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WO2025006055A1 - Light guide tip for fluorescence microscopes - Google Patents

Light guide tip for fluorescence microscopes Download PDF

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Publication number
WO2025006055A1
WO2025006055A1 PCT/US2024/027054 US2024027054W WO2025006055A1 WO 2025006055 A1 WO2025006055 A1 WO 2025006055A1 US 2024027054 W US2024027054 W US 2024027054W WO 2025006055 A1 WO2025006055 A1 WO 2025006055A1
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WO
WIPO (PCT)
Prior art keywords
light
light guide
mirror
light beam
guide tip
Prior art date
Application number
PCT/US2024/027054
Other languages
French (fr)
Inventor
Christopher Thomas BAUMANN
Bryan William Heck
Joel Carter Smith
Paul Maddox
Original Assignee
Mizar Imaging, Llc
The University Of North Carolina At Chapel Hill
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mizar Imaging, Llc, The University Of North Carolina At Chapel Hill filed Critical Mizar Imaging, Llc
Publication of WO2025006055A1 publication Critical patent/WO2025006055A1/en

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Classifications

    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/0004Microscopes specially adapted for specific applications
    • G02B21/002Scanning microscopes
    • G02B21/0024Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052Optical details of the image generation
    • G02B21/0076Optical details of the image generation arrangements using fluorescence or luminescence
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00Microscopes
    • G02B21/16Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
    • G01N21/6456Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458Fluorescence microscopy

Definitions

  • This disclosure generally relates to fluorescence microscopes and, more particularly, to light guide tips for fluorescence microscopes.
  • LSFM Light Sheet Fluorescence Microscopy
  • LSFM light sheet fluorescence microscope
  • sample containers may include different materials (e.g., materials with different refraction indexes), shapes, sizes (e.g., thickness), and so on, which then requires readjustment of settings when there is a sample change. Additional variations may be further introduced due to likely manufacturing defects in mass production. For example, the sidewall of a container may not have the same thickness and curvature throughout the sidewall. Furthermore, handling of the sample containers may also introduce certain variations, e.g., fingerprints on the sidewall. All of these variations may increase the complexity of manipulating the LSFM in sample detection.
  • the existing LSFM may be not purported for sample imaging for samples included in multi-well cell culture plates.
  • many wells are not on the edge of the plate and thus may be not accessible by the LSFM from the sidewall.
  • a cell culture plate may have an opaque sidewall, which further prevents the application of LSFM in sample detection in such a cell culture plate, even for a well located along the edge of the plate.
  • the disclosure describes methods and systems for imaging a sample using light guide tips configured for fluorescence microscopes.
  • the light guide tips can be placed inside a well of a cell culture plate, which then does not require an illuminating light sheet to pass through a sidewall of the well in sample detection. This then improves the accessibility, consistency, and efficiency of sample detection using fluorescence microscopes.
  • the disclosure provides a method for imaging a sample using fluorescence microscopy.
  • the method includes positioning a light guide tip, having a first mirror, over the sample.
  • the method further includes directing a light beam into the light guide tip. where at a first time instance, a first portion of the light beam is directed towards the first mirror, and where the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample.
  • the method further includes receiving, at an objective lens, the fluorescent emission for imaging the sample.
  • the disclosure provides a system for imaging a sample using fluorescence microscopy.
  • the system includes a light source configured to propagate a light beam, and an illumination system including a light guide tip.
  • the light guide tip is positioned over a sample.
  • a first portion of the light beam is directed towards a first mirror included in the light guide tip. and the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample.
  • the example system further includes an objective lens for receiving the fluorescent emission for imaging the sample.
  • the disclosure provides an illumination system for imaging a sample using fluorescence microscopy.
  • the illumination system includes a light guide tip in one of a column or cylinder shape, the light guide tip including a mirror disposed along a bottom edge of the light guide tip and a hemisphere dome disposed on a bottom surface of the light guide tip, where the mirror guides an incoming light beam at a first direction to a second direction perpendicular to the first direction.
  • the illumination system further includes a photomask coupled to the light guide tip, the photomask including an outlet for creating an interference pattern for the incoming light beam.
  • FIG. 1 illustrates an example system for detecting a sample using an existing LSFM, according to one embodiment.
  • FIG. 2 illustrates an example system for detecting a sample using an LSFM including a light guide tip, according to one embodiment.
  • FIG. 3 illustrates a schematic diagram of an illumination system including a light guide tip and a photomask, according to one embodiment.
  • FIG. 4A illustrates an example off-axis paraboloidal mirror for guiding a collimated light sheet, according to one embodiment.
  • FIG. 4B illustrates an example planar mirror for guiding a collimated light sheet in sample detection, according to one embodiment.
  • FIGS. 5A-5B illustrate various example shapes of a light guide tip, according to one embodiment.
  • FIG. 6 illustrates an example photomask, according to one embodiment.
  • FIG. 7 is a flow chart of an example method for imaging a sample using fluorescence microscopy, according to one embodiment.
  • FIGS. 8A-8B illustrate a couple of application scenarios for directing a light beam through a photomask, according to one embodiment.
  • FIG. 1 illustrates an exemplary system 100 for detecting a sample using an existing LSFM.
  • System 100 includes a detection objective 104 of the LSFM.
  • Sample 102 is mounted in view of detection objective 104.
  • a source light 106 is directed toward sample 102 (e.g., along a plane of the interest of the sample) from outside a sample container through the sidewall 110 of the container.
  • the source light 106 may be a light sheet generated through a collimation system of the LSFM, and the focal point of the source light is coplanar with a focal plane of detection objective 104 (or the plane of the interest of the sample) in sample detection. As shown in FIG.
  • two interfaces 112a between air and sidewall 1 10) and 112b (betw een sidewall 110 and cell culture media 114) may be formed along the light path, and refraction generated therefrom may affect the imaging quality and operation of the LSFM in sample detection as described earlier.
  • FIG. 2 illustrates an example system 200 for detecting a sample using an LSFM including a light guide tip, according to one embodiment.
  • the source light is guided through a tip, which changes the direction of the light within the tip, so as to focus the light on the sample without requiring the source light to pass through the sidew all of a sample container.
  • the tip disclosed herein may be also referred to as a “light guide tip.” which may be applied to an LSFM microscope disclosed herein, or may be applied to another different type of microscope that requires to detect the samples across the sidewall of a sample container.
  • the source light 206a/206b may be propagated in a vertical direction from the top of a sample container, which can be guided to change the horizontal direction by the light guide tip.
  • the source light does not need to pass through the interfaces between the air and the sidewall of the sample container or between the sidewall of the sample container and the cell culture media, thereby variations caused by these interfaces and the refraction generated therefrom can be avoided.
  • the refraction generated therefrom may be minimized by selecting the tip materials (e g., certain inertia chemicals) with refraction indexes close to cell culture media and/or air.
  • the tip can be controlled during the manufacturing process to minimize the variation (e.g., by making the tip surface as flat as possible).
  • the tip may be formed in a cylinder shape, another column shape, or other different shapes, which allow the source light to be directed toward an imaging sample 202 from more than one direction, thereby increasing the detection sensitivity in sample detection, as will be described in detail later.
  • different mechanisms/ structures e.g., an off-axis paraboloidal trough mirror or a planar mirror
  • FIG. 3 illustrates a schematic diagram of an example illumination system 300 for an LSFM disclosed herein, according to one embodiment.
  • the illumination system 300 includes a light guide tip 302 configured to change the direction of the source light through light guide structures 310a and 310b (together or individually referred to as “light guide structure 310’').
  • the light guide structure 310 is configured to change the direction of the source light from a first direction (e.g., a direction perpendicular to the substrate of the cell culture plate) to a second direction (e.g., a direction along the plane of the interest of the sample) substantially perpendicular to the first direction.
  • substantially perpendicular includes perfectly perpendicular (i.e., 90 degrees), and also other angles within a small tolerance (e.g., a fraction of a degree, 1. 2, 5, degrees, etc.) of 90 degrees.
  • the first direction is generally a vertical direction and the second direction is generally a horizontal direction substantially perpendicular to the first direction.
  • the first and second directions may be different from the illustrated embodiment, but the two directions are still substantially perpendicular to each other.
  • the first direction may be not exactly the vertical direction but can be in another different direction, at an angle to the horizontal direction, or can even be the horizontal direction.
  • the light guide structure 310 may be configured in different shapes.
  • the source light propagated into the light guide tip is a light sheet collimated in parallel
  • the light guide structure 310 may include an off-axis paraboloidal mirror that not only changes the light direction but also focuses the collimated source light along its height, generating a converging line, or “sheet”, of light used to illuminate the sample.
  • the off-axis paraboloidal mirror may be disposed along the bottom edge of the light guide tip and positioned so that the marginal ray of the converging light closet to the sample surface may travel in parallel to the plane of the interest of the sample (or along the focal plane of the objective lenses).
  • FIG. 4A One exemplary light guiding and converging mechanism is shown in FIG. 4A, which includes an off-axis paraboloidal mirror 402a for guiding and converging the collimated light.
  • the off- axis paraboloidal mirror may be disposed along the bottom edge of the light guide tip and may be positioned to have a focal point of the paraboloidal mirror to be coplanar with the focal plane of the objective lens (or the plane of the interest of the sample).
  • the source light propagated into the light guide tip is not a collimated light but rather a converging light sheet or line.
  • the light guide structure of the light guide tip may then be a planar mirror 402b instead, as shown in FIG. 4B.
  • the planar mirror may be also disposed along the bottom edge of the light guide tip and positioned to form an approximately 45-degree angle (with a tolerance of a fraction of a degree, 1, 2, 6 degrees, etc.), with the focal plane of the objective lens (or the plane of the interest of the sample).
  • the converging light may have one side (e.g., side 404 shown in FIG.
  • the light guide structure may have other different shapes than those shown in FIGS. 4A-4B.
  • the light guide structure may allow the focusing light beam after passing through the light guide structure to be directed toward the sample from different directions in sample detection, thereby increasing the imaging quality in sample detection. For example, by imaging a sample from different directions, it may reduce shadowing, fluorescence polarization, and so on, thereby improving the imaging quality in sample detection.
  • a light guide tip 302 may be a cylinder shape as shown in FIG. 3, and the light guide structure 310 may be also a circular structure (e.g., a circular paraboloidal mirror) that allows the light to be directed toward the sample from surrounding 360 degrees.
  • the light guide structures 310a and 310b are actually different portions of the same circular light guide structure (e.g., a single paraboloidal mirror).
  • the light guide structure 310 may include several off-axis parabolic mirrors (e.g., 4, 8, 16, 32, 64, 128. etc.) arranged in a circular pattern.
  • edges may be formed between these off-axis parabolic mirrors.
  • the light guide tip and the corresponding light guide structure(s) may also have other different shapes, as further illustrated in FIGS. 5A-5B.
  • FIGS. 5A-5B illustrate other example shapes for a light guide tip, according to one embodiment.
  • two light guide tips are depicted, each having a hexagon column shape, where each side of the respective hexagon has the same length.
  • another light guide tip is constructed to have a hexagon shape, where the six sides do not have the same length (e.g., the lengths of two sides are greater than the lengths of the other four sides).
  • the shapes of light guide tips in FIGS. 5A-5B are also provided for exemplary purposes and not for limitation.
  • a light guide tip may also have other different shapes (e.g., an octagon column, an enneagon column, a decagon column, a dodecagon column, a circular column, an ovular column, etc.).
  • other different shapes e.g., an octagon column, an enneagon column, a decagon column, a dodecagon column, a circular column, an ovular column, etc.
  • a light guide structure is generally disposed along the bottom edge of the light guide tip. Accordingly, a light guide structure generally has a shape matching the overall shape of a light guide tip. For example, if a light guide tip is a cylinder, the light guide structure of the tip also has a circular structure (e g., a paraboloidal minor or a plurality of parabolic mirrors arranged in a circular or arcuate pattern). Similarly, if a light guide tip is a hexagon column, the light guide structure of the light guide tip may also have six sides (e.g., having six, or fewer or more mirrors), that each can be an off-axis parabolic mirror or a planar mirror.
  • a circular structure e.g., a paraboloidal minor or a plurality of parabolic mirrors arranged in a circular or arcuate pattern.
  • the light guide structure of the light guide tip may also have six sides (e.g., having six, or fewer or more mirrors), that each can be an off
  • a light guide tip may have a predefined height.
  • the height of the light guide tip may have a predefined value or value range in order for the converging light sheet to be focused on the sample in imaging.
  • a light guide tip may have a flexible height.
  • the source light propagated into the light guide tip is a light sheet that is collimated but not converging (e.g., parallel light as shown in FIG. 4A)
  • the light guide tip may have a flexible height. For instance, as shown in FIG.
  • a light guide tip included in an LSFM be a tall light guide tip or a short light guide tip shown in the figure.
  • a hexagon column may also have a predefined height or height range or may have different heights depending on the source light and/or other design constraints.
  • different sides may have different heights.
  • one side of a hexagon-shaped light guide tip may be taller or shorter than another side of the same light guide tip.
  • it may break the coherence of the source light if the source light is coherent.
  • the source light is incoherent, the different sides of a light guide tip may have the same height.
  • light sources that are relatively incoherent limit their interference to the focal plane of the objective lens of LSFM, while highly coherent sources generate reflections from virtually every dust particle and imperfection in the optical system, and thus are less desirable.
  • a light guide tip may have an empty inside structure. That is, the source light passes through the air within the tip before being directed toward the cell culture media in sample detection.
  • a light guide tip may be filled with certain materials instead, which can be a light-transmitting material and/or non-transmitting materials.
  • the light-transmitting materials it may be selected based on the refraction index of the materials. For example, a light-transmitting material that has a refraction index close to the cell culture media may be selected, to minimize refraction generated in the interface between the light guide tip and cell culture media.
  • non-transmitting materials may be used instead in these sections (e.g., a central portion 312 of the cylinder shape light guide tip 302 shown in FIG. 3).
  • a light guide tip may have a sealed bottom with an open top (unfilled) or a sealed top with filled materials inside the tip.
  • the outside surface of a light guide tip may be further salinized, to increase the hydrophobicity of the tip surface to prevent contamination of the tip by proteins, cellular residues, or other materials inside a cell culture media.
  • the illumination system 300 disclosed herein may further include a photomask 304 that is configured to direct and/or distribute the source light along a light path that matches the shape of the light guide tip and the corresponding light guide structure. For example, before a source light 306 (coherent or incoherent) is propagated into the light guide tip 302, the majority of source light 306 may be blocked by photomask 304 with only a small portion 308 of the source light passing through the photomask.
  • a photomask 304 that is configured to direct and/or distribute the source light along a light path that matches the shape of the light guide tip and the corresponding light guide structure. For example, before a source light 306 (coherent or incoherent) is propagated into the light guide tip 302, the majority of source light 306 may be blocked by photomask 304 with only a small portion 308 of the source light passing through the photomask.
  • photomask 304 disclosed herein includes certain slits that allow at least a portion of the source light to pass through the photomask.
  • these slits may be preferably disposed on the edge of the photomask.
  • these slits may be configured at positions that correspond to the location(s) of the light guide structure(s) 310.
  • photomask 304 may be controlled to spin during sample detection. Accordingly, even though these slits do not form a circular or arcuate pattern when stationary, when the photomask spins, the source light may still form a circular ring after continuously passing through the spinning photomask. For example, as illustrated in FIG. 3, after source light 306 passes through spinning photomask 304, a light in the circular ring shape may be propagated into the light guide tip 302 that also has a circular structure (e.g., cylinder shape).
  • a circular structure e.g., cylinder shape
  • FIG. 6 illustrates an example photomask 604, according to one embodiment.
  • photomask 604 includes a certain number of sets (e.g., four sets) of slits disposed close to the edge of the photomask. These slits allow at least a portion of the source light to pass through, while the remaining portion of the source light is blocked by the non-slit portion of photomask 604.
  • these slits are configured to have a specific pattern and dimensions, so as to create an interfering, diffraction-limited light sheet that lengths the narrow beam “waist’ ’ of the light sheet. In one example, in each set of shts.
  • slits also referred to as “quadruple-slit” shaped for elongating the light sheet, where the slits included in each quadruple-slit are arranged approximately parallel to one another, orthogonal to the incident collimated light, and parallel to the edge of photomask 604, which allows the creation of a set (e.g., four) of interfering, diffraction-limited light sheets.
  • the interference pattern created by the photomask may have a beneficial effect, e.g., creating a pseudo or quasi-non-diffracting beam.
  • the interference pattern created by the photomask may have a moire pattern, which is a type of visual interference pattern that occurs when two similar patterns are overlaid or superimposed on each other with a slight angle or displacement.
  • the resulting pattern is a complex pattern that may appear as a series of dark and light lines, curves, or circles and that may create desirable effects, such as the ability to perform super-resolution imaging (a way to increase resolution beyond the theoretical diffraction limit).
  • the source light may be distributed in a circular pattern that matches the shape of the light guide structure as shown in FIG. 3.
  • a photomask may not have a circular shape as shown in FIG. 6, but rather has a shape that matches the shape of a light guide tip.
  • a photomask may have a hexagon shape that has six sides with the same or different lengths.
  • the photomask may not spin but rather keep static during a sample detection process.
  • a photomask may have differentially added materials, such as quarter wave plates.
  • Galvo scanners are motorized mirror mounts and systems used for light beam (e.g., laser beam) steering or scanning applications. They are ideal for moving small light beams fast, with great accuracy and precision. Galvo scanners are highly dynamic electro-optical components that use a rotatable low-inertia mirror to position a light beam with high precision and repeatability.
  • a Galvo scanner may be used to direct the source light in a pattern that matches the light guide structure of a light guide tip.
  • the Galvo scanner may control the source light to flow according to a circular pattern, a hexagon pattern, or another different pattern, depending on the shape of the light guide structure included in a light guide tip.
  • the mirror included in a Galvo scanner may have a planar surface, so that the Galvo scanner is configured to merely direct the source light into different directions following a predefined pattern.
  • the mirror included in a Galvo scanner may have a curved surface (e.g., an off-axis paraboloidal mirror), which may be configured to converge a parallel light sheet into a converging light sheet when directing the source light according to a predefined pattern.
  • other different mechanisms may be employed to distribute/direct/converge the source light in an LSFM disclosed herein.
  • no photomask, Galvo scanner, or another different component is disposed over the light guide tip as shown in FIG. 3. That is, the source light may be directly propagated into the light guide tip without passing through a photomask. Galvo scanner, or the like.
  • the light guide tip may be filled with nontransmitting materials, which may block the source light from entering into portions of the light guide tip other than the light path.
  • the center dark portion 312 of the light guide tip 302 may be filled with non-transmitting materials, so that only light corresponding to the light guide structures 310a/310b can eventually reach the sample in detection.
  • a light guide tip may further include a hemisphere structure (e.g., a hemisphere dome) 314 disposed on the bottom surface of the light guide tip.
  • a hemisphere structure e.g., a hemisphere dome
  • the configuration of such a structure is to make sure light rays being directed tow ard the sample are generally normal to the hemisphere surface (or the tension of the cell culture media), to minimize the refraction generated in the interface between the light guide tip and the cell culture media.
  • the hemisphere dome 314 may be centered around the focal point of the LSFM.
  • the hemisphere dome 314 is clear and may be made of any appropriate materials, such as glass, plastic, or a polymer (e.g., poly dimethylsiloxane).
  • materials with refraction indexes close to cell culture media may be used instead, to minimize the refraction produced at the interface between the tip and the cell culture media.
  • the hemisphere dome 314 is of a uniform thickness.
  • the hemisphere dome may further include a vent 316 at its top so that the hemisphere can be filled with a cell culture media suitable for live samples.
  • vent 316 may provide space for cell culture media to freely flow inside the vent when a light guide tip moves to different positions in sample detection.
  • vent 316 may have an opening on the top or side surface of the light guide tip. When the opening is disposed on the side surface of a light guide tip, the opening may be located at a position higher than the cell culture media. This may allow air to get into cell culture media, so as to provide air to the live samples if the sample detection takes a long time period to process.
  • an LSFM (or imaging system) disclosed herein may include additional components not described above.
  • an LSFM disclosed herein may further include a detection objective (e.g., objective lens illustrated in FIGS. 1-2) for imaging the samples.
  • the detection objective may be aligned to have an imaging axis perpendicular to the plane of the interest of the sample in sample detection, or the focal plane of the detection objective is coplanar with the plane of the interest of the sample in sample detection.
  • an LSFM disclosed herein may further include a light source for generating a light beam, for example, for emitting a radially symmetric, Gaussian beam, or another different light beam.
  • an LSFM disclosed herein may further include a light sheet collimator that collimates the light beam generated by the light source into a light sheet.
  • the light sheet collimator may include a first off-axis paraboloidal mirror for collimating the light beam on a first axis and a second off-axis paraboloidal mirror for collimating the light beam on a second axis.
  • the collimated light beam may be then propagated into the illumination system of the LSFM. including the photomask and the light guide tip discussed above.
  • the collimated light sheet may be converged first by an optical component before being propagated into the illumination system including the disclosed light guide tip.
  • a light source may be configured to generate a light disc in the disclosed LSFM.
  • the specific mechanism and structure for generating a light disc may refer to U.S. Patent No. US 11,314,074 B2, which is hereby incorporated by reference in its entirety.
  • FIG. 7 is a flow diagram of an example method 700 for imaging a sample using fluorescence microscopy, according to one embodiment.
  • Method 700 includes step 702 for positioning a light guide tip, having a first mirror, over the sample.
  • positing the light guide tip includes positioning the hemisphere dome surrounding the sample.
  • ‘’surrounding” does not mean surrounding the sample on all sides, but rather, on the top side, as shown in FIG. 3.
  • the light guide tip may be controlled to move from the top toward the bottom of the cell culture plate, where the bottom portion of the light guide tip including the hemisphere dome may be immersed into the cell culture media and in contact with (or close to) the bottom surface of the cell culture plate.
  • the imaging sample may be positioned under the hemisphere dome for detection.
  • the light guide tip of the fluorescence microscope may be controlled to move toward the top surface of the microscope slide.
  • Method 700 further includes step 704 for directing a light beam into the light guide tip, where at a first time instance, a first portion of the light beam is directed towards the first mirror, and where the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample.
  • the light beam can be a collimated light beam or a converging light beam.
  • the first portion of the light beam can be partial of the light beam or can be nearly all of the light beam.
  • directing the light beam to the light guide tip includes directing, at the first time instance, a second portion of the light beam towards a second mirror within the light guide tip, w here the second mirror reflects the second portion of the light beam as a second collimated light sheet over the sample. That is, at the first time instance, the light beam may be directed to the first portion of the light beam to the first mirror of the light guide tip and the second portion of the light beam to the second mirror of the light guide tip.
  • the first portion 802a of the light beam may be directed to the first mirror 810a of the light guide tip 808 through the first outlet 806a
  • the second portion 802b of the light beam may be directed to the second mirror 810b of the light guide tip 808 through the second outlet 806b
  • the first outlet 806a and the second outlet 806b may have the slit structures described earlier (e.g., quadruple-slit structures in FIG. 6).
  • the first outlet 806a may include one or more quadruple-slit structures
  • the second outlet 806b may include one or more quadruple-slit structures.
  • method 700 may further include directing, at a second time instance, the first portion of the collimated light towards a second mirror within the light guide tip, where the second mirror reflects the first portion of the collimated light as a second focusing light beam over the sample.
  • a photomask 854 may have a single outlet 856 that spins during the sample examination. Accordingly, at the first time instance (shown on the left of FIG. 8A), the first portion 852a of the light beam is directed to the first mirror 860a of the light guide tip 858. After the photomask spins for a time period, outlet 856 of the photomask 854 moves to a new position as shown on the right side of FIG. 8B. Accordingly, at the second time instance, the second portion 852b of the light beam is directed to the second mirror 860b of the light guide tip 858.
  • the light guide tip includes a plurality' of mirrors (e.g., 4, 8, 16, 32, 64, etc.), including the first and second mirrors described above.
  • a plurality' of mirrors e.g., 4, 8, 16, 32, 64, etc.
  • the plurality of mirrors may be arranged in a circular pattern. Accordingly, when spinning the photomask containing the single outlet (or even multiple outlets), respective reflections of the first portion of the light beam from the plurality of mirrors may create a plurality' of focusing light beams originating in a circular pattern (e.g., forming a light disc), where each focusing light beam is directed over the sample.
  • method 700 further includes step 706 for receiving, at an objective lens, the fluorescent emission for imaging the sample.
  • the objective lens may be positioned under the sample holder and have a focal plane that is coplanar with the plane of the interest of the sample.

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Abstract

Methods and systems relate to imaging a sample using fluorescence microscopy. An example system includes a light source configured to propagate a light beam, and an illumination system comprising a light guide tip. The light guide tip is positioned over a sample. When the light beam is directed into the light guide tip at a first time instance, a first portion of the light beam is directed towards a first mirror included in the light guide tip, and the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample. The example system further includes an objective lens for receiving the fluorescent emission for imaging the sample.

Description

LIGHT GUIDE TIP FOR FLUORESCENCE MICROSCOPES
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/511,524, filed June 30, 2023, the entire contents of which are incorporated by reference herein.
GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under Award Nos. DE- SC0022507 and DE-SC0021598, awarded by U.S. Department of Energy (DoE), Office of Science, Office of Advanced Scientific Computing Research. The government has certain rights in the invention.
TECHNICAL FIELD
[0003] This disclosure generally relates to fluorescence microscopes and, more particularly, to light guide tips for fluorescence microscopes.
BACKGROUND
[0004] Light Sheet Fluorescence Microscopy (LSFM) has emerged as a powerful approach to restricting the excitation volume of fluorescence microscopy by forming a thin pseudo-non-diffracting “sheet” of light. This sheet is typically thinner than samples so that out-of-focus fluorescence is not generated and photobleaching is significantly reduced. With LSFM, cell biologists have been able to drastically extend the imaging lifetime for their living fluorescent organisms without introducing unnecessary photobleaching of fluorophores or phototoxicity of the samples.
[0005] One current obstacle that limits the application of LSFM is its directing of source light toward a sample from outside a sample container through the sidewall of the container. This necessarily introduces two interfaces along the light path from the source light of a light sheet fluorescence microscope (also referred to as “LSFM”) to the sample, including one interface formed between the outside air and the sidewall of the container, and another interface formed between the sidewall and cell culture media inside the container. Due to the likely different refraction indexes between the air and sidewall and between the sidewall and cell culture media, refraction may be generated when the source light passes through these interfaces, which then affects the sample detection by the LSFM. For example, different sample containers may include different materials (e.g., materials with different refraction indexes), shapes, sizes (e.g., thickness), and so on, which then requires readjustment of settings when there is a sample change. Additional variations may be further introduced due to likely manufacturing defects in mass production. For example, the sidewall of a container may not have the same thickness and curvature throughout the sidewall. Furthermore, handling of the sample containers may also introduce certain variations, e.g., fingerprints on the sidewall. All of these variations may increase the complexity of manipulating the LSFM in sample detection.
[0006] In addition, in sample detections by an existing LSFM, when changing the target positions of a sample, due to the movement of the sample container, the ratios between the air and sidewall and/or between the sidewall and cell culture media along the light path may also change, which then requires readjustment of the LSFM after each position change.
[0007] Furthermore, the existing LSFM may be not purported for sample imaging for samples included in multi-well cell culture plates. For example, for a 96-well cell culture plate, many wells are not on the edge of the plate and thus may be not accessible by the LSFM from the sidewall. Under certain circumstances, a cell culture plate may have an opaque sidewall, which further prevents the application of LSFM in sample detection in such a cell culture plate, even for a well located along the edge of the plate.
[0008] Accordingly, there is a need for an improved LSFM to address the above problems and other problems of existing LSFMs.
SUMMARY
[0009] The disclosure describes methods and systems for imaging a sample using light guide tips configured for fluorescence microscopes. The light guide tips can be placed inside a well of a cell culture plate, which then does not require an illuminating light sheet to pass through a sidewall of the well in sample detection. This then improves the accessibility, consistency, and efficiency of sample detection using fluorescence microscopes.
[0010] In one aspect, the disclosure provides a method for imaging a sample using fluorescence microscopy. The method includes positioning a light guide tip, having a first mirror, over the sample. The method further includes directing a light beam into the light guide tip. where at a first time instance, a first portion of the light beam is directed towards the first mirror, and where the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample. The method further includes receiving, at an objective lens, the fluorescent emission for imaging the sample.
[0011] In another aspect, the disclosure provides a system for imaging a sample using fluorescence microscopy. The system includes a light source configured to propagate a light beam, and an illumination system including a light guide tip. The light guide tip is positioned over a sample. When the light beam is directed into the light guide tip at a first time instance, a first portion of the light beam is directed towards a first mirror included in the light guide tip. and the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample. The example system further includes an objective lens for receiving the fluorescent emission for imaging the sample.
[0012] In yet another aspect, the disclosure provides an illumination system for imaging a sample using fluorescence microscopy. The illumination system includes a light guide tip in one of a column or cylinder shape, the light guide tip including a mirror disposed along a bottom edge of the light guide tip and a hemisphere dome disposed on a bottom surface of the light guide tip, where the mirror guides an incoming light beam at a first direction to a second direction perpendicular to the first direction. The illumination system further includes a photomask coupled to the light guide tip, the photomask including an outlet for creating an interference pattern for the incoming light beam.
[0013] The above and other preferred features, including various novel details of implementation and combination of elements, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular methods and apparatuses are shown by way of illustration only and not as limitations. As will be understood by those skilled in the art, the principles and features explained herein may be employed in various and numerous embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The disclosed embodiments have advantages and features which will be more readily apparent from the detailed description, the appended claims, and the accompanying figures (or drawings). A brief introduction of the figures is below.
[0015] FIG. 1 illustrates an example system for detecting a sample using an existing LSFM, according to one embodiment.
[0016] FIG. 2 illustrates an example system for detecting a sample using an LSFM including a light guide tip, according to one embodiment.
[0017] FIG. 3 illustrates a schematic diagram of an illumination system including a light guide tip and a photomask, according to one embodiment.
[0018] FIG. 4A illustrates an example off-axis paraboloidal mirror for guiding a collimated light sheet, according to one embodiment.
[0019] FIG. 4B illustrates an example planar mirror for guiding a collimated light sheet in sample detection, according to one embodiment.
[0020] FIGS. 5A-5B illustrate various example shapes of a light guide tip, according to one embodiment.
[0021] FIG. 6 illustrates an example photomask, according to one embodiment.
[0022] FIG. 7 is a flow chart of an example method for imaging a sample using fluorescence microscopy, according to one embodiment.
[0023] FIGS. 8A-8B illustrate a couple of application scenarios for directing a light beam through a photomask, according to one embodiment.
DETAILED DESCRIPTION
[0024] The Figures (FIGS.) and the following description relate to specific embodiments by way of illustration only. It should be noted that from the following discussion, alternative embodiments of the structures and methods disclosed herein will be readily recognized as viable alternatives that may be employed without departing from the principles of the present disclosure.
[0025] Reference will now be made in detail to several embodiments, examples of w hich are illustrated in the accompanying figures. It is to be noted that wherever practicable, similar or like reference numbers may be used in the figures and may indicate similar or like functionality. The figures depict embodiments of the disclosed systems (or methods) for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles described herein.
[0026] FIG. 1 illustrates an exemplary system 100 for detecting a sample using an existing LSFM. System 100 includes a detection objective 104 of the LSFM. Sample 102 is mounted in view of detection objective 104. A source light 106 is directed toward sample 102 (e.g., along a plane of the interest of the sample) from outside a sample container through the sidewall 110 of the container. The source light 106 may be a light sheet generated through a collimation system of the LSFM, and the focal point of the source light is coplanar with a focal plane of detection objective 104 (or the plane of the interest of the sample) in sample detection. As shown in FIG. 1, two interfaces 112a (between air and sidewall 1 10) and 112b (betw een sidewall 110 and cell culture media 114) may be formed along the light path, and refraction generated therefrom may affect the imaging quality and operation of the LSFM in sample detection as described earlier.
[0027] FIG. 2 illustrates an example system 200 for detecting a sample using an LSFM including a light guide tip, according to one embodiment. In the disclosed system 200, the source light is guided through a tip, which changes the direction of the light within the tip, so as to focus the light on the sample without requiring the source light to pass through the sidew all of a sample container. The tip disclosed herein may be also referred to as a “light guide tip.” which may be applied to an LSFM microscope disclosed herein, or may be applied to another different type of microscope that requires to detect the samples across the sidewall of a sample container. In one example, as shown in FIG. 2, the source light 206a/206b may be propagated in a vertical direction from the top of a sample container, which can be guided to change the horizontal direction by the light guide tip.
[0028] As illustrated in FIG. 2, in the disclosed system 200, the source light does not need to pass through the interfaces between the air and the sidewall of the sample container or between the sidewall of the sample container and the cell culture media, thereby variations caused by these interfaces and the refraction generated therefrom can be avoided.
[0029] It should be noted, that while there are still some interfaces (e.g., interfaces between air and the tip and between the tip and the cell culture media) in the disclosed system 200, the refraction generated therefrom may be minimized by selecting the tip materials (e g., certain inertia chemicals) with refraction indexes close to cell culture media and/or air. In addition, the tip can be controlled during the manufacturing process to minimize the variation (e.g., by making the tip surface as flat as possible).
[0030] In some embodiments, to improve the image quality, the tip may be formed in a cylinder shape, another column shape, or other different shapes, which allow the source light to be directed toward an imaging sample 202 from more than one direction, thereby increasing the detection sensitivity in sample detection, as will be described in detail later. In addition, depending on the source light propagating into the light guide tip, different mechanisms/ structures (e.g., an off-axis paraboloidal trough mirror or a planar mirror) may be used to guide the source light through the light guide tip, as further described in detail below in FIG. 3.
[0031] FIG. 3 illustrates a schematic diagram of an example illumination system 300 for an LSFM disclosed herein, according to one embodiment. As illustrated, the illumination system 300 includes a light guide tip 302 configured to change the direction of the source light through light guide structures 310a and 310b (together or individually referred to as “light guide structure 310’'). For example, the light guide structure 310 is configured to change the direction of the source light from a first direction (e.g., a direction perpendicular to the substrate of the cell culture plate) to a second direction (e.g., a direction along the plane of the interest of the sample) substantially perpendicular to the first direction. As used herein, substantially perpendicular includes perfectly perpendicular (i.e., 90 degrees), and also other angles within a small tolerance (e.g., a fraction of a degree, 1. 2, 5, degrees, etc.) of 90 degrees. In the illustrated embodiment, the first direction is generally a vertical direction and the second direction is generally a horizontal direction substantially perpendicular to the first direction. In actual applications, the first and second directions may be different from the illustrated embodiment, but the two directions are still substantially perpendicular to each other. For example, the first direction may be not exactly the vertical direction but can be in another different direction, at an angle to the horizontal direction, or can even be the horizontal direction.
[0032] In some embodiments, depending on the light source propagated into the light guide tip. the light guide structure 310 may be configured in different shapes. For example, if the source light propagated into the light guide tip is a light sheet collimated in parallel, the light guide structure 310 may include an off-axis paraboloidal mirror that not only changes the light direction but also focuses the collimated source light along its height, generating a converging line, or “sheet”, of light used to illuminate the sample. The off-axis paraboloidal mirror may be disposed along the bottom edge of the light guide tip and positioned so that the marginal ray of the converging light closet to the sample surface may travel in parallel to the plane of the interest of the sample (or along the focal plane of the objective lenses). One exemplary light guiding and converging mechanism is shown in FIG. 4A, which includes an off-axis paraboloidal mirror 402a for guiding and converging the collimated light. The off- axis paraboloidal mirror may be disposed along the bottom edge of the light guide tip and may be positioned to have a focal point of the paraboloidal mirror to be coplanar with the focal plane of the objective lens (or the plane of the interest of the sample).
[0033] In some embodiments, the source light propagated into the light guide tip is not a collimated light but rather a converging light sheet or line. The light guide structure of the light guide tip may then be a planar mirror 402b instead, as shown in FIG. 4B. The planar mirror may be also disposed along the bottom edge of the light guide tip and positioned to form an approximately 45-degree angle (with a tolerance of a fraction of a degree, 1, 2, 6 degrees, etc.), with the focal plane of the objective lens (or the plane of the interest of the sample). The converging light may have one side (e.g., side 404 shown in FIG. 4B) that is incident at an approximately 45-degree angle with the planar mirror 402b, so that the marginal ray 406 on the side of the converging light, after reflection by the planar mirror, may travel substantially along (or substantially in parallel with) the focal plane of the objective lens. In some embodiments, depending on the source light propagated into the light guide tip. the light guide structure may have other different shapes than those shown in FIGS. 4A-4B. [0034] Referring back to FIG. 3. in some embodiments, by properly shaping a light guide structure 310, it may allow the focusing light beam after passing through the light guide structure to be directed toward the sample from different directions in sample detection, thereby increasing the imaging quality in sample detection. For example, by imaging a sample from different directions, it may reduce shadowing, fluorescence polarization, and so on, thereby improving the imaging quality in sample detection.
[0035] In one example, a light guide tip 302 may be a cylinder shape as shown in FIG. 3, and the light guide structure 310 may be also a circular structure (e.g., a circular paraboloidal mirror) that allows the light to be directed toward the sample from surrounding 360 degrees. In such a configuration, light guide structures 310a and 310b are actually different portions of the same circular light guide structure (e.g., a single paraboloidal mirror). In some embodiments, instead of providing a single paraboloidal shaped mirror, the light guide structure 310 may include several off-axis parabolic mirrors (e.g., 4, 8, 16, 32, 64, 128. etc.) arranged in a circular pattern. Compared to the paraboloidal structure that has an overall smooth surface, edges may be formed between these off-axis parabolic mirrors. In some embodiments, the light guide tip and the corresponding light guide structure(s) may also have other different shapes, as further illustrated in FIGS. 5A-5B.
[0036] FIGS. 5A-5B illustrate other example shapes for a light guide tip, according to one embodiment. In FIG. 5A, two light guide tips are depicted, each having a hexagon column shape, where each side of the respective hexagon has the same length. In FIG. 5B, another light guide tip is constructed to have a hexagon shape, where the six sides do not have the same length (e.g., the lengths of two sides are greater than the lengths of the other four sides). It is to be noted that the shapes of light guide tips in FIGS. 5A-5B are also provided for exemplary purposes and not for limitation. In real applications, a light guide tip may also have other different shapes (e.g., an octagon column, an enneagon column, a decagon column, a dodecagon column, a circular column, an ovular column, etc.).
[0037] It should be noted that a light guide structure is generally disposed along the bottom edge of the light guide tip. Accordingly, a light guide structure generally has a shape matching the overall shape of a light guide tip. For example, if a light guide tip is a cylinder, the light guide structure of the tip also has a circular structure (e g., a paraboloidal minor or a plurality of parabolic mirrors arranged in a circular or arcuate pattern). Similarly, if a light guide tip is a hexagon column, the light guide structure of the light guide tip may also have six sides (e.g., having six, or fewer or more mirrors), that each can be an off-axis parabolic mirror or a planar mirror.
[0038] In some embodiments, a light guide tip may have a predefined height. For example, when the source light propagated into the light guide tip is a converging light sheet (as shown in FIG. 4B), the height of the light guide tip may have a predefined value or value range in order for the converging light sheet to be focused on the sample in imaging. In other embodiments, a light guide tip may have a flexible height. For example, when the source light propagated into the light guide tip is a light sheet that is collimated but not converging (e.g., parallel light as shown in FIG. 4A), the light guide tip may have a flexible height. For instance, as shown in FIG. 5A, a light guide tip included in an LSFM be a tall light guide tip or a short light guide tip shown in the figure. Although not shown in FIG. 5B, a hexagon column may also have a predefined height or height range or may have different heights depending on the source light and/or other design constraints.
[0039] In some embodiments, even within a single light guide tip, different sides may have different heights. For example, one side of a hexagon-shaped light guide tip may be taller or shorter than another side of the same light guide tip. In applications, by including different heights within the same light guide tip, it may break the coherence of the source light if the source light is coherent. If the source light is incoherent, the different sides of a light guide tip may have the same height. In general, light sources that are relatively incoherent limit their interference to the focal plane of the objective lens of LSFM, while highly coherent sources generate reflections from virtually every dust particle and imperfection in the optical system, and thus are less desirable.
[0040] Referring back to FIG. 3, in some embodiments, a light guide tip may have an empty inside structure. That is, the source light passes through the air within the tip before being directed toward the cell culture media in sample detection. In some embodiments, a light guide tip may be filled with certain materials instead, which can be a light-transmitting material and/or non-transmitting materials. For the light-transmitting materials, it may be selected based on the refraction index of the materials. For example, a light-transmitting material that has a refraction index close to the cell culture media may be selected, to minimize refraction generated in the interface between the light guide tip and cell culture media. In some embodiments, for sections that are not positioned along the light path within a light guide tip, non-transmitting materials may be used instead in these sections (e.g., a central portion 312 of the cylinder shape light guide tip 302 shown in FIG. 3).
[0041] In some embodiments, a light guide tip may have a sealed bottom with an open top (unfilled) or a sealed top with filled materials inside the tip. In some embodiments, to keep the tip clean during sample detection, the outside surface of a light guide tip may be further salinized, to increase the hydrophobicity of the tip surface to prevent contamination of the tip by proteins, cellular residues, or other materials inside a cell culture media.
[0042] Still referring to FIG. 3, in some embodiments, the illumination system 300 disclosed herein may further include a photomask 304 that is configured to direct and/or distribute the source light along a light path that matches the shape of the light guide tip and the corresponding light guide structure. For example, before a source light 306 (coherent or incoherent) is propagated into the light guide tip 302, the majority of source light 306 may be blocked by photomask 304 with only a small portion 308 of the source light passing through the photomask.
[0043] In one embodiment, photomask 304 disclosed herein includes certain slits that allow at least a portion of the source light to pass through the photomask. In some embodiments, these slits may be preferably disposed on the edge of the photomask. For example, these slits may be configured at positions that correspond to the location(s) of the light guide structure(s) 310. In some embodiments, photomask 304 may be controlled to spin during sample detection. Accordingly, even though these slits do not form a circular or arcuate pattern when stationary, when the photomask spins, the source light may still form a circular ring after continuously passing through the spinning photomask. For example, as illustrated in FIG. 3, after source light 306 passes through spinning photomask 304, a light in the circular ring shape may be propagated into the light guide tip 302 that also has a circular structure (e.g., cylinder shape).
[0044] FIG. 6 illustrates an example photomask 604, according to one embodiment. As illustrated, photomask 604 includes a certain number of sets (e.g., four sets) of slits disposed close to the edge of the photomask. These slits allow at least a portion of the source light to pass through, while the remaining portion of the source light is blocked by the non-slit portion of photomask 604. In some embodiments, these slits are configured to have a specific pattern and dimensions, so as to create an interfering, diffraction-limited light sheet that lengths the narrow beam “waist’ ’ of the light sheet. In one example, in each set of shts. there are four slits (also referred to as “quadruple-slit”) shaped for elongating the light sheet, where the slits included in each quadruple-slit are arranged approximately parallel to one another, orthogonal to the incident collimated light, and parallel to the edge of photomask 604, which allows the creation of a set (e.g., four) of interfering, diffraction-limited light sheets.
[0045] The interference pattern created by the photomask may have a beneficial effect, e.g., creating a pseudo or quasi-non-diffracting beam. In some embodiments, the interference pattern created by the photomask may have a moire pattern, which is a type of visual interference pattern that occurs when two similar patterns are overlaid or superimposed on each other with a slight angle or displacement. The resulting pattern is a complex pattern that may appear as a series of dark and light lines, curves, or circles and that may create desirable effects, such as the ability to perform super-resolution imaging (a way to increase resolution beyond the theoretical diffraction limit).
[0046] It should be noted that, while only four sets of slits (i.e., four quadruple-slits) are disposed in a small portion of the photomask, when the photomask spins, the source light may be distributed in a circular pattern that matches the shape of the light guide structure as shown in FIG. 3.
[0047] It should be also noted that the number of sets of slits and the dimension and patterns of the slits in each set are provided in FIG. 6 for exemplar} purposes. In actual applications, these slits may have other different dimensions, numbers, and patterns. For example, when a light guide tip is a hexagon column, there may be six quadruple-slits, where a quadruple-slit structure may be disposed in positions corresponding to the light guide structures having six different sides. In addition, in some embodiments, a photomask may not have a circular shape as shown in FIG. 6, but rather has a shape that matches the shape of a light guide tip. For example, a photomask may have a hexagon shape that has six sides with the same or different lengths. In some embodiments, if a light guide tip does not have a circular structure, the photomask may not spin but rather keep static during a sample detection process. In addition, although not shown, a photomask may have differentially added materials, such as quarter wave plates.
[0048] In some embodiments, instead of using a photomask, a Galvanometer optical scanner (also called Galvo or Galvo scanner) (and re-collimator lens) or another optical element may be used in distributing/directing the source light. Galvo scanners are motorized mirror mounts and systems used for light beam (e.g., laser beam) steering or scanning applications. They are ideal for moving small light beams fast, with incredible accuracy and precision. Galvo scanners are highly dynamic electro-optical components that use a rotatable low-inertia mirror to position a light beam with high precision and repeatability. In the LSFM disclosed herein, a Galvo scanner may be used to direct the source light in a pattern that matches the light guide structure of a light guide tip. For example, the Galvo scanner may control the source light to flow according to a circular pattern, a hexagon pattern, or another different pattern, depending on the shape of the light guide structure included in a light guide tip. In some embodiments, the mirror included in a Galvo scanner may have a planar surface, so that the Galvo scanner is configured to merely direct the source light into different directions following a predefined pattern. In some embodiments, the mirror included in a Galvo scanner may have a curved surface (e.g., an off-axis paraboloidal mirror), which may be configured to converge a parallel light sheet into a converging light sheet when directing the source light according to a predefined pattern. In some embodiments, other different mechanisms may be employed to distribute/direct/converge the source light in an LSFM disclosed herein.
[0049] In some embodiments, no photomask, Galvo scanner, or another different component is disposed over the light guide tip as shown in FIG. 3. That is, the source light may be directly propagated into the light guide tip without passing through a photomask. Galvo scanner, or the like. In such situations, the light guide tip may be filled with nontransmitting materials, which may block the source light from entering into portions of the light guide tip other than the light path. For example, as shown in FIG. 3, the center dark portion 312 of the light guide tip 302 may be filled with non-transmitting materials, so that only light corresponding to the light guide structures 310a/310b can eventually reach the sample in detection.
[0050] In some embodiments, a light guide tip may further include a hemisphere structure (e.g., a hemisphere dome) 314 disposed on the bottom surface of the light guide tip. The configuration of such a structure is to make sure light rays being directed tow ard the sample are generally normal to the hemisphere surface (or the tension of the cell culture media), to minimize the refraction generated in the interface between the light guide tip and the cell culture media.
[0051] In some embodiments, the hemisphere dome 314 may be centered around the focal point of the LSFM. In some embodiments, the hemisphere dome 314 is clear and may be made of any appropriate materials, such as glass, plastic, or a polymer (e.g., poly dimethylsiloxane). In some embodiments, materials with refraction indexes close to cell culture media may be used instead, to minimize the refraction produced at the interface between the tip and the cell culture media. In some embodiments, the hemisphere dome 314 is of a uniform thickness. In addition, as illustrated in FIG. 3, in some embodiment, the hemisphere dome may further include a vent 316 at its top so that the hemisphere can be filled with a cell culture media suitable for live samples. Under certain circumstances, vent 316 may provide space for cell culture media to freely flow inside the vent when a light guide tip moves to different positions in sample detection. In some embodiments, vent 316 may have an opening on the top or side surface of the light guide tip. When the opening is disposed on the side surface of a light guide tip, the opening may be located at a position higher than the cell culture media. This may allow air to get into cell culture media, so as to provide air to the live samples if the sample detection takes a long time period to process.
[0052] In some embodiments, an LSFM (or imaging system) disclosed herein may include additional components not described above. For example, an LSFM disclosed herein may further include a detection objective (e.g., objective lens illustrated in FIGS. 1-2) for imaging the samples. The detection objective may be aligned to have an imaging axis perpendicular to the plane of the interest of the sample in sample detection, or the focal plane of the detection objective is coplanar with the plane of the interest of the sample in sample detection.
[0053] In addition, an LSFM disclosed herein may further include a light source for generating a light beam, for example, for emitting a radially symmetric, Gaussian beam, or another different light beam. In some embodiments, an LSFM disclosed herein may further include a light sheet collimator that collimates the light beam generated by the light source into a light sheet. The light sheet collimator may include a first off-axis paraboloidal mirror for collimating the light beam on a first axis and a second off-axis paraboloidal mirror for collimating the light beam on a second axis. The collimated light beam may be then propagated into the illumination system of the LSFM. including the photomask and the light guide tip discussed above. In some embodiments, the collimated light sheet may be converged first by an optical component before being propagated into the illumination system including the disclosed light guide tip. In some embodiments, a light source may be configured to generate a light disc in the disclosed LSFM. The specific mechanism and structure for generating a light disc may refer to U.S. Patent No. US 11,314,074 B2, which is hereby incorporated by reference in its entirety. [0054] FIG. 7 is a flow diagram of an example method 700 for imaging a sample using fluorescence microscopy, according to one embodiment.
[0055] Method 700 includes step 702 for positioning a light guide tip, having a first mirror, over the sample. In some embodiments, positing the light guide tip includes positioning the hemisphere dome surrounding the sample. Here, ‘’surrounding” does not mean surrounding the sample on all sides, but rather, on the top side, as shown in FIG. 3. For example, when imaging cells in a cell culture plate, the light guide tip may be controlled to move from the top toward the bottom of the cell culture plate, where the bottom portion of the light guide tip including the hemisphere dome may be immersed into the cell culture media and in contact with (or close to) the bottom surface of the cell culture plate. The imaging sample may be positioned under the hemisphere dome for detection. In conditions where a sample is placed onto a microscope slide, the light guide tip of the fluorescence microscope may be controlled to move toward the top surface of the microscope slide.
[0056] Method 700 further includes step 704 for directing a light beam into the light guide tip, where at a first time instance, a first portion of the light beam is directed towards the first mirror, and where the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample. The light beam can be a collimated light beam or a converging light beam. The first portion of the light beam can be partial of the light beam or can be nearly all of the light beam.
[0057] In some embodiments, directing the light beam to the light guide tip includes directing, at the first time instance, a second portion of the light beam towards a second mirror within the light guide tip, w here the second mirror reflects the second portion of the light beam as a second collimated light sheet over the sample. That is, at the first time instance, the light beam may be directed to the first portion of the light beam to the first mirror of the light guide tip and the second portion of the light beam to the second mirror of the light guide tip.
[0058] For instance, as shown in FIG. 8A, when a photomask 804 has a first outlet 806a and a second outlet 806, at the first time instance, the first portion 802a of the light beam may be directed to the first mirror 810a of the light guide tip 808 through the first outlet 806a, and the second portion 802b of the light beam may be directed to the second mirror 810b of the light guide tip 808 through the second outlet 806b. Here, the first outlet 806a and the second outlet 806b may have the slit structures described earlier (e.g., quadruple-slit structures in FIG. 6). For example, the first outlet 806a may include one or more quadruple-slit structures, and the second outlet 806b may include one or more quadruple-slit structures.
[0059] In some embodiments, method 700 may further include directing, at a second time instance, the first portion of the collimated light towards a second mirror within the light guide tip, where the second mirror reflects the first portion of the collimated light as a second focusing light beam over the sample.
[0060] For instance, as shown in FIG. 8B, a photomask 854 may have a single outlet 856 that spins during the sample examination. Accordingly, at the first time instance (shown on the left of FIG. 8A), the first portion 852a of the light beam is directed to the first mirror 860a of the light guide tip 858. After the photomask spins for a time period, outlet 856 of the photomask 854 moves to a new position as shown on the right side of FIG. 8B. Accordingly, at the second time instance, the second portion 852b of the light beam is directed to the second mirror 860b of the light guide tip 858.
[0061] In some embodiments, the light guide tip includes a plurality' of mirrors (e.g., 4, 8, 16, 32, 64, etc.), including the first and second mirrors described above. For example, in FIG. 8B. there may be a plurality of mirrors 860a . .. 860n. The plurality of mirrors may be arranged in a circular pattern. Accordingly, when spinning the photomask containing the single outlet (or even multiple outlets), respective reflections of the first portion of the light beam from the plurality of mirrors may create a plurality' of focusing light beams originating in a circular pattern (e.g., forming a light disc), where each focusing light beam is directed over the sample.
[0062] In some embodiments, method 700 further includes step 706 for receiving, at an objective lens, the fluorescent emission for imaging the sample. The objective lens may be positioned under the sample holder and have a focal plane that is coplanar with the plane of the interest of the sample.
[0090] Although specific examples and features have been described above, these examples and features are not intended to limit the scope of the present disclosure, even where only a single example is described with respect to a particular feature. Examples of features provided in the disclosure are intended to be illustrative rather than restrictive unless stated otherwise. The above description is intended to cover such alternatives, modifications, and equivalents as would be apparent to a person skilled in the art having the benefit of this disclosure. [0091] The scope of the present disclosure includes any feature or combination of features disclosed in this specification (either explicitly or implicitly), or any generalization of features disclosed, whether or not such features or generalizations mitigate any or all of the problems described in this specification. Accordingly, new claims may be formulated during the prosecution of this application (or an application claiming priority to this application) to any such combination of features. In particular, with reference to the appended claims, features from dependent claims may be combined with those of the independent claims, and features from respective independent claims may be combined in any appropriate manner and not merely in the specific combinations enumerated in the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method for imaging a sample using fluorescence microscopy, the method comprising: positioning a light guide tip, having a first mirror, over the sample; directing a light beam into the light guide tip, wherein at a first time instance, a first portion of the light beam is directed towards the first mirror, and wherein the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample: and receiving, at an objective lens, the fluorescent emission for imaging the sample.
2. The method of claim 1, wherein the light guide tip has one of a column or cylinder shape, and the first mirror is disposed along a bottom edge of the light guide tip.
3. The method of claim 2, wherein the first mirror comprises an off-axis mirror, and the first mirror is disposed along the bottom edge of the light guide tip by positioning the first mirror to have a focal point of the off-axis mirror to be coplanar with a focal plane of the objective lens.
4. The method of claim 2, wherein the first mirror comprises a planar mirror, and the first mirror is disposed along the bottom edge of the light guide tip by positioning the first mirror to form a 45-degree angle with a focal plane of the objective lens.
5. The method of claim 1, wherein the light guide tip comprises a hemisphere dome on a bottom surface of the light guide tip, and wherein positing the light guide tip comprises positioning the hemisphere dome surrounding the sample.
6. The method of claim 1 , wherein directing the light beam comprises directing at the first time instance, a second portion of the light beam towards a second mirror within the light guide tip, and wherein the second mirror reflects the second portion of the light beam as a second collimated light sheet over the sample.
7. The method of claim 6, wherein directing the light beam into the light guide tip comprises directing the light beam to a photomask having a first outlet and a second outlet, wherein the first portion of the light beam comprises light emitted from the first outlet and the second portion of the light beam comprises light emitted from the second outlet.
8. The method of claim 1, further comprising: directing, at a second time instance, the first portion of the collimated light towards a second mirror within the light guide tip, wherein the second mirror reflects the first portion of the collimated light as a second focusing light beam over the sample.
9. The method of claim 8, wherein directing the light beam into the light guide tip comprises directing the light beam to a photomask having an outlet through which the first portion of the light beam is emitted, and the method further comprises: spinning the photomask, such that at the first instance of time, the first portion of the light beam is directed towards the first mirror and at the second instance of time, the first portion of the light beam is directed towards the second mirror.
10. The method of claim 9. wherein: the light guide tip comprises a plurality of mirrors, comprising the first and second mirrors; spinning the photomask directs the first portion of the light beam to the plurality of mirrors at respective time instances comprising the first and second instances; and respective reflections of the first portion of the light beam from the plurality of mirrors create a plurality of focusing light beams, comprising the first and second focusing light beams, and originating in a circular pattern, each focusing light beam being directed over the sample.
11. A system for imaging a sample using fluorescence microscopy, the system comprising: a light source configured to propagate a light beam; an illumination system comprising a light guide tip. wherein the light guide tip is positioned over a sample, when the light beam is directed into the light guide tip at a first time instance, a first portion of the light beam is directed towards a first mirror included in the light guide tip, and the first mirror reflects the first portion of the light beam as a first focusing light beam over the sample, causing a fluorescent emission from the sample; and an objective lens for receiving the fluorescent emission for imaging the sample.
12. The system of claim 11, wherein the light guide tip has one of a column or cylinder shape, and the first mirror is disposed along a bottom edge of the light guide tip.
13. The system of claim 12, wherein the first mirror comprises an off-axis mirror, and the first mirror is disposed along the bottom edge of the light guide tip by positioning the first mirror to have a focal point of the off-axis mirror to be coplanar with a focal plane of the objective lens.
14. The system of claim 12, wherein the first mirror comprises a planar mirror, and the first mirror is disposed along the bottom edge of the light guide tip by positioning the first mirror to form a 45 -degree angle with a focal plane of the objective lens.
15. The system of claim 11, wherein the light guide tip comprises a hemisphere dome on a bottom surface of the light guide tip. and wherein positing the light guide tip comprises positioning the hemisphere dome surrounding the sample.
16. The system of claim 11, wherein when the light beam is directed at the first time instance, a second portion of the light beam is directed towards a second mirror within the light guide tip, and wherein the second mirror reflects the second portion of the light beam as a second focusing light beam over the sample.
17. The system of claim 16, wherein, when the light beam is directed into the light guide tip, the light beam is directed to a photomask having a first outlet and a second outlet, wherein the first portion of the light beam comprises light emitted from the first outlet and the second portion of the light beam comprises light emitted from the second outlet.
18. The system of claim 11 , wherein, at a second time instance, the first portion of the collimated light is directed towards a second mirror within the light guide tip, wherein the second mirror reflects the first portion of the collimated light as a second focusing light beam over the sample.
19. The system of claim 18, wherein, when the light beam is directed into the light guide tip. the light beam is directed to a photomask having an outlet through which the first portion of the light beam is emitted, wherein the photomask is configured to spin, such that at the first instance of time, the first portion of the light beam is directed towards the first mirror and at the second instance of time, the first portion of the light beam is directed towards the second mirror.
20. The system of claim 19, wherein: the light guide tip comprises a plurality of mirrors, comprising the first and second mirrors; spinning the photomask directs the first portion of the light beam to the plurality of mirrors at respective time instances comprising the first and second instances; and respective reflections of the first portion of the light beam from the plurality of mirrors create a plurality of focusing light beams, comprising the first and second focusing light beams, and originating in a circular pattern, each focusing light beam being directed over the sample.
21. An illumination system for imaging a sample using fluorescence microscopy, the illumination system comprising: a light guide tip in one of a column or cylinder shape, the light guide tip comprising a mirror disposed along a bottom edge of the light guide tip and a hemisphere dome disposed on a bottom surface of the light guide tip, wherein the mirror guides an incoming light beam at a first direction to a second direction perpendicular to the first direction; and a photomask coupled to the light guide tip, the photomask comprising an outlet for creating an interference pattern for the incoming light beam.
PCT/US2024/027054 2023-06-30 2024-04-30 Light guide tip for fluorescence microscopes WO2025006055A1 (en)

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US63/511,524 2023-06-30

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