure 2D, the microfluidic tubing 262 defined by the microfluidic tubing material 260 can include a plurality of channels 264 (two are shown, but more can be possible), and a plurality of isolation docks 266 are fluidically connected to the plurality of channels 264.

Each isolation dock 266 can include an isolation structure 272, an isolation region 270 within the isolation structure 272, and a connection region 268. The connection region 268 fluidly connects the channel 264 to the isolation region 270 from a proximal opening 274 at the channel 264 to a distal opening 276 at the isolation structure 272. Generally, as described above with respect to FIG. 2B and FIG. 2C , flow 278 of the first fluid medium 254 in the channel 264 can create a secondary flow 282 of the first fluid medium 254 from the channel 264 into and/or out of the corresponding connection region 268 of the isolation dock 266.

As shown in FIG2E , the connection region 268 of each isolation dock 266 generally includes an area extending between a proximal opening 274 to the channel 264 and a distal opening 276 to the isolation structure 272. The length Lcon of the connection region 268 can be greater than the maximum penetration depth Dp of the secondary stream 282, in which case the secondary stream 282 will extend into the connection region 268 without being redirected toward the isolation region 270 (as shown in FIG2D ). Alternatively, as shown in FIG2F , the connection region 268 can have a length Lcon that is less than the maximum penetration depth Dp, in which case the secondary stream 282 will extend through the connection region 268 and be redirected toward the isolation region 270. In the latter case, the sum of the lengths Lc1 and Lc2 of the connection region 268 is greater than the maximum penetration depth Dp, so that the secondary stream 282 will not extend into the isolation region 270. Regardless of whether the length Lcon of the connecting region 268 is greater than the penetration depth Dp, or whether the sum of the lengths Lc1 and Lc2 of the connecting region 268 is greater than the penetration depth Dp, the fluid 278 of the first medium 254 in the channel 264 that does not exceed the maximum velocity Vmax will generate a secondary flow having a penetration depth Dp, and the micro-objects (not shown but which may be the same as or substantially similar to the micro-objects 246 shown in FIG. 2C ) in the isolated region 270 of the isolation dock 266 will not be drawn out of the isolated region 270 by the fluid 278 of the first medium 254 in the channel 264. The fluid 278 in the channel 264 also does not draw contaminant material (not shown) from the channel 264 into the isolated region 270 of the isolation dock 266. Therefore, diffusion is the only mechanism by which components in the first medium 254 in the channel 264 can move from the channel 264 to the second medium 258 in the isolated region 270 of the isolation dock 266. Likewise, diffusion is the only mechanism by which components in the second medium 258 in the isolation region 270 of the isolation dock 266 can move from the isolation region 270 to the first medium 254 in the channel 264. The first medium 254 can be the same medium as the second medium 258, or the first medium 254 can be a different medium than the second medium 258. Alternatively, the first medium 254 and the second medium 258 can be the same at the beginning and then become different, for example, by adjusting the second medium by one or more units in the isolation region 270, or by changing the medium flowing through the channel 264.

As shown in FIG2E , the width Wch of the channel 264 in the channel 264 (i.e., transverse to the direction of flow of the fluid medium through the channel, as indicated by arrow 278 in FIG2D ) can be substantially perpendicular to the width _Wcon1 of the proximal opening 274 and substantially parallel to the width _Wco2 of the distal opening 276. However, the width _Wcon1 of the proximal opening 274 and the width _Wcon2 of the distal opening 276 do not need to be substantially perpendicular to each other. For example, the angle between the axis (not shown) along which the width _Wcon1 of the proximal opening 274 is oriented and the other axis along which the width _Wcon2 of the distal opening 276 is oriented may not be perpendicular and therefore not be 90 degrees. Examples of alternatively oriented angles include angles in any of the following ranges: about 30 degrees to about 90 degrees, about 45 degrees to about 90 degrees, about 60 degrees to about 90 degrees, etc.

In various embodiments of the isolation dock (e.g., 124, 126, 128, 130, 224, 226, 228, or 266), the isolation region (e.g., 240 or 270) is configured to contain a plurality of micro-objects. In other embodiments, the isolation region can be configured to contain only one, two, three, four, five, or similar relatively small numbers of micro-objects. Thus, the volume of the isolation region can be, for example, at least 5×10 ⁵ , 8×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ , 4×10 ⁶ , 6×10 ⁶ cubic microns, or more.

In various embodiments of the isolation dock, the width Wch of the channel (e.g., 122) at the proximal opening (e.g., 234) can be any of the following ranges: approximately 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, and 100-120 microns. In some other embodiments, the width _W of the channel (e.g., 122) at the proximal opening (e.g., 234) can be in the range of approximately 200-800 microns, 200-700 microns, or 200-600 microns. The foregoing is merely an example, and the width _W of the channel 122 can be in other ranges (e.g., a range defined by any of the endpoints listed above). Furthermore, _{the width} W of the channel 122 can be selected to be within any of these ranges in the region of the channel excluding the proximal opening of the isolation dock.

In some embodiments, the height of the isolation dock is about 30 to about 200 microns, or about 50 to about 150 microns. In some embodiments, the isolation dock has a cross-sectional area of about 1×10 ⁴ to about 3×10 ⁶ square microns, about 2×10 ⁴ to about 2×10 ⁶ square microns, about 4×10 ⁴ to about 1×10 ⁶ square microns, about 2×10 ⁴ to about 5×10 ⁵ square microns, about 2×10 ⁴ to about 1×10 ⁵ square microns, or about 2×10 ⁵ to about 2×10 ⁶ square microns. In some embodiments, the connection region has a cross-sectional width of about 20 to about 100 microns, about 30 to about 80 microns, or about 40 to about 60 microns.

In various embodiments of the isolation dock, the height _Hch of the channel (e.g., 122) at the proximal opening (e.g., 234) can be in any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are merely examples, and the height _Hch of the channel (e.g., 122) can be in other ranges (e.g., a range defined by any of the endpoints listed above). The height _Hch of the channel 122 can be selected to be in any of these ranges in the region of the channel other than the proximal opening of the isolation dock.

In various embodiments of the isolation dock, the cross-sectional area of the channel (e.g., 122) at the proximal opening (e.g., 234) can be in any of the following ranges: 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20 ...5,000 square microns, 1,000-20,000 square microns, 1,000-25,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000-25,000 square microns, 1,000-25,000 square microns, 1,000-25,000 square microns, 0-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000-5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000 square microns, 2,000-7,500 square microns, 2,000-6,000 square microns, 3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500 square microns, or 3,000-6,000 square microns. The foregoing are examples only, and the cross-sectional area of the passageway (eg, 122 ) at the proximal opening (eg, 234 ) may be within other ranges (eg, ranges defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the length _Lcon of the connection region (e.g., 236) can be any of the following ranges: about 20 to about 300 microns, about 40 to about 250 microns, about 60 to about 200 microns, about 80 to about 150 microns, about 20 to about 500 microns, about 40 to about 400 microns, about 60 to about 300 microns, about 80 to about 200 microns, or about 100 to about 150 microns. The foregoing are merely examples, and the length _Lcon of the connection region (e.g., 236) can be in a range different from the foregoing examples (e.g., a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the width _Wcon of the connection region (e.g., 236) at the proximal opening (e.g., 234) can be any of the following ranges: about 20 to about 150 microns, about 20 to about 100 microns, about 20 to about 80 microns, about 20 to about 60 microns, about 30 to about 150 microns, about 30 to about 100 microns, about 30 to about 80 microns, about 30 to about 60 microns, about 40 to about 150 microns, about 40 to about 100 microns, about 40 to about 80 microns, about 40 to about 60 microns, about 50 to about 150 microns, about 50 to about 100 microns, about 50 to about 80 microns, about 60 to about 150 microns, about 60 to about 100 microns, about 60 to about 80 microns, about 70 to about 150 microns, about 70 to about 100 microns, about 80 to about 150 microns, and about 80 to about 100 microns. The foregoing are merely examples, and the width _Wcon of the connection region (eg, 236) at the proximal opening (eg, 234) may be different from the foregoing examples (eg, a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the width _Wcon of the connection region (e.g., 236) at the proximal opening (e.g., 234) can be at least as large as the maximum dimension of the micro-object (e.g., a biological cell, which can be an immune cell, such as a B cell or a T cell, or a hybridoma cell, etc.) for which the isolation dock is to be placed. For example, the width Wcon of the connection region 236 at the proximal opening 234 of the isolation dock where the immune cell (e.g., B cell) will be placed can be any of the following: about 20 microns, about 25 microns, about 30 microns, about 35 microns, about 40 microns, about 45 microns, about 50 microns, about 55 microns, about 60 microns, about 65 microns, about 70 microns, about 75 microns, or about 80 microns. The foregoing are merely examples, and the width _Wcon of the connection region (e.g., 236) at the proximal opening (e.g., 234) can be different from the foregoing examples (e.g., a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the ratio of the length _Lcon of the connection region (e.g., 236) to the width _Wcon of the connection region (e.g., 236) at the proximal opening 234 can be greater than or equal to any of the following ratios: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. The foregoing are merely examples, and the ratio of the length _Lcon of the connection region 236 to the width _Wcon of the connection region 236 at the proximal opening 234 can be different from the foregoing examples.

In various embodiments of the microfluidic devices 100, 200, 230, 250, 280, 290, 320, _Vmax can be set to approximately 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 μL/sec.

In various embodiments of a microfluidic device having an isolation dock, the volume of the isolation region (e.g., 240) of the isolation dock can be, for example, at least 5×10 ⁵ , 8×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ , 4×10 ⁶ , 6×10 ⁶ , 8×10 ⁶ , 1×10 ⁷ cubic microns or more. In various embodiments of a microfluidic device having an isolation dock, the volume of the isolation dock can be about 5×10 ⁵ , 6×10 ⁵ , 8×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ , 4×10 ⁶ , 8×10 ⁶ , 1×10 ⁷ cubic microns or more. In some other embodiments, the volume of the isolation dock can be about 0.5 nanoliters to about 10 nanoliters, about 1.0 nanoliters to about 5.0 nanoliters, about 1.5 nanoliters to about 4.0 nanoliters, about 2.0 nanoliters to about 3.0 nanoliters, about 2.5 nanoliters, or any range bounded by two of the foregoing endpoints.

In various embodiments, the microfluidic device has an isolation dock configured as in any embodiment described herein, wherein the microfluidic device has about 5 to about 10 isolation docks, about 10 to about 50 isolation docks, about 100 to about 500 isolation docks, about 200 to about 1000 isolation docks, about 500 to about 1500 isolation docks, about 1000 to about 2000 isolation docks, or about 1000 to about 3500 isolation docks. The isolation docks need not all be the same size and can include a variety of configurations (e.g., different widths, different features within the isolation docks).

In some other embodiments, the microfluidic device has isolation docks configured as in any of the embodiments described herein, wherein the microfluidic device has about 1500 to about 3000 isolation docks, about 2000 to about 3500 isolation docks, about 2500 to about 4000 isolation docks, about 3000 to about 4500 isolation docks, about 3500 to about 5000 isolation docks, about 4000 to about 5500 isolation docks, about 4500 to about 6000 isolation docks, about 5000 to about 6500 isolation docks, about 5500 to about 7000 isolation docks, 0 isolation docks, about 6000 to about 7500 isolation docks, about 6500 to about 8000 isolation docks, about 7000 to about 8500 isolation docks, about 7500 to about 9000 isolation docks, about 8000 to about 9500 isolation docks, about 8500 to about 10,000 isolation docks, about 9000 to about 10,500 isolation docks, about 9500 to about 11,000 isolation docks, about 10,000 to about 11,500 isolation docks, about 10,500 to about 12,000 isolation docks, about 11,000 to about 12,500 isolation docks, about 11,500 to about 13,000 isolation docks, about 12,000 to about 13,500 isolation docks, about 12,500 to about 14,000 isolation docks, about 13,000 to about 14,500 isolation docks, about 13,500 to about 15,000 isolation docks, about 14,000 to about 15,500 isolation docks, about 14,500 to about 16,000 isolation docks, about 15,000 to about 16,500 isolation docks, about 15,500 to about 17,000 isolation docks, about 16,000 to about 17,500 isolation docks, about 16,500 to about 18,000 isolation docks, about 17,000 to about 18,500 isolation docks, about 17,500 to about 19,000 isolation docks, about 18,000 to about 19,500 isolation docks, about 18,500 to about 20,000 isolation docks, about 19,000 to about 20,500 isolation docks, about 19,500 to about 21,000 isolation docks, or about 20,000 to about 21,500 isolation docks.

Control System Elements. Figures 3A-3B illustrate various embodiments of a system 150 of the present invention that can be used to operate and observe a microfluidic device (e.g., 100, 200, 230, 280, 250, 290, 320). As shown in Figure 3A, the system 150 can include a structure ("nest") 300 configured to hold a microfluidic device 320, or any other microfluidic device described herein. The nest 300 can include a receptacle 302 that can interface with the microfluidic device 320 (e.g., the optically actuated electromotive device 100) and provide an electrical connection from the power source 192 to the microfluidic device 320. The nest 300 can also include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 can be configured to provide a bias voltage to the receptacle such that when the receptacle 302 is held, a bias voltage is applied across a pair of electrodes in the microfluidic device 320. Thus, the electrical signal generation subsystem 304 can be part of the power source 192. The ability to apply a bias voltage to the microfluidic device 320 does not mean that the bias voltage will be applied at all times while the microfluidic device 320 is held by the receptacle 302. Rather, in most cases, the bias voltage will be applied intermittently, e.g., only as needed to promote the generation of electrokinetic forces in the microfluidic device 320, such as dielectrophoresis or electrowetting.

3A , the nest 300 may include a printed circuit board assembly (PCBA) 322. The electrical signal generating subsystem 304 may be mounted on and electrically integrated into the PCBA 322. The exemplary support also includes a socket 302 mounted on the PCBA 322.

Typically, the electrical signal generating subsystem 304 includes a waveform generator (not shown). The electrical signal generating subsystem 304 may also include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) that is configured to amplify the waveform received from the waveform generator. The oscilloscope (if present) may be configured to measure the waveform supplied to the microfluidic device 320 held by the socket 302. In certain embodiments, the oscilloscope measures the waveform at the position of the microfluidic device 320 proximal end (and away from the waveform generator) to ensure higher accuracy when measuring the waveform actually applied to the device. The data obtained from the oscilloscope measurement can, for example, be provided to the waveform generator as feedback, and the waveform generator can be configured to adjust its output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is RedPitaya ^™ .

In certain embodiments, the nest 300 further includes a controller 308, such as a microprocessor for sensing and/or controlling the electrical signal generating subsystem 304. Examples of suitable microprocessors include Arduino ^™ microprocessors, such as Arduino Nano ^™ microprocessors. The controller 308 can be used to perform functions and analyses, or can communicate with an external main controller 154 (shown in FIG1A ) to perform functions and analyses. In the embodiment shown in FIG3A , the controller 308 communicates with the main controller 154 via an interface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can include an electrical signal generation subsystem 304 that includes a RedPitaya ^™ waveform generator/oscilloscope unit ("Red Pitaya unit") and a waveform amplification circuit that amplifies the waveform generated by the RedPitaya ^™ unit and passes the amplified voltage to the microfluidic device 100. In some embodiments, the RedPitaya ^™ unit is configured to measure the amplified voltage at the microfluidic device 320 and then adjust its own output voltage as needed so that the measured voltage at the microfluidic device 320 is a desired value. In some embodiments, the waveform amplification circuit can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on PCBA 322, resulting in a signal of up to 13Vpp at the microfluidic device 100.

As shown in FIG3A , the support structure 300 may further include a thermal control subsystem 306. The thermal control subsystem 306 may be configured to regulate the temperature of the microfluidic device 320 held by the support structure 300. For example, the thermal control subsystem 306 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device may have a first surface configured to engage with at least one surface of the microfluidic device 320. The cooling unit may be, for example, a cooling block (not shown), such as a liquid-cooled aluminum block. The second surface of the Peltier thermoelectric device (e.g., the surface opposite the first surface) may be configured to engage with the surface of such a cooling block. The cooling block may be connected to a fluid path 314, which is configured to circulate a cooling fluid through the cooling block. In the embodiment shown in FIG3A , the support structure 300 includes an inlet 316 and an outlet 318 to receive a cooling fluid from an external reservoir (not shown), introduce the cooling fluid into the fluid path 314 and pass through the cooling block, and then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, cooling unit, and/or fluid path 314 can be mounted on the housing 312 of the support structure 300. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Peltier thermoelectric device to achieve a target temperature of the microfluidic device 320. For example, temperature regulation of the Peltier thermoelectric device can be achieved by a thermoelectric power source such as a Pololu™ thermoelectric power source (Pololu Robotics and Electronics Corp.). The thermal control subsystem 306 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal control subsystem 306 having a feedback circuit that is an analog voltage divider circuit (not shown) that includes a resistor (e.g., having a resistance of 1 kΩ +/- 0.1%, a temperature coefficient of +/- 0.02 ppm/C0) and an NTC thermistor (e.g., having a nominal resistance of 1 kΩ +/- 0.01%). In some cases, the thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as an input to an onboard PID control loop algorithm. The output of the PID control loop algorithm can drive, for example, direction and pulse width modulated signal pins on a Pololu ^™ motor driver (not shown) to actuate the thermoelectric power supply, thereby controlling the Peltier thermoelectric device.

The nest 300 may include a serial port 324 that allows the microprocessor of the controller 308 to communicate with the external host controller 154 via the interface 310 (not shown). Additionally, the microprocessor of the controller 308 may communicate with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (e.g., via a Plink tool (not shown)). Thus, via the combination of the controller 308, the interface 310, and the serial port 324, the electrical signal generation subsystem 304 and the thermal control subsystem 306 may communicate with the external host controller 154. In this manner, the host controller 154 may assist the electrical signal generation subsystem 304 by, among other things, performing scaling calculations for output voltage adjustment. A graphical user interface (GUI) (not shown) provided via the display device 170 coupled to the external host controller 154 may be configured to plot temperature and waveform data obtained from the thermal control subsystem 306 and the electrical signal generation subsystem 304. Alternatively or additionally, the GUI may allow for updates to the controller 308 , the thermal control subsystem 306 , and the electrical signal generation subsystem 304 .

FIG3B is an optical schematic diagram of an optical device 350 of the system 150 for a microfluidic device. In some embodiments, the optical device 350 may include a structured light modulator 330. The structured light modulator 330 may include a digital mirror device (DMD) or a micro-gate array system (MSA), either of which may be configured to receive light from a light source 332 and transmit a subset of the received light to the optical device 350. Alternatively, the structured light modulator 330 may include a device that generates its own light (and therefore does not require a light source 332), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon (FLOS), or a transmissive liquid crystal display (LCD). The structured light modulator 330 may be, for example, a projector. Thus, the structured light modulator 330 is capable of emitting both structured light and unstructured light. In certain embodiments, the imaging module and/or motion module of the system may control the structured light modulator 330.

In some embodiments, the optical device 350 can have a microscope configuration. In such embodiments, the nest 300 and the structured light modulator 330 can be separately configured to be integrated into the microscope configuration of the optical device 350. In some embodiments, the optical device 350 can also include one or more image sensors or detectors 348. In some embodiments, the image sensor 348 is controlled by the imaging module. The image sensor 348 can include an eyepiece, a charge coupled device (CCD), a camera (e.g., a digital camera), or any combination thereof. If there are at least two image sensors 348, one image sensor can be, for example, a fast frame rate camera, while the other detector can be a high sensitivity camera. In addition, the optical device 350 can be configured to receive reflected and/or emitted light from the microfluidic device 320 and focus at least a portion of the reflected and/or emitted light on the one or more image sensors 348.

In some embodiments, the optical device 350 is configured to use at least two light sources. For example, a first light source 332 can be used to generate structured light (e.g., via the light modulation subsystem 330), and a second light source 334 can be used to provide unstructured light. The first light source 332 can generate structured light for optically actuated electrodynamic and/or fluorescence excitation, and the second light source 334 can be used to provide bright field illumination. In these embodiments, the motion module 164 can be used to control the first light source 332, and the imaging module 164 can be used to control the second light source 334. The optical device 350 can be configured to receive structured light from the structured light modulator 330 and project the structured light onto at least a first area in a microfluidic device (such as an optically actuated electrodynamic device) while the device is held by the nest 300, and to receive reflected and/or emitted light from the microfluidic device and image at least a portion of such reflected and/or emitted light onto the image sensor 348. The optical device 350 can also be configured to receive unstructured light from a second light source and project the unstructured light onto at least a second region of the microfluidic device when the device is held by the nest 300. In some embodiments, the first region and the second region of the microfluidic device 320 can be overlapping regions. For example, the first region can be a subset of the second region.

In FIG3B , a first light source 332 is shown supplying light to a structured light modulator 330, which provides structured light to the microfluidic device 320. A second light source 334 is shown providing unstructured light via a beam splitter 336. The structured light from the light modulator 330 and the unstructured light from the second light source 334 travel together from the beam splitter 336 to a second beam splitter (or dichroic filter 338, depending on the light provided by the light modulator 330), where the light is reflected downwardly through an objective lens 340 toward the microfluidic device 320. Reflected and/or emitted light from the microfluidic device 320 then travels upwardly through the objective lens 340, passes through the beam splitter and/or dichroic filter 338, and reaches a dichroic filter 346. Only a portion of the light that reaches the dichroic filter 346 passes through and reaches a detector 348.

In some embodiments, the second light source 334 emits blue light. With an appropriate dichroic filter 346, the blue light reflected from the microfluidic device 320 is able to pass through the dichroic filter 346 and reach the detector 348. Conversely, the structured light from the light modulator 330 reflects from the microfluidic device 320 but does not pass through the dichroic filter 346. In this example, the dichroic filter 346 filters out visible light with wavelengths greater than 495 nm. If the light emitted from the light modulator does not include any wavelengths shorter than 495 nm, only this filtering of the light from the light modulator 330 will be completed (as shown). In fact, if the light from the light modulator 330 includes wavelengths shorter than 495 nm (e.g., blue wavelengths), some of the light from the light modulator will pass through the filter 346 and reach the image sensor 348. In such embodiments, the filter 346 is used to change the balance between the amount of light reaching the image sensor 348 from the first light source 332 and the second light source 334. This is beneficial if first light source 332 is significantly more powerful than second light source 334. In other embodiments, second light source 334 can emit red light, and dichroic filter 346 can filter out visible light other than red light (e.g., visible light having a wavelength less than 650 nm).

In some embodiments, the first light source 332 can emit a broad spectrum of wavelengths (e.g., "white" light). The first light source 332 can emit, for example, at least one wavelength suitable for exciting a fluorophore. The first light source 332 can be sufficiently powerful so that the structured light emitted by the light modulator 330 can activate photoactivated electrophoresis in the photoactivated microfluidic device 320. In some embodiments, the first light source 332 can include a high-intensity discharge arc lamp, such as those comprising metal halides, ceramic discharge, sodium, mercury, and/or xenon. In other embodiments, the first light source 332 can include one or more LEDs (e.g., an array of LEDs such as a 2×2 array of 4 LEDs or a 3×3 array of 9 LEDs). The LEDs can include broad-spectrum, white-light-resistant LEDs (e.g., UHP-T-LED-White manufactured by PRIZMATIX) or various narrow-band wavelength LEDs (e.g., emitting wavelengths of approximately 380 nm, 480 nm, or 560 nm). In other embodiments, the first light source 332 can include a laser configured to emit light at a selectable wavelength (e.g., for OET and/or fluorescence).

In some embodiments, the second light source 334 is suitable for bright field illumination. Thus, the second light source 334 may include one or more LEDs (e.g., an LED array, such as a 2×2 array of 4 LEDs or a 3×3 array of 9 LEDs). In some embodiments, the LEDs may be configured to emit white light (i.e., broad spectrum) light, blue light, red light, etc., and the second light source 334 may emit light having a wavelength of 495 nm or less. For example, the second light source 622 may emit light having a wavelength of substantially 480 nm, substantially 450 nm, or substantially 380 nm. In other embodiments, the second light source 334 may emit light having a wavelength of 650 nm or longer. For example, the second light source 334 may emit light having a wavelength of substantially 750 nm. In other embodiments, the second light source 334 may emit light having a wavelength of substantially 560 nm.

In some embodiments, the optical device 350 includes a dichroic filter 346 that at least partially filters out visible light having a wavelength greater than 495 nm. In other embodiments, the optical device 350 includes a dichroic filter 346 that at least partially filters out visible light having a wavelength less than 650 nm (or less than 620 nm). More generally, the optical device 350 may also include a dichroic filter 346 configured to reduce or substantially prevent structured light from the first light source 332 from reaching the detector 348. Such a filter 346 may be located proximal to the detector 346 (along the optical device). Alternatively, the optical device 350 may include one or more dichroic filters 346 configured to balance the amount of structured light (e.g., visible structured light) from the light modulator 330 and the amount of unstructured light (e.g., visible unstructured light) from the second light source 334 that reaches the detector 348. Such a balance may be used to ensure that structured light does not overwhelm unstructured light at detector 348 (or in an image obtained by detector 348 ).

In some embodiments, the optical device 350 may further include at least one tube lens 381 positioned between the objective lens 340 and the image sensor 348 in the imaging path of the device 350. The objective lens 340 no longer projects the intermediate image directly into the intermediate image plane. Instead, the objective lens 340 is configured so that light exiting the rear aperture of the objective lens 340 is focused to infinity, and the tube lens 381 is configured to form an image at the focal plane of the tube lens 381. The light beam exiting the infinitely focusing objective lens 340 is collimated, allowing the beam splitter 338, filter 346, polarizer, and other components requiring a parallel beam to be easily introduced into the imaging path. After passing through these auxiliary optical devices, the parallel beam can be configured to be focused by the tube lens 381 and form an image of the microfluidic device 320. Without the tube lens 381, the insertion of a beam splitter and other components into the imaging path would introduce spherical aberration and possible "ghost" image effects due to the focused beam passing through the beam splitter. Objective lens 340 and tube lens 381 together can produce an image at image sensor 348. The region between objective lens 340 and tube lens 381 (infinite space) provides a path for a parallel light beam in which the composite optical components can be positioned without introducing spherical aberration or modifying the working distance of objective lens 340.

FIG4A is an optical schematic diagram of a system 1000 for imaging and manipulating micro-objects. System 1000 may include a microfluidic device 1320, such as a light-activated microfluidic (or "LAMF") device, and an optical device 1350. Microfluidic device 1320 may be any microfluidic device described herein or known in the art. For example, the microfluidic device may include a housing configured to hold one or more micro-objects in a fluid medium and a substrate 1320c. FIG4B provides an image of a portion (or field of view) or exemplary microfluidic device 1320. Substrate 1320c of the LAMF device may include a surface 1120 and a plurality of dielectrophoresis (DEP) electrodes disposed on (or formed from or integrated with) the surface. Microfluidic device 1320 may also include a flow region 1122 and one or more (e.g., a plurality) of isolation docks 1226. As shown in FIG4B , each isolation dock 1226 may be fluidically connected to a flow region 1122. Flow region 1122 and the plurality of isolation docks 1226 may be disposed on surface 1120 of the substrate of microfluidic device 1320. The microfluidic device 1320 may further include a cover 1320a. The cover 1320a may include a ground electrode. As shown in FIG4B , the cover 1320a may be transparent to visible light.

The optical device 1350 can be configured to perform imaging, analysis, and manipulation of one or more micro-targets within the housing of the microfluidic device 1320. As shown in FIG4A , the optical device 1350 can include a structured light modulator 1330, a first tube lens 1381, an objective lens 1340, a dichroic beam splitter 1338, a second tube lens 1382, and an image sensor 1348. The optical device 1350 can also include a first light source 1332.

In general, the structured light modulator 1330 can be configured to receive an unstructured light beam from the first light source 1332 and transmit the structured light beam to the first tube lens 1381. As described in more detail above, the structured light beam can be used to selectively activate one or more of the plurality of dielectrophoresis (DEP) electrodes on the surface 1120. The first tube lens 1381 is configured to capture the structured light beam from the structured light modulator 1330. The objective lens 1340 is configured to image at least a portion of the plurality of isolation docks 1226 of the microfluidic device 1320 within a field of view. For example, the field of view can be larger than 10 mm x 10 mm, 11 mm x 11 mm, 12 mm x 12 mm, 13 mm x 13 mm, 14 mm x 14 mm, 15 mm x 15 mm, etc.

The dichroic beam splitter 1338 is configured to reflect or transmit the light beam from the first tube lens 1381 to the objective lens 1340, and transmit or reflect the light beam received from the objective lens 1340 to the second tube lens 1382. The second tube lens 1382 is configured to receive the light beam from the dichroic beam splitter 1338 and transmit the light beam to the image sensor 1348. The image sensor 1348 is configured to receive the light beam from the second tube lens and generate therefrom an image of at least a portion of the plurality of isolation docks 1226 within a field of view.

In some embodiments, the structured light modulator 1330 may include a digital mirror device (DMD) or a micro-gate array system (MSA), either of which can be configured to receive light from the light source 332 and selectively transmit a subset of the received light. An exemplary DMD suitable for any of the optical devices disclosed herein (including the optical device 1350) is the DLP-9000 (Texas Instruments). Alternatively, the structured light modulator 1330 may include a device that generates its own light (and therefore does not require the light source 1332), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon (FLOS) device, or a transmissive liquid crystal display (LCD). The structured light modulator 1330 may be, for example, a projector. Thus, the structured light modulator 1330 is capable of emitting both structured and unstructured light.

In some embodiments, the structured light modulator 1330 can be configured to modulate the light beam received from the first light source 1332 and transmit a plurality of illumination light beams as structured beams. The structured light beams can include the plurality of illumination light beams. The plurality of illumination light beams can be selectively activated to generate a plurality of illumination patterns. In some embodiments, the structured light modulator 1330 can be configured to generate a movable and adjustable illumination pattern. The optical device 1350 can further include a control unit (not shown) configured to adjust the illumination pattern to selectively activate one or more of the plurality of DEP electrodes and generate a DEP force to move one or more micro-objects within the plurality of isolation docks 1226.

For example, multiple illumination patterns can be adjusted over time in a controlled manner to manipulate micro-objects in the microfluidic device 1320. For example, each of the multiple illumination patterns can be shifted to move the location of the generated DEP force and move the structured light from one location to another to move the micro-objects within the housing of the microfluidic device 1320.

4A , in some embodiments, the optical device 1350 is configured so that each of the plurality of isolated docks 1226 within the field of view is simultaneously focused on the image sensor 1348 and the structured light modulator 1330. For example, the optical device 1350 can have a confocal configuration or confocal properties. The optical device 1350 can also be configured so that only the interior area of the flow region within the field of view and/or each of the plurality of isolated docks 1226 is imaged onto the image sensor 1348 to reduce overall noise and increase image contrast and resolution.

For example, the structured light modulator 1330 may be located at a conjugate plane of the image sensor 1348. The structured light modulator 1330 may receive an unstructured light beam from the first light source 1332 and modulate the light beam to generate a plurality of illumination light beams, which are structured light beams. The effective area of the structured light modulator may be at least 10 mm × 10 mm (e.g., at least 10.5 mm × 10.5 mm, 11 mm × 11 mm, 11.5 mm × 11.5 mm, 12 mm × 12 mm, 12.5 mm × 12.5 mm, 13 mm × 13 mm, 13.5 mm × 13.5 mm, 14 mm × 14 mm, 14.5 mm × 14.5 mm, 15 mm × 15 mm, or larger). The first tube lens 1381 may have a large clear aperture, for example, a diameter greater than 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, or 50 mm, and thus, the first tube lens 1381 may have an aperture large enough to capture all (or substantially all) of the light beams emitted from the structured light modulator.

FIG4C illustrates that the first tube lens 1381 of the optical device 1350 in FIG4A is configured to capture all light beams from the structured light modulator 1330. The structured light modulator 1330 may have a plurality of mirrors. Each of the plurality of mirrors may have a size of 5 microns by 5 microns, 6 microns by 6 microns, 7 microns by 7 microns, 8 microns by 8 microns, 9 microns by 9 microns, 10 microns by 10 microns, or any value therebetween. The structured light modulator 1330 may include a mirror (or pixel) array that is 2000×1000, 2580×1600, 3000×2000, or any value therebetween. For a mirror size of 7.6 microns by 7.6 microns, the structured light modulator 1330 may have a size of 15.2 mm by 7.6 mm, 19.6 mm by 12.2 mm, 22.8 mm by 15.2 mm, or any value therebetween. As shown in FIG4C , in some embodiments, only a portion of the illumination area 1330a of the structured light modulator 1330 is used. For example, 50%, 60%, 80%, or any value therebetween of the illumination area 1330a of the structured light modulator 1330 is used. The first tube lens 1381 can be configured to have a large field of view 1381a that is larger than the illumination area 1330a of the structured light modulator 1330. The first tube lens 1381 can be configured to capture all light beams from the structured light modulator 1330.

4A , in some embodiments, the first tube lens 1381 can be configured to generate a collimated light beam and send the collimated light beam to the objective lens 1340. The objective lens 1340 can receive the collimated light beam from the first tube lens 1381 and focus the collimated light beam into each interior region of the flow region, and each of the plurality of isolated docks 1226 within the field of view of the image sensor 1348 or the optical device 1350. In some embodiments, the first tube lens 1381 can be configured to generate a plurality of collimated light beams and send the plurality of collimated light beams to the objective lens 1340. The objective lens 1340 can receive the plurality of collimated light beams from the first tube lens 1381 and focus the plurality of collimated light beams into each of the plurality of isolated docks 1226 within the field of view of the image sensor 1348 or the optical device 1350.

In some embodiments, the optical device 1350 can be configured to illuminate at least a portion of the isolation dock with a plurality of illumination points. The objective lens 1340 can receive the plurality of collimated light beams from the first tube lens 1381 and project the plurality of illumination points onto each of the plurality of isolation docks 1226 within the field of view. For example, each of the plurality of illumination points can have a size of approximately 10 microns by 30 microns, 30 microns by 60 microns, 60 microns by 120 microns, 80 microns by 100 microns, 100 microns by 140 microns, and any values therebetween. For example, each of the plurality of illumination points can have an area of approximately 4,000 to approximately 10,000, 5,000 to approximately 15,000, 7,000 to approximately 20,000, 8,000 to approximately 22,000, 10,000 to approximately 25,000 square microns, and any values therebetween.

In some embodiments, the optical device 1350 can be configured to perform confocal imaging. For example, the structured light modulator 1330 can be configured to generate a thin strip that can be scanned across the plurality of isolated docks 1226 within the field of view to reduce out-of-focus light and thereby reduce overall noise. As another example, the structured light modulator 1330 can be configured to generate multiple illumination points within the diffraction limit. As another example, the structured light modulator 1330 can be configured to move along the optical axis of the optical device 1350 to obtain multiple images along the optical axis, and the multiple images along the optical axis can be combined to reconstruct a three-dimensional image of the micro-objects in the plurality of isolated docks 1226 in the microfluidic device 1320.

A second tube lens 1382 is positioned between the objective lens 1340 and the image sensor 1348 in the imaging path of the device 1350. The objective lens 1340 is configured to focus light emitted from a back aperture of the objective lens 1340 to infinity, and the second tube lens 1382 is configured to form an image of the micro-objects in the plurality of isolation docks 1226 at the focal plane of the tube lens 1382. The light beam exiting the infinity-focusing objective lens 1340 can be configured to be collimated so that the beam splitter 1338 and other components can be easily introduced into the imaging path of the optical device 1350 without introducing spherical aberration or modifying the working distance of the objective lens 1340.

In some embodiments, optical device 1350 may further include nest 1300. Nest 1300 may be configured to hold microfluidic device 1320 and provide electrical connections to the housing. Nest 1300 may be integrated with optical device 1350 and be part of device 1350. Nest 1300 may also be configured to provide fluidic connections to the housing. A user may simply load microfluidic device 1320 into nest 1300. In some other embodiments, nest 1300 may be a separate component from optical device 1350.

FIG5A illustrates multiple light sources that can be used in an optical device 5350 and a light-actuated microfluidic device in some other embodiments. As discussed above, a variety of light sources can be used as a first light source 5332 to provide structured light to a DMD tube lens 5460. In some embodiments, the first light source 5332 can be a light-emitting diode (LED). The light source 5332 can emit light 505 having a broad spectrum of wavelengths, which, when illuminating the DMD 5440, provides structured light 515 to a DMD fold mirror 5336, which can be a dichroic fold mirror. The DMD fold mirror 5336 redirects the structured light 515 toward the DMD tube lens 5460. In some embodiments, the optical device can also include a second light source 5334 configured to provide unstructured brightfield illumination 525 through the DMD fold mirror 5336 to the DMD tube lens 5460. In some other embodiments, the optical device may further include a third light source 5335, such as a laser light source, providing light illumination 535, which may be configured to heat the plurality of isolated docks in the microfluidic device. FIG5B illustrates an example of light being transmitted through a dichroic folding mirror 5336, as for the plurality of light sources configured in FIG5A.

The structured light 515 arriving from the DMD 5440 can have a wavelength of from about 400 nm to about 710 nm and can be used in any microfluidic device for photoactivation of a DEP or OEW structure within a microfluidic device as described herein after passing through the DMD tube lens 5460. The structured light 515 having a wavelength of from about 400 nm to about 710 nm can additionally or alternatively provide fluorescence excitation illumination to the microfluidic device. In some embodiments, the structured light 515 can have a wavelength of from about 400 nm to about 650 nm, from about 400 nm to about 600 nm, from about 400 nm to about 550 nm, from about 400 nm to about 500 nm, from about 450 nm to about 710 nm, from about 450 nm to about 600 nm, or from about 450 nm to about 550 nm.

Unstructured brightfield illumination 525 from second light source 5334 reaches DMD fold mirror 5336 and can pass through mirror 5336 at substantially (e.g., within about 10%) the same wavelength and/or at substantially (e.g., within about 10%) the same intensity before impinging on the mirror. Alternatively, mirror 5336 can be folded to allow brightfield illumination 525 to pass through, enter tube lens 5460, and further travel to enter the microfluidic device, which can be any microfluidic device as described herein. Brightfield illumination light 525 can have any suitable wavelength, and in some embodiments, can have a wavelength of about 400 nm to about 760 nm. In some embodiments, brightfield illumination light 525 can have a wavelength greater than about 5336 nm and less than about 760 nm, greater than about 600 nm and less than about 750 nm, or about 650 nm and less than about 750 nm. In some embodiments, the bright field illumination light can have a wavelength of about 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm, or about 750 nm.

The third illumination light 535 can pass through the DMD mirror 5336, or the DMD mirror 5336 can be folded to allow the illumination light 535 to pass through and enter the tube lens 5460 and further travel to the microfluidic device, which can be any microfluidic device as described herein. The third illumination light 535 can be a laser and can be configured to heat a portion of one or more isolation docks within the microfluidic device. The laser irradiation 535 can be configured to heat a fluid medium, a microobject, a wall of an isolation dock or a portion of a wall of an isolation dock, a metal target disposed within a microfluidic channel or isolation dock of a microfluidic channel, or a photoreversible physical barrier within the microfluidic device. In other embodiments, the laser irradiation 535 can be configured to induce photofragmentation of a surface modified portion of a modified surface of a microfluidic device or photofragmentation of a portion that provides adhesion functionality for microobjects within an isolation dock within a microfluidic device. The laser irradiation 535 can have any suitable wavelength. In some embodiments, the laser irradiation 535 can have a wavelength of about 350 nm to about 900 nm, about 370 nm to about 850 nm, about 390 nm to about 825 nm, about 400 nm to about 800 nm, about 450 nm to about 750 nm, or any value therebetween. In some embodiments, the laser irradiation 535 can have a wavelength of about 700 nm, about 710 nm, about 720 nm, about 730 nm, about 740 nm, about 760 nm, about 770 nm, about 780 nm, about 790 nm, about 800 nm, about 810 nm, or greater.

FIG5C is a schematic diagram of a system 5000 including an optical device 5350, which includes a first light source 5335, a second light source 5334, and a third light source 5332. The first light source 5335 can transmit light to a structured light modulator 5330, which can include a digital mirror device (DMD) or a micro-gate array system (MSA), either of which can be configured to receive light from the first light source 5335 and selectively transmit a subset of the received light to the optical device 5350. Alternatively, the structured light modulator 5330 can include a device that generates its own light (and thus does not require a light source 5335), such as an organic light-emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon (FLOS) device, or a transmissive liquid crystal display (LCD). The structured light modulator 5330 can be, for example, a projector. Thus, the structured light modulator 5330 is capable of emitting both structured and unstructured light. In certain embodiments, the imaging module and/or motion module of the system can control the structured light modulator 5330. The structured light modulator 5330 can transmit a subset of the light to a first dichroic beam splitter 5338 , which can reflect the light to a first tube lens 5381 .

Second light source 5334 can send light to second dichroic beam splitter 5336, which also receives light from third light source 5332. Third light source 5332 can transmit light to reflector 5003 through paired relay lens 5001. Additionally, beam splitter 5338 can receive light from third light source 5332 and second light source 5334 and transmit it to first tube lens 5381. Light from the first, second, and third light sources passes through first tube lens 5381 and is transmitted to third dichroic beam splitter 5339 and filter switch 5005. The third dichroic beam splitter can reflect a portion of the light and transmit it through one or more filters in filter switch 5005 to objective lens 5340, which can be a nosepiece having multiple different objective lenses that can be switched as needed. Some light can pass through third dichroic beam splitter 5339 and be terminated or absorbed by beam block 5007. Light reflected from third dichroic beam splitter 5339 passes through objective lens 5340 to illuminate sample plane 5320, which can be a microfluidic device, such as part of an isolation dock as described herein. The combined light can be used to illuminate, heat, and/or excite the sample in sample plane 5320. Light can be reflected and/or emitted from sample plane 5320 to pass through nosepiece 5340, through filter changer 5005, and through third dichroic beam splitter 5339 back to second tube lens 5382. The light can pass through second tube lens 5382 (or imaging tube lens 5382) and reflect from mirror 5015 to imaging sensor 5348. Stray light baffles 5009 , 5011 , and 5013 may be placed between the first tube lens 5381 and the third dichroic beam splitter 5339 , between the third dichroic beam splitter 5339 and the second tube lens 5382 , and between the second tube lens 5382 and the imaging sensor 5348 .

6A is a schematic diagram of a system 2000 including an optical device 2350 having an excitation filter 2346a and an emission filter 2346b according to some other embodiments of the present disclosure. The excitation filter and the emission filter can be inserted into the optical path of the optical device 2350. The first tube lens 2381, the second tube lens 2382, and the objective lens 2340 form an infinity-corrected optical configuration such that the beam splitter 2338, the excitation filter 2346A, and the emission filter 2346B can be easily introduced into the optical path of the optical device 2350 without introducing spherical aberration.

6B is a schematic diagram of a system 3000 including an optical device 3350 according to some other embodiments of the present disclosure, wherein a beam splitter 3338 is configured to reflect a light beam from a first light source 3332. As shown in FIG4A and FIG6B , the beam splitter can be configured to transmit or reflect the light beam from the first light source and to reflect or transmit the light beam from the objective lens, respectively.

Referring back to FIG4A , the optical device may include an objective lens 1340 specifically designed and configured for observing and manipulating micro-objects within the microfluidic device 1320. For example, a conventional microscope objective lens is designed to observe micro-objects on a glass slide or in an aqueous fluid with a total length of 5 mm. The micro-objects within the microfluidic device 1320 are within a plurality of isolation docks 1226 having a depth of 20, 30, 40, 50, 60, 70, 80 microns, or any value therebetween. In some embodiments, a transparent cover 1320a, such as a glass or ITO cover having a thickness of approximately 750 microns, may be placed on top of the plurality of isolation docks 1226. Therefore, images of the micro-objects obtained using a conventional microscope objective lens may have large aberrations, such as spherical aberration and chromatic aberration, which may reduce the quality of the image. The objective lens 1340 of the optical device 1350 may be configured to correct for spherical aberration and chromatic aberration in the optical device 1350.

Fig. 6 C is the schematic diagram of the system 4000 that comprises the optical device 4350 with correction lens 4340b to compensate for the aberration from object lens 4340 of some other embodiments of the present disclosure.Object lens 4340 can be a conventional microscope objective lens, for example, has the objective lens of magnification 4X, 10X, 20X etc. from Olympus (Olympus) or Nikon (Nikon).Due to the complexity of optical design, redesigning microscope objective lens may be very challenging and expensive.In some embodiments, correction lens 4340b can be used for compensating, correcting and minimizing the residual aberration produced by using conventional microscope objective lens 4340.For example, correction lens 4340b can be inserted between object lens 4340 and beam splitter 1338.For another example, correction lens can be inserted between object lens and microfluidic device.In some other embodiments, the first tube lens and the second tube lens can be configured to minimize the residual aberration of conventional microscope objective lens.

Referring again to FIG4A , due to the limitations of available space, the optical device 1350 of the system 1000 for imaging and manipulating micro-objects is typically mechanically constrained. The tube lenses 1381, 1382 of the optical device 1350 must be specifically designed and configured to meet mechanical and optical requirements. In some embodiments, the first tube lens can have a focal length of approximately 155 mm or approximately 162 mm, and the second tube lens can have a focal length of approximately 180 mm. In some other embodiments, the first tube lens can have a focal length of approximately 180 mm and the second tube lens can have a focal length of approximately 200 mm.

The conjugates of the front and back focal points of the tube lenses 1381 and 1382 of the optical device 1350 differ from conventional tube lens arrangements. Typically, for conventional tube lenses, the back focal length (BFL) and front focal length (FFL) are approximately equal. The conjugates of the front and back focal points of conventional tube lenses are typically symmetrical and equally spaced from the midpoint of the tube lens. However, for the optical device 1350, the "infinite space" between the objective lens 1340 and the first tube lens 1381 must be configured to meet mechanical constraints. In some embodiments, this "infinite space" must be maximized. In some embodiments, this "infinite space" must be minimized. In some embodiments, the conjugate point corresponding to the front focal point of the first tube lens 1381 must be positioned as far away from the edge of the tube lens 1381 as possible to ensure available mechanical space. In some embodiments, the other conjugate point corresponding to the back focal point of the first tube lens 1381 must be positioned as close to the edge of the tube lens 1381 as possible to minimize the distance from the tube lens to the structured light modulator. Therefore, the BFL of the tube lens 1381 must be designed or configured to be minimized. In some other embodiments, the BFL of the tube lens 1381 must be designed or configured to be maximized.

Similarly, in some embodiments, the "infinite space" between objective lens 1340 and second tube lens 1382 must be maximized. In other embodiments, the "infinite space" between objective lens 1340 and second tube lens 1382 must be minimized. For example, if second tube lens 1382 has an effective focal length (EFL) of 180 mm, then in a conventional tube lens design, the conjugates are the front and back focal points, and the distance from the midpoint of tube lens 1382 on either side would be 180 mm. In optical device 1350, to maximize the "infinite space" between objective lens 1340 and second tube lens 1382, the BFL of tube lens 1382 can be configured or designed to be minimized and as short as possible. In other embodiments, the BFL of tube lens 1382 can be configured or designed to be maximized as much as possible. Consequently, the conjugates of the front and back focal points of tube lenses 381, 1382 of optical device 1350 are not equidistant from the midpoint and are asymmetrical.

Fig. 7 A is the optical schematic diagram of the tube lens 7381 of the optical device with an EFL of 155mm.There is no commercially available tube lens with an EFL shorter than 162mm at present.It is difficult to design a tube lens with a short EFL of 155mm because the light beam passing through the tube lens is bent at a large angle, and therefore produces large aberrations.Special consideration must be given in order to minimize the "infinite" space between the tube lens and the object lens.The front focus and the back focus of the tube lens are not equidistantly spaced apart and asymmetric from the midpoint of the tube lens.The BFL of the tube length is minimized.For example, in some embodiments, the BFL of the tube length is about 133mm, 134mm, 135mm or 136mm.

For example, a tube lens with an EFL of 155 mm may include: a first surface having a convex shape and a positive radius of curvature of approximately 91 mm; a second surface having a convex shape and a positive radius of curvature of approximately 42 mm; a third surface having a concave shape and a negative radius of curvature of approximately -62 mm; and a fourth surface having a concave shape and a negative radius of curvature of approximately -116 mm. The tube lens may have a clear aperture diameter greater than 44, 45, 46, 47, 48, 49, or 50 mm. For example, the tube lens may have a clear aperture diameter of approximately 48 mm.

Figure 7B is an optical schematic diagram of a tube lens 7831e of an optical device having an EFL of 162 mm. The tube lens may include: a first surface having a convex shape and a positive radius of curvature of approximately 95 mm; a second surface having a convex shape and a positive radius of curvature of approximately 54 mm; a third surface having a concave shape and a negative radius of curvature of approximately -56 mm; and a fourth surface having a concave shape and a negative radius of curvature of approximately -105 mm. The tube lens has a clear aperture diameter greater than 44, 45, 46, 47, 48, 49, or 50 mm. For example, the tube lens may have a clear aperture diameter of approximately 48 mm. The front and rear focal points of the tube lens are unequally spaced from the midpoint and are asymmetrical. The BFL of the tube length is minimized. For example, in some embodiments, the BFL of the tube length is approximately 144 mm, 145 mm, 146 mm, or 147 mm.

Figure 7C is an optical schematic diagram of a tube lens 7831 of an optical device having an EFL of 180 mm. The tube lens may include: a first surface having a convex shape and a positive radius of curvature of approximately 95 mm; a second surface having a convex shape and a positive radius of curvature of approximately 64 mm; a third surface having a concave shape and a negative radius of curvature of approximately -60 mm; and a fourth surface having a concave shape and a negative radius of curvature of approximately -126 mm. The tube lens has a clear aperture diameter greater than 44, 45, 46, 47, 48, 49, or 50 mm. For example, the tube lens may have a clear aperture diameter of approximately 48 mm. The front and rear focal points of the tube lens are unequally spaced from the midpoint and are asymmetrical. The BFL of the tube length is minimized. For example, in some embodiments, the BFL of the tube length is 161 mm, 162 mm, 163 mm, 164 mm, or 165 mm.

7D is an optical schematic of a tube lens 7381″′ of an optical device having an EFL of 200 mm. The tube lens may include: a first surface having a convex shape and a positive radius of curvature of approximately 160 mm; a second surface having a concave shape and a negative radius of curvature of approximately -62 mm; a third surface having a concave shape and a negative radius of curvature of approximately -80 mm; and a fourth surface having a concave shape and a negative radius of curvature of approximately -109 mm. The tube lens has a clear aperture having a diameter greater than 44, 45, 46, 47, 48, 49, 50 mm. For example, the tube lens may have a clear aperture having a diameter of approximately 48 mm. The front and back focal points of the tube lens are not equidistant from the midpoint and are asymmetric. The BFL of the barrel length is minimized. For example, in some embodiments, the BFL of the barrel length is 189 mm, 190 mm, 191 mm, or 192 mm. For example, the BFL of the barrel length may be 191.08 mm.

Table 1 summarizes examples of BFLs for optical equipment tube lenses. Table 2 shows examples of lens data for a tube lens with an EFL of 155 mm for optical equipment. Table 3 shows examples of lens data for a tube lens with an EFL of 162 mm for optical equipment. Table 4 shows examples of lens data for a tube lens with an EFL of 180 mm for optical equipment. Table 5 shows examples of lens data for a tube lens with an EFL of 200 mm for optical equipment.

Table 1: Examples of BFLs of tube lenses of optical devices

Table 2: Example of an optical setup with a tube lens having an EFL of 155 mm

Table 3: Example of an optical setup with a tube lens having an EFL of 162 mm

Table 4: Examples of optical setups with tube lenses having an EFL of 180 mm

Table 5: Examples of tube lenses for an optical setup with an EFL of 200 mm

FIG8A illustrates another embodiment of an optical configuration that can be used with optical system 8000. A first light source 8332 (i.e., a laser) can transmit light to lens relay 8001. The light can pass through the lens relay to a first reflector 8003, which can reflect the light through a first dichroic beam splitter 8336. The first dichroic beam splitter 8336 also receives light from a second light source 8334 (i.e., a bright field LED) and reflects the light, along with the light from the first light source, through a second dichroic beam splitter 8338. The second dichroic beam splitter 8338 can also receive light from a third light source 8335, which can first transmit light to a structured light modulator 8330, which can reflect all or a portion of the light to the second dichroic beam splitter 8338. An intermediate laser focal plane 8017 for lens relay 8001 can be located between the first dichroic beam splitter 8336 and the second dichroic beam splitter 8338. The second dichroic beam splitter reflects light from the third light source 8335 and passes light from the first light source 8332 and the second light source 8334 to the first tube lens 8381. The combined light passes through the first tube lens 8381 to the first filter 8346 and then through the third dichroic beam splitter 8339, which can reflect the light to the objective 8340, which focuses the light onto the sample plane 8320. The sample plane 8320 is illuminated, heated, and/or excited by the combined light and can emit light in response to the excitation, which can pass through the objective 8340 and then through the third dichroic beam splitter 8339, through the second filter 8347, through the second tube lens 8382, and to the imaging sensor (i.e., camera).

FIG8B shows another embodiment of an optical configuration that can be used by an optical system 8000′ having a first light source 8332 (i.e., a laser), a second light source 8334 (i.e., a bright field LED), and a third light source 8335. The second light source 8334 can transmit light toward a first reflector 8003, which can reflect the light toward a first dichroic beam splitter 8336 and through the first dichroic beam splitter 8336. The first dichroic beam splitter 8336 can also receive light from a third light source 8335, which can first transmit light toward a structured light modulator 8330, which can reflect all or a portion of the light toward the first dichroic beam splitter 8336. The light is reflected or transmitted through the first dichroic beam splitter 8336 to a first filter 8346, and then to a second dichroic beam splitter 8338, which can reflect the light toward an objective lens 8340. The first light source 8332 can emit light and pass it through the collimating lens 8019 to the third dichroic beam splitter 8339. The third dichroic beam splitter 8339 reflects the light and passes it through the second filter, through the second dichroic beam splitter 8338, and reaches the objective lens 8340. The combined light from all light sources is focused by the objective lens 8340 onto the sample plane, and the sample can emit light, which can return after excitation through the objective lens 8340, through the second dichroic beam splitter 8338, through the second filter 8347, through the third dichroic beam splitter 8339, through the second tube lens 8382, and reach the imaging sensor 8348 (i.e., camera).

8C illustrates another embodiment of an optical configuration that can be used by an optical system 8000″ having a first light source 8332 (i.e., a laser), a second light source 8334 (i.e., a bright field LED), and a third light source 8335. The second light source 8334 can transmit light toward the first reflector 8003, which can reflect the light toward a first dichroic beam splitter 8336 and through the first dichroic beam splitter 8336. The first dichroic beam splitter 8336 can also receive light from the third light source 8335, which can first transmit light toward a structured light modulator 8330, which can reflect all or a portion of the light toward the first dichroic beam splitter 8336. The light is reflected or transmitted through the first dichroic beam splitter 8336, through the first pipeline 8381, and through the second dichroic beam splitter 8338. The second dichroic beam splitter 8338 can also receive light from the first light source 8332, which can first emit light through the collimating lens 8019 before reflecting from the second dichroic beam splitter 8338. Light reflected and transmitted through the second dichroic beam splitter 8338 is transmitted through the first filter 8346 to the third dichroic beam splitter 8339, which reflects the light to the objective 8340, which focuses the light onto the sample plane 8320. The sample can emit light from the excitation and also reflect light back through the objective 8340, through the third dichroic beam splitter 8339, through the second filter 8347, through the second tube lens 8382, and to the imaging sensor 8348 (i.e., camera).

8D illustrates another embodiment of an optical configuration that can be used by an optical system 8000′″ having a first light source 8332 (i.e., a laser), a second light source 8334 (i.e., a bright field LED), and a third light source 8335. The second light source 8334 can transmit light toward a first reflector 8003, which can reflect the light toward and through a first dichroic beam splitter 8336. The first dichroic beam splitter 8336 can also receive light from a third light source 8335, which can first transmit light toward a structured light modulator 8330, which can reflect all or a portion of the light toward the first dichroic beam splitter 8336. Light passes through a first dichroic beam splitter 8336, through a first line 8381, through a first filter 8346, and to a second dichroic beam splitter 8338. The second dichroic beam splitter 8338 reflects or transmits the light, allowing the light to pass through a third dichroic beam splitter 8339 and reach the objective lens 8340. The first light source 8332 can transmit light through a collimating lens 8019 and reach the third dichroic beam splitter 8339, which can reflect the light to the objective lens 8340. The combined light can be focused by the objective lens onto the sample plane 8320 to illuminate, heat, and/or excite the sample. The light can be reflected and transmitted back through the objective lens 8340, through the third dichroic beam splitter 8339, through the second dichroic beam splitter 8338, through the second tube lens 8382, and reach the imaging sensor 8348 (i.e., camera).

Figures 9A and 9B illustrate the use of angular imaging techniques, also known as Fourier stack microscopy (FPM), that can be incorporated into any embodiment described herein. Angular imaging techniques can be used to increase image resolution without increasing the power of the objective lens. For example, a 10X objective lens can be used to achieve 20X resolution. FPM works by taking multiple relatively low-resolution images from multiple different angles. An iterative process that switches between the spatial domain and the Fourier domain is used to computationally generate a higher-resolution image from the multiple images.

In step 1, the FPM method begins by taking an initial low-resolution image, designating it as the initial high-resolution image, and applying a Fourier transform to the image to create a broad spectrum in the Fourier domain.

In step 2, a small sub-region of the spectrum is selected by applying a low-pass filter, and then a Fourier transform is applied to generate a new low-resolution target image in the spatial domain. The low-pass filter shape is a circular pupil corresponding to the coherence transfer function of the objective lens. The position of the low-pass filter is selected to correspond to the illumination angle of the image being processed.

In step 3, the amplitude component of the target image is replaced by the square root of the low-resolution measurement obtained at the current illumination angle to form an updated low-resolution target image. A Fourier transform is applied to the updated low-resolution target image, which is used to replace the corresponding subregion of the initial high-resolution Fourier space.

In step 4, steps 2 and 3 are repeated for other sub-regions, ensuring that the sub-regions overlap with adjacent sub-regions to ensure focus, and the process is repeated for all images.

In step 5, steps 2 to 4 are repeated until a self-consistent solution is achieved in Fourier space. A Fourier transform is then applied to bring the converged solution back to the spatial domain, which is the final high-resolution image.

Figure 9A shows a simplified portion of an optical train including a structured light modulator 9330, a tube lens 9381, an objective lens 9340, and a sample plane 9320. In Figure 9A, light between the tube lens 9381 and the objective lens 9340 is collimated, and the objective lens 9340 then focuses the collimated light onto the sample plane 9320. Figure 9B illustrates the addition of a sliding lens 9001 between the structured light modulator 9330 and the tube lens 9381, which is used for FPM. The sliding lens 9001 can be slidably inserted and removed from the optical train. In some embodiments, the system can have one or more different sliding lenses 9001, each of which can be slidably inserted and removed. In some embodiments, the position of the sliding lens can be adjusted to produce different images from different angles. The insertion of the sliding lens causes (i) light traveling from the tube lens 9381 to the objective lens 9340 to arrive at a focal point between the tube lens 9381 and the objective lens 9340 (rather than being collimated), and (ii) light traveling from the objective lens 9340 to the sample plane 9320 to be collimated rather than arriving at a focal point. By selectively illuminating different portions of the structured light modulator 9330, light striking the sample plane 9320 will arrive at different angles. Images of the sample plane 9320 illuminated by light arriving from several angles are then combined as described above to produce a higher resolution image. The structured light modulator can be divided into at least eight different portions (so that at least eight images with light arriving at the sample plane at different angles are produced) to achieve higher resolution. Dividing the structured light modulator into even more portions, such as 12, 16, 20, 24, etc., to produce different angles/images will produce even better resolution.

The system may include a computing device having a processor and memory programmed to perform the FPM calculations described above.

Various embodiments of methods for manipulating one or more micro-objects of a sample are disclosed herein. The methods may include loading a sample containing the one or more micro-objects into a microfluidic device having a housing. For example, the microfluidic device may include a substrate having a surface and a plurality of dielectrophoresis (DEP) electrodes on the surface and a flow region, and a plurality of isolated docks, each fluidically connected to the flow region.

The method may include the step of applying a voltage potential across the microfluidic device.The method may include the step of selectively activating a DEP force adjacent to at least one micro-object located within the microfluidic device using an optical device.

The optical apparatus may be configured to project structured light onto a first location on the surface of the substrate of the microfluidic device, wherein the first location is adjacent to a second location on the surface of the substrate, the second location being located below the at least one micro-object.

The optical device may include a first light source, a structured light modulator, a first and second tube lenses, an objective lens, a dichroic beam splitter, and an image sensor. The structured light modulator is configured to receive unstructured light from the first light source and transmit structured light suitable for selectively activating one or more of the plurality of DEP electrodes on the surface of the substrate of the microfluidic device. The first tube lens is configured to capture structured light from the structured light modulator and transmit the structured light to the objective lens. The objective lens is configured to receive the structured light transmitted from the first tube lens and project the structured light into a housing of the microfluidic device, and the objective lens is further configured to receive light reflected or emitted from at least a portion of the housing within a field of view of the objective lens. The dichroic beam splitter may be located between the first tube lens and the objective lens, wherein the dichroic beam splitter is configured to transmit the structured light received from the first tube lens to the objective lens and reflect the light received from the objective lens to the second tube lens. The second tube lens is configured to receive reflected light from the dichroic beam splitter and transmit the reflected light to the image sensor. The image sensor is configured to receive reflected light from the second tube lens and record an image of at least a portion of the housing within the field of view of the objective lens.

The method may include the step of moving a position of a DEP force generated adjacent to at least one micro-object by using the optical apparatus to move the structured light from the first position on the surface of the substrate of the light-actuated microfluidic device to a third position on the surface of the substrate.

In some embodiments, the method may further comprise the step of capturing the image of the at least a portion of the housing of the microfluidic device using the image sensor. In some embodiments, the imaged portion of the housing of the microfluidic device comprises at least one isolation dock and at least one micro-object.

In some embodiments, the optical device includes a second light source that generates unstructured light, and wherein the method further includes projecting the unstructured light from the second light source into a housing of the microfluidic device using the optical device to provide bright field illumination within the housing.

In some embodiments, the optical device comprises a laser light source, and wherein the method further comprises projecting laser light from the laser light source onto a surface of a substrate of the housing of the microfluidic device using the optical device.

In some embodiments, the optical device further includes a second dichroic beam splitter positioned between the structured light modulator and the first tube lens, and wherein the structured light transmitted by the structured light modulator is reflected by the second dichroic beam splitter into the first tube lens.

In some embodiments, the unstructured light generated by the second light source is transmitted to the first tube lens through a second dichroic beam splitter. In some embodiments, the laser light generated by the laser light source is transmitted to the emitter of the first tube lens through a second dichroic beam splitter.

In some embodiments, the structured light projected onto the first location on the substrate surface includes a plurality of illumination points. In some embodiments, the first location on the substrate surface is located in a flow region of the microfluidic device, and wherein the third location on the substrate surface is located within one of the plurality of isolated bays.

In some embodiments, the structured light projected onto the first location on the substrate surface includes a shape like a line segment or a symbol. In some embodiments, the structured light projected onto the first location on the substrate surface has a shape similar to the outline of a polygon.

In some embodiments, the method may further include a step of selectively activating DEP forces adjacent to a plurality of micro-objects located within the microfluidic device by projecting structured light onto a plurality of first locations on the surface of a substrate of the microfluidic device using an optical device, wherein each of the plurality of first locations is located near a corresponding second location on the surface of the substrate, and the corresponding second location is located below a corresponding micro-object in the plurality of first locations.

In some embodiments, the method may further include the step of shifting positions of DEP forces generated adjacent to the plurality of micro-objects by moving the imaged structured light from a plurality of first positions on the substrate surface to a plurality of corresponding third positions on the substrate surface using an optical device.

In some embodiments, the method may further include the step of capturing an image of at least a portion of the housing, the step including imaging only the flow region within the imaged portion of the housing and the interior region of each isolator dock, thereby reducing overall noise to achieve high image quality. In some embodiments, the method may further include the step of analyzing the image to provide feedback and adjustments to the first position.

Disclosed herein is a method for imaging one or more micro-objects of a sample. The method may include loading the sample containing the one or more micro-objects into a microfluidic device having a housing including a flow region.

The method may include capturing a plurality of images of at least a portion of the housing containing the one or more micro-objects using a plurality of corresponding illumination patterns projected into the at least a portion of the housing, wherein each illumination pattern of the plurality of illumination patterns is generated using structured light and is different from other illumination patterns of the plurality of illumination patterns, and wherein the plurality of images are captured using an optical device.

The optical device may include a first light source, a structured light modulator, a first and second tube lenses, an objective lens, a dichroic beam splitter, and an image sensor. The structured light modulator is configured to receive unstructured light from the first light source and transmit structured light corresponding to any one of the plurality of illumination patterns. The first tube lens is configured to capture structured light from the structured light modulator and transmit the structured light to the objective lens. The objective lens is configured to receive structured light transmitted from the first tube lens and the protrusion, and the objective lens is further configured to receive light reflected or emitted from at least a portion of the housing. The dichroic beam splitter is located between the first tube lens and the objective lens, and is configured to transmit the structured light received from the first tube lens to the objective lens and reflect light received from the objective lens to the second tube lens. The second tube lens is configured to receive reflected light from the dichroic beam splitter and transmit the reflected light to the image sensor. The image sensor is configured to receive reflected light from the second tube lens and record an image therefrom. The method may further include combining the plurality of images to produce a single image of the one or more micro-objects located in the portion of the housing, wherein the combining step includes processing each of the plurality of images to remove out-of-focus background light.

In some embodiments, the microfluidic device comprises a flow region, and wherein the one or more micro-objects are located in the flow region. In some embodiments, the microfluidic device comprises a flow region and a plurality of isolation docks, each isolation dock in the plurality of isolation docks being fluidically connected to the flow region, and wherein the one or more micro-objects are located in one or more of the plurality of isolation docks and/or the flow region.

In some embodiments, a plurality of corresponding illumination patterns projected into at least a portion of the housing and a corresponding image captured at the image sensor are simultaneously focused. In some embodiments, the plurality of corresponding illumination patterns are configured to scan the field of view within the housing.

Although particular embodiments of the disclosed invention have been shown and described herein, it will be understood by those skilled in the art that they are not intended to limit the invention, and that various changes and modifications (for example, the dimensions of the various parts) may be made without departing from the scope of the disclosed invention, which will be defined only by the appended claims and their equivalents. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

Description of embodiments of the present disclosure.

1. An optical apparatus for imaging a micro-object in a housing of a microfluidic device, the optical apparatus comprising:

a structured light modulator configured to receive an unstructured light beam from a first light source and reflect or transmit a structured light beam suitable for illuminating a micro-object located in a housing of a microfluidic device;

a first tube lens configured to capture and transmit the structured light beam from the structured light modulator;

an objective lens configured to capture and transmit an image beam from a field of view encompassing at least a portion of the housing of the microfluidic device;

a first dichroic beam splitter configured to receive and reflect or transmit the structured beam from the first tube lens, and further configured to receive and transmit or reflect the image beam from the objective lens;

a second tube lens configured to receive and transmit the image beam from the first dichroic beam splitter; and

An image sensor is configured to receive the image light beam from the second tube lens, wherein the image sensor forms an image of the field of view based on the image light beam received from the second tube lens.

2. The optical device of embodiment 1, wherein the structured light modulator comprises an active area of at least 15 mm. In some embodiments, the structured light modulator can comprise an active area of at least 15.5 mm, 16.0 mm, 16.5 mm, 17.0 mm, or more.

3. The optical device of embodiment 1 or 2, wherein the first tube lens has a clear aperture of at least 45 mm.

4. The optical device of embodiment 3, wherein the first tube lens has a clear aperture configured to capture substantially all light beams from the structured light modulator (eg, all or substantially all structured light beams from the structured light modulator).

5. The optical device of any one of embodiments 1 to 4, wherein the first tube lens has an effective focal length of approximately 162 mm (eg, 162 mm +/- 0.8 mm) or less.

6. The optical device of any one of embodiments 1 to 4, wherein the first tube lens has an effective focal length of approximately 155 mm (eg, 155 +/- 0.8 mm).

7. The optical device of any one of embodiments 1 to 5, wherein the first tube lens has a numerical aperture of about 0.071 to about 0.085. In some embodiments, the first tube lens can have a numerical aperture of about 0.074 to about 0.082 or about 0.076 to about 0.080.

8. The optical device of any one of embodiments 1 to 7, wherein the second tube lens has an effective focal length of 180 mm +/- 0.9 mm (or greater).

9. The optical device of any one of embodiments 1 to 7, wherein the second tube lens has an effective focal length of 200 mm +/- 1 mm.

10. The optical device of any one of embodiments 1 to 9, wherein the second tube lens has a numerical aperture of about 0.063 to about 0.077. In some embodiments, the second tube lens can have a numerical aperture of about 0.066 to about 0.074 or about 0.068 to about 0.072.

11. The optical device of any one of embodiments 1 to 10, wherein the image sensor comprises an active area of at least 16.5 mm. In some embodiments, the image sensor can comprise an active area of at least 17.0 mm, 17.5 mm, 18.0 mm, 18.5 mm, 19.0 mm, or more.

12. The optical device of any one of embodiments 1 to 11, wherein the device is characterized by an aperture stop at the back surface of the objective lens, wherein the aperture stop is at least 25 mm. In some embodiments, the aperture stop is at least 26 mm, 27 mm, 28 mm, 29 mm or more, or 24 mm to 26 mm.

13. An optical device according to any one of embodiments 1 to 12, wherein the first dichroic beam splitter is configured to (i) reflect the light beam from the first tube lens to the objective lens, and (ii) transmit the light beam from the objective lens to the second tube lens.

14. An optical device according to any one of embodiments 1 to 12, wherein the first dichroic beam splitter is configured to (i) transmit the light beam from the first tube lens to the objective lens, and (ii) reflect the light beam from the objective lens to the second tube lens.

15. An optical device according to any one of embodiments 1 to 14, wherein the objective lens is configured to minimize aberrations in the image of the field of view formed by the image sensor.

16. The optical device of embodiment 16, wherein the second tube lens is configured to correct residual aberrations of the objective lens.

17. The optical device according to embodiment 15 or 16 further includes a correction lens configured to correct residual aberrations of the objective lens.

18. The optical device of any one of embodiments 1 to 17, wherein the structured light modulator is disposed at a conjugate plane of the image sensor.

19. An optical device according to any one of embodiments 1 to 18, wherein the device is configured to perform confocal imaging.

20. The optical device according to any one of embodiments 1 to 17 further includes a sliding lens, which is slidably positioned between the structured light modulator and the first tube lens, wherein the sliding lens is configured to support a stacked microscopy technique.

21. The optical device according to any one of embodiments 1 to 20, further comprising a first light source.

22. The optical device of claim 21, wherein the first light source has a power of at least 10 watts.

23. An optical device according to embodiment 21 or 22, wherein the structured light beam reflected or transmitted by the structured light modulator is suitable for selectively activating one or more of a plurality of dielectrophoresis (DEP) electrodes on the surface of the substrate of the microfluidic device or composed of the surface of the substrate of the microfluidic device.

24. An optical device according to claim 21 or 22, further comprising a second light source (eg, an LED or a laser).

25. An optical device according to embodiment 24, wherein the second light source is configured to provide unstructured bright field illumination.

26. An optical device according to embodiment 24 or 25, wherein the second light source comprises a laser.

27. The optical device of any one of embodiments 1 to 26, further comprising a second dichroic beam splitter. (For example, the second dichroic beam splitter can be configured to reflect the structured light beam from the structured light modulator to the first tube lens; alternatively, the second dichroic beam splitter can also transmit an unstructured light beam from a second light source to the first tube lens.)

28. The optical device according to any one of embodiments 24 to 27 further includes a third light source.

29. An optical device according to embodiment 28, wherein the third light source comprises a laser, and optionally, wherein the laser of the third light source is configured to heat the inner surface of the microfluidic device and/or the liquid medium located within the housing of the microfluidic device (for example, the laser can be configured to heat a sufficient amount to generate bubbles within the housing of the microfluidic device).

30. The optical apparatus of any one of embodiments 1 to 29, further comprising a nest, wherein the nest is configured to hold the microfluidic device.

31. The optical apparatus of embodiment 30, wherein the nest is further configured to provide at least one electrical connection to the microfluidic device.

32. The optical apparatus of embodiment 30 or 31, wherein the nest is further configured to provide a fluid connection to the microfluidic device.

33. An optical device as described in any of embodiments 1 to 32, wherein the microfluidic device includes a cover, the cover includes glass, and wherein the cover has a thickness of about 600 microns or greater (for example, the cover may have a thickness of about 600 microns to about 1000 microns, about 625 microns to about 850 microns, or about 640 microns to about 700 microns).

34. The optical device according to any one of embodiments 1 to 33 further includes a control unit for providing instructions to the structured light modulator, wherein the instructions cause the structured light modulator to generate one or more illumination patterns.

35. The optical device of claim 34, wherein the illumination pattern varies over time (e.g., a first pattern is replaced by a second pattern, a second pattern is replaced by a third pattern, etc., such that the pattern appears to move as a function of time).

36. A system for imaging micro-objects, the system comprising:

a microfluidic device comprising a housing, wherein the housing comprises a substrate having a plurality of dielectrophoresis (DEP) electrodes disposed on or consisting of a surface of the substrate;

an optical device configured to image the micro-objects in the housing of the microfluidic device, the optical device comprising:

a structured light modulator configured to receive unstructured light from a first light source and reflect or transmit a structured light beam suitable for illuminating a micro-object located in a housing of the microfluidic device;

a first tube lens configured to capture and transmit the structured light beam from the structured light modulator;

an objective lens configured to capture and transmit an image beam from a field of view encompassing at least a portion of the housing of the microfluidic device;

a first dichroic beam splitter configured to receive and reflect or transmit the structured beam from the first tube lens, and further configured to receive and transmit or reflect the image beam from the objective lens;

a second tube lens configured to receive and transmit the image beam from the first dichroic beam splitter;

an image sensor configured to receive the image beam from the second tube lens, wherein the image sensor forms an image of the field of view based on the image beam received from the second tube lens; and

A nest is provided for holding the microfluidic device in a position that allows the microfluidic device to be imaged by the optical apparatus.

37. A system according to embodiment 36, wherein the optical device is configured according to any one of embodiments 2 to 29.

38. The system of embodiment 36 or 37, wherein the nest provides at least one electrical connection to the microfluidic device.

39. The system of any one of embodiments 36 to 38, wherein the nest provides a fluid connection to the microfluidic device.

40. The system of any one of embodiments 36 to 39, further comprising a control unit for providing instructions to the structured light modulator, wherein the instructions cause the structured light modulator to generate one or more illumination patterns.

41. The system of claim 40, wherein the illumination pattern varies over time (e.g., a first pattern is replaced by a second pattern, the second pattern is replaced by a third pattern, etc., such that the pattern appears to move as a function of time).

42. A method of manipulating one or more micro-objects of a sample, the method comprising:

loading the sample containing the one or more micro-objects into a microfluidic device having a housing comprising a substrate, wherein the substrate comprises a plurality of light-actuated dielectrophoresis (DEP) electrodes located on or consisting of a surface of the substrate;

applying a voltage potential across the microfluidic device;

The method comprises: projecting structured light onto a first location on a surface of a substrate of a microfluidic device using an optical device to selectively activate a DEP force adjacent to at least one micro-object located within the microfluidic device, wherein the first location includes one or more of the plurality of light-actuated DEP electrodes and is located near a second location on the surface of the substrate, the second location being located below the at least one micro-object, and wherein the optical device comprises: a first light source; a structured light modulator configured to receive an unstructured light beam from the first light source and transmit a structured light beam suitable for selectively activating the one or more DEP electrodes at the first location on the surface of the substrate of the microfluidic device; a first tube lens configured to capture and transmit the structured light beam from the structured light modulator; and an objective lens configured to capture the structured light beam transmitted from the first tube lens and transmit the structured light beam to the objective lens. the structured light beam being projected onto the first location on the surface of the substrate of the microfluidic device, and wherein the objective lens is further configured to capture and transmit an image light beam reflected or emitted from a field of view encompassing at least a portion of the housing of the microfluidic device, the field of view surrounding the first location and the second location on the surface of the substrate; a first dichroic beam splitter configured to reflect or transmit the structured light beam received from the first tube lens to the objective lens, and further configured to transmit or reflect the image light beam received from the objective lens; a second tube lens configured to receive and transmit the image light beam from the first dichroic beam splitter; and an image sensor configured to receive the image light beam from the second tube lens, wherein the image sensor records an image of the field of view based on the image light beam received from the second tube lens; and

The position of the DEP force generated adjacent to at least one micro-object is shifted by moving the projected structured light from the first position on the surface of the substrate of the microfluidic device to a third position on the surface of the substrate using the optical device, wherein the third position also includes one or more of the plurality of light-actuated DEP electrodes.

43. The method of claim 42, wherein the third position is surrounded by the field of view.

44. A method according to embodiment 42 or 43, wherein the third position overlaps with or surrounds the second position.

45. The method of any one of embodiments 42 to 44 further comprises recording an image of the field of view using the image sensor.

46. The method of any one of embodiments 42 to 45, wherein the housing of the microfluidic device comprises a flow region, and at least one isolation dock is fluidically connected to the flow region.

47. The method of embodiment 46, wherein the field of view surrounds an isolation dock in the at least one isolation dock and at least a portion of the flow area.

48. A method according to any one of embodiments 42 to 47, wherein the optical device includes a second light source that generates unstructured light, and wherein the method further comprises: using the optical device to project the unstructured light from the second light source into the housing of the microfluidic device, thereby providing bright field illumination within the housing.

49. A method according to any one of embodiments 42 to 48, wherein the optical device includes a laser light source, and wherein the method further comprises: using the optical device to project laser light from the laser light source onto a surface within the housing of the microfluidic device (e.g., a fourth position on the substrate surface).

50. A method according to any one of embodiments 42 to 49, wherein the optical device further includes a second dichroic beam splitter positioned between the structured light modulator and the first tube lens, and wherein the structured light beam transmitted by the structured light modulator is reflected by the second dichroic beam splitter into the first tube lens.

51. The method of embodiment 50, wherein the unstructured light generated by the second light source is transmitted to the first tube lens through the second dichroic beam splitter.

52. The method of embodiment 50 or 51, wherein the laser light generated by the laser light source is transmitted to the first tube lens through the second dichroic beam splitter.

53. A method according to any one of embodiments 42 to 52, wherein the structured light projected onto the first position on the substrate surface includes a plurality of illumination points.

54. A method according to embodiment 46, wherein the first position on the substrate surface is located in the flow area of the microfluidic device, and wherein the third position on the substrate surface is located within one of the isolation docks in the plurality of isolation docks.

55. The method of any one of embodiments 42 to 54, wherein the structured light projected onto the first position on the substrate surface comprises a shape resembling a line segment or a symbol.

56. The method of claim 55, wherein the structured light projected onto the first location on the substrate surface has a shape similar to the outline of a polygon. In some embodiments, the shape can have the outline of a quadrilateral polygon, such as a square, rectangle, diamond, or pentagon.

57. The method of any one of embodiments 42 to 56, further comprising:

selectively activating a DEP force adjacent to a plurality of micro-objects located within the microfluidic device by projecting structured light onto a plurality of first locations on the surface of the substrate of the microfluidic device using the optical apparatus, wherein each of the plurality of first locations comprises one or more of the plurality of light-actuated DEP electrodes and is located adjacent to a corresponding second location on the surface of the substrate, the corresponding second location being located beneath the plurality of micro-objects; and

The positions of the DEP forces generated adjacent to the plurality of micro-objects are shifted by moving the projected structured light from the plurality of first positions on the substrate surface to a plurality of corresponding third positions on the substrate surface using the optical device.

58. The method of claim 47, wherein recording an image of the field of view comprises imaging only the flow region and an interior region of each isolation dock located in the field of view (e.g., thereby reducing overall noise to achieve high image quality).

59. The method of claim 45, further comprising analyzing the recorded images to provide feedback and adjustments to the first position.

60. A method of imaging one or more micro-objects of a sample, the method comprising:

loading a sample comprising the one or more micro-objects into a housing of a microfluidic device;

capturing a plurality of images of a field of view encompassing at least a portion of the housing containing the one or more micro-objects using a plurality of corresponding illumination patterns projected into the field of view, wherein each illumination pattern in the plurality of illumination patterns is generated using structured light and is different from other illumination patterns in the plurality of illumination patterns, and wherein the plurality of images are captured using an optical device comprising: a first light source; a structured light modulator configured to receive an unstructured light beam from the first light source and to transmit a structured light beam corresponding to any one of the plurality of illumination patterns; a first tube lens configured to capture and transmit the structured light beam from the structured light modulator; and an objective lens configured to capture the structured light beam transmitted from the first tube lens. and projecting the structured light beam into the at least a portion of the housing of the microfluidic device surrounded by the field of view, wherein the objective lens is further configured to receive an image light beam reflected or emitted from within the field of view; a first dichroic beam splitter configured to reflect or transmit the structured light beam received from the first tube lens to the objective lens, and further configured to transmit or reflect the image light beam received from the objective lens; a second tube lens configured to receive and transmit the image light beam from the first dichroic beam splitter; and an image sensor configured to receive the image light beam from the second tube lens, wherein the image sensor records an image of the field of view based on the image light beam received from the second tube lens; and

The plurality of digital images are combined to generate a confocal image of the one or more micro-objects located in the field of view, wherein the combining step includes processing each of the plurality of images to remove out-of-focus background light.

61. The method of embodiment 60, wherein the microfluidic device comprises a flow region, and wherein the one or more micro-objects are located in the flow region.

62. A method according to embodiment 60, wherein the microfluidic device includes a flow area and a plurality of isolation docks, each isolation dock in the plurality of isolation docks is fluidically connected to the flow area, and wherein the one or more micro-objects are located in one or more of the plurality of isolation docks and/or the flow area.

63. A method according to any one of embodiments 60 to 62, wherein the multiple corresponding illumination patterns projected into the field of view and the corresponding images captured at the image sensor are focused simultaneously.

64. A method according to any one of embodiments 60 to 63, wherein the plurality of corresponding illumination patterns are configured to scan through the field of view.

65. A tube lens of an optical device for imaging micro-objects in a microfluidic device, the tube lens comprising:

a first surface having a convex shape and a first radius of curvature;

a second surface having a second radius of curvature;

a third surface having a concave shape and a third radius of curvature;

a fourth surface having a concave shape and a fourth radius of curvature; and

A clear aperture of at least 45 mm in diameter;

wherein the first radius of curvature is positive, the third radius of curvature is negative, and the fourth radius of curvature is negative, and wherein the front and back focal points of the tube lens are not equidistantly spaced from a midpoint of the tube lens and/or are asymmetric.

66. The tube lens of embodiment 65, wherein the back focal length (BFL) of the tube lens is minimized.

67. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 155 mm (e.g., 155 mm +/- 1 mm) and a back focal length (BFL) of approximately 135 mm (e.g., 135 mm +/- 1 mm).

68. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 162 mm (e.g., 162 mm +/- 1 mm) and a back focal length (BFL) of approximately 146 mm (e.g., 146 mm +/- 1 mm).

69. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 180 mm (e.g., 180 mm +/- 1 mm) and a back focal length (BFL) of approximately 164 mm (e.g., 164 mm +/- 1 mm).

70. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 200 mm (e.g., 200 mm +/- 1 mm) and a back focal length (BFL) of approximately 191 mm (e.g., 191 mm +/- 1 mm).

71. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 155 mm (e.g., 155 mm +/- 0.78 mm), wherein the first radius of curvature is approximately 91 mm (e.g., 91 mm +/- 0.45 mm), the second radius of curvature is approximately 42 mm (e.g., 42 mm +/- 0.21 mm), the third radius of curvature is approximately -62 mm (e.g., -62 mm +/- 0.31 mm), and the fourth radius of curvature is approximately -116 mm, e.g., approximately -116 mm (-116 mm +/- 0.58 mm).

72. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 162 mm (e.g., 162 mm +/- 0.81 mm), wherein the first radius of curvature is approximately 95 mm (e.g., 95 mm +/- 0.48 mm), the second radius of curvature is approximately 54 mm (e.g., 54 mm +/- 0.27 mm), the third radius of curvature is approximately -56 mm (e.g., -56 mm +/- 0.28 mm), and the fourth radius of curvature is approximately -105 mm (e.g., -105 mm +/- 0.53 mm).

73. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 180 mm (e.g., 180 mm +/- 0.90 mm), wherein the first radius of curvature is approximately 95 mm (e.g., 95 mm +/- 0.48 mm), the second radius of curvature is approximately 64 mm (e.g., 64 mm +/- 32 mm), the third radius of curvature is approximately -60 mm (e.g., -60 mm +/- 0.30 mm), and the fourth radius of curvature is approximately -126 mm (e.g., -126 mm +/- 0.63 mm).

74. A tube lens according to embodiment 65, wherein the tube lens has an effective focal length (EFL) of approximately 200 mm (e.g., 200 mm +/- 1.0 mm), wherein the first radius of curvature is approximately 160 mm (e.g., 160 mm +/- 0.80 mm), the second radius of curvature is approximately -62 mm (e.g., -62 mm +/- 0.31 mm), the third radius of curvature is approximately -80 mm (e.g., -80 mm +/- 0.40 mm), and the fourth radius of curvature is approximately -109 mm (e.g., -109 mm +/- 0.55 mm).

75. A method of imaging one or more micro-objects of a sample, the method comprising:

loading a sample comprising the one or more micro-objects into a housing of a microfluidic device;

The method further comprises capturing a plurality of images of a field of view including at least a portion of the housing containing the one or more micro-objects using a corresponding plurality of light illumination angles projected into the field of view, wherein the plurality of images are captured using an optical device comprising: a first light source; a structured light modulator configured to receive an unstructured light beam from the first light source and transmit a structured light beam corresponding to any one of the plurality of illumination patterns; a first tube lens configured to capture and transmit the structured light beam from the structured light modulator; and an objective lens configured to capture the structured light beam transmitted from the first tube lens and project the structured light beam onto all portions of the microfluidic device surrounded by the field of view. wherein the objective lens is further configured to receive an image beam reflected or emitted from within the field of view; a first dichroic beam splitter is configured to reflect or transmit the structured light beam received from the first tube lens to the objective lens, and is further configured to transmit or reflect the image beam received from the objective lens; a second tube lens is configured to receive and transmit the image beam from the first dichroic beam splitter; an image sensor is configured to receive the image beam from the second tube lens; and a sliding lens is positioned between the structured light modulator and the first tube lens, wherein the sliding lens is configured to support a stacked microscopy technique; and

The plurality of captured images are iteratively combined to produce a composite image having a higher resolution than any of the captured images.

76. A method according to embodiment 75, wherein the microfluidic device includes a flow area, and wherein the one or more micro-objects are located in the flow area.

77. A method according to embodiment 75, wherein the microfluidic device includes a flow area and a plurality of isolation docks, each isolation dock in the plurality of isolation docks is fluidically connected to the flow area, and wherein the one or more micro-objects are located in one or more of the plurality of isolation docks and/or the flow area.

78. The method of any one of embodiments 75 to 77, wherein the plurality of captured images comprises at least 8 images. In some embodiments, the plurality of captured images comprises at least 10, 12, 16, 20, 24, or more images.

79. The method of any one of embodiments 75 to 78, wherein the plurality of light illumination angles are generated by structured light from corresponding plurality of different portions of the structured light modulator.

80. The method of embodiment 79, wherein the different portions of the structured light modulator are non-overlapping (or substantially non-overlapping).

Claims (33)

1. An optical device for imaging micro-objects within the housing of a microfluidic device, the optical device comprising: A structured light modulator is configured to receive an unstructured light beam from a first light source and reflect or transmit a structured light beam suitable for illuminating micro-objects located in the housing of the microfluidic device. A first barrel lens is configured to capture and transmit the structured light beam from the structured light modulator, including... The first surface has a raised shape and a first radius of curvature; The second surface has a second radius of curvature; The third surface has a concave shape and a third radius of curvature; A fourth surface, having a concave shape and a fourth radius of curvature; and A light-transmitting aperture with a diameter of at least 45 mm; Wherein the first radius of curvature is positive, the third radius of curvature is negative, and the fourth radius of curvature is negative, and The front focal point and rear focal point of the tube lens are not equidistant from the midpoint of the tube lens; The objective lens is configured to capture and transmit an image beam from a field of view including at least a portion of the housing of the microfluidic device; The first dichroic beam splitter is configured to receive and reflect or transmit the structure beam from the first barrel lens, and is also configured to receive and transmit or reflect the image beam from the objective lens. A second barrel lens is configured to receive and transmit the image beam from the first dichroic beam splitter; and An image sensor is configured to receive the image beam from the second lens barrel, wherein the image sensor forms an image of the field of view based on the image beam received from the second lens barrel. 2. The optical device according to claim 1, wherein the structured light modulator has an effective area of at least 15 mm. 3. The optical device according to claim 1, wherein the first barrel lens has an effective focal length of 162 mm or less. 4. The optical device according to claim 1, wherein the first barrel lens has an effective focal length of 155 mm. 5. The optical device according to claim 4, wherein the first barrel lens has a numerical aperture of 0.071 to 0.085. 6. The optical device according to claim 1, wherein the second barrel lens has an effective focal length of 180 mm or longer. 7. The optical device according to claim 6, wherein the second barrel lens has an effective focal length of 200 mm. 8. The optical device according to claim 1, wherein the second barrel lens has a numerical aperture of 0.063 to 0.077. 9. The optical device of claim 1, wherein the image sensor comprises an effective area of at least 18.0 mm. 10. The optical device according to claim 1, wherein the device is characterized by an aperture stop at the back of the objective lens, wherein the aperture stop is at least 25 mm. 11. The optical device of claim 1, wherein the objective lens is configured to minimize aberrations in the image of the field of view formed by the image sensor. 12. The optical device of claim 11, wherein the second barrel lens is configured to correct the residual aberration of the objective lens. 13. The optical device of claim 11, further comprising a correction lens configured to correct residual aberrations of the objective lens. 14. The optical device of claim 1, wherein the structured light modulator is located at the conjugate plane of the image sensor. 15. The optical device according to claim 1, further comprising a first light source. 16. The optical device of claim 15, wherein the first light source has a power of at least 10 watts. 17. The optical device according to claim 15, further comprising a second light source. 18. The optical device of claim 17, wherein the second light source is configured to provide unstructured bright field illumination, or wherein the second light source comprises a laser. 19. The optical device according to claim 1, further comprising a second dichroic beam splitter. 20. The optical device of claim 17 further includes a third light source, wherein the third light source includes a laser, and wherein the laser of the third light source is configured to heat an inner surface of the microfluidic device or a liquid medium located within the housing of the microfluidic device. 21. The optical device of claim 1, further comprising a nest, wherein the nest is configured to retain the microfluidic device, provide at least one electrical connection to the microfluidic device, or provide a fluid connection to the microfluidic device. 22. The optical device of claim 1, further comprising a control unit for providing instructions to the structured light modulator, wherein the instructions cause the structured light modulator to generate one or more illumination patterns. 23. A method for manipulating one or more micro-objects of a sample, the method comprising: The sample containing the one or more micro-objects is loaded into a microfluidic device having a housing including a substrate, wherein the substrate includes a plurality of photoactuated dielectrophoretic (DEP) electrodes disposed on or formed by the surfaces of the substrate. A voltage potential is applied to the microfluidic device; By projecting structured light onto a first location on the surface of a substrate of a microfluidic device using an optical device, a DEP force adjacent to at least one micro-object located within the microfluidic device is selectively activated, wherein the first location includes one or more of the plurality of photoactuated DEP electrodes and is located near a second location on the surface of the substrate, the second location being located below the at least one micro-object, and wherein the optical device comprises: First light source; A structured light modulator is configured to receive an unstructured light beam from the first light source and transmit a structured light beam suitable for selectively activating one or more DEP electrodes at a first location on the surface of the substrate of the microfluidic device. A first barrel lens, configured to capture and transmit the structured light beam from the structured light modulator, and includes: The first surface has a raised shape and a first radius of curvature; The second surface has a second radius of curvature; The third surface has a concave shape and a third radius of curvature; A fourth surface, having a concave shape and a fourth radius of curvature; and A light-transmitting aperture with a diameter of at least 45 mm; Wherein the first radius of curvature is positive, the third radius of curvature is negative, and the fourth radius of curvature is negative, and The front focal point and rear focal point of the tube lens are not equidistant from the midpoint of the tube lens; An objective lens is configured to capture the structured beam transmitted from the first barrel lens and project the structured beam onto a first position on the surface of the substrate of the microfluidic device, wherein the objective lens is further configured to capture and transmit an image beam reflected or emitted from a field of view including at least a portion of the housing containing the microfluidic device, the field of view surrounding the first and second positions on the surface of the substrate. The first dichroic beam splitter is configured to reflect or transmit the structured beam received from the first barrel lens to the objective lens, and is further configured to transmit or reflect the image beam received from the objective lens. The second lens barrel is configured to receive the image beam from the first dichroic beam splitter and transmit the image beam from the first dichroic beam splitter; and An image sensor is configured to receive the image beam from the second lens barrel, wherein the image sensor records an image of the field of view based on the image beam received from the second lens barrel; and The position of the DEP force generated by shifting the projected structured light from a first position on the surface of the substrate of the microfluidic device to a third position on the surface of the substrate by using the optical device, wherein the third position further includes one or more of the plurality of photoactuated DEP electrodes. 24. The optical device of claim 1, wherein the back focal length (BFL) of the barrel lens is minimized. 25. The optical device of claim 1, wherein the barrel lens has an effective focal length (EFL) of 155 mm and a back focal length (BFL) of 135 mm. 26. The optical device of claim 1, wherein the barrel lens has an effective focal length (EFL) of 162 mm and a back focal length (BFL) of 146 mm. 27. The optical device of claim 1, wherein the barrel lens has an effective focal length (EFL) of 180 mm and a back focal length (BFL) of 164 mm. 28. The optical device according to claim 1, wherein the barrel lens has an effective focal length (EFL) of 200 mm and a back focal length (BFL) of 191 mm. 29. The optical device according to claim 25, wherein the effective focal length (EFL) of the lens barrel is in the range of 155 mm +/- 1 mm, and the back focal length (BFL) of the lens barrel is in the range of 135 mm +/- 1 mm. 30. The optical device according to claim 26, wherein the effective focal length (EFL) of the lens barrel is in the range of 162 mm +/- 1 mm, and the back focal length (BFL) of the lens barrel is in the range of 146 mm +/- 1 mm. 31. The optical device according to claim 27, wherein the effective focal length (EFL) of the lens barrel is in the range of 180 mm +/- 1 mm, and the back focal length (BFL) of the lens barrel is in the range of 164 mm +/- 1 mm. 32. The optical device according to claim 28, wherein the effective focal length (EFL) of the lens barrel is in the range of 200 mm +/- 1 mm, and the back focal length (BFL) of the lens barrel is in the range of 191 mm +/- 1 mm. 33. An optical device for imaging micro-objects within the housing of a microfluidic device, the optical device comprising: A structured light modulator is configured to receive an unstructured light beam from a first light source and reflect or transmit a structured light beam suitable for illuminating micro-objects located in the housing of the microfluidic device. The first barrel lens is configured to capture and transmit the structured beam from the structured light modulator; The objective lens is configured to capture and transmit an image beam from a field of view including at least a portion of the housing of the microfluidic device; The first dichroic beam splitter is configured to receive and reflect or transmit the structure beam from the first barrel lens, and is also configured to receive and transmit or reflect the image beam from the objective lens. A second barrel lens is configured to receive and transmit the image beam from the first dichroic beam splitter, wherein the second barrel lens comprises: The first surface has a raised shape and a first radius of curvature; The second surface has a second radius of curvature; The third surface has a concave shape and a third radius of curvature; A fourth surface, having a concave shape and a fourth radius of curvature; and A light-transmitting aperture with a diameter of at least 45 mm; Wherein the first radius of curvature is positive, the third radius of curvature is negative, and the fourth radius of curvature is negative. The front and rear focal points of the tube lens are not equidistant from the midpoint of the tube lens; and An image sensor is configured to receive the image beam from the second lens barrel, wherein the image sensor forms an image of the field of view based on the image beam received from the second lens barrel.