阅读说明：本技术 用于显微镜样本保持器的设备、系统及方法 (Apparatus, system, and method for microscope sample holder ) 是由 A·K·格拉泽 J·T·C·刘 N·雷德 于 2020-02-12 设计创作，主要内容包括：本申请案涉及用于模块化样本保持器的设备、系统及方法。显微镜可沿着照明路径引导照明光朝向样本且沿着集光路径收集来自所述样本的光。沿着所述照明路径的光可穿过浸没流体及所述样本保持器的材料以到达所述样本。沿着集光路径的光可穿过所述样本保持器的所述材料及所述浸没流体。所述样本保持器可具有大体上垂直于所述照明路径的光学轴的沿着所述照明路径的第一光学表面。所述样本保持器可具有大体上垂直于所述集光路径的光学轴的沿着所述集光路径的第二光学表面。所述样本保持器可为模块化的。所述样本保持器可将所述样本容纳在封闭通道中。(The present application relates to apparatuses, systems, and methods for modular sample holders. The microscope can direct illumination light along an illumination path toward a sample and collect light from the sample along a collection path. Light along the illumination path may pass through immersion fluid and material of the sample holder to reach the sample. Light along a light collection path may pass through the material of the sample holder and the immersion fluid. The sample holder can have a first optical surface along the illumination path that is substantially perpendicular to an optical axis of the illumination path. The sample holder can have a second optical surface along the light collection path that is substantially perpendicular to an optical axis of the light collection path. The sample holder may be modular. The sample holder can contain the sample in a closed channel.)

1. An apparatus, comprising:

illumination optics configured to direct illumination light to a sample along an illumination path;

a collection optic configured to receive light from the sample along a collection path;

an immersion chamber configured to hold an immersion medium; and

a sample holder configured to support the sample, comprising a first surface and a second surface opposite the first surface, wherein the second surface is adjacent to the immersion medium, wherein the second surface comprises: a first optical surface along the illumination path that is substantially perpendicular to the illumination path; and a second optical surface along the light collection path that is substantially perpendicular to the light collection path.

2. The apparatus of claim 1, further comprising a support member configured to support the sample holder in an orientation relative to the illumination path and the collection path.

3. The apparatus of claim 2, wherein the sample holder is a modular sample holder removably positioned in a receptacle of the support member.

4. The apparatus of claim 1, wherein the first optical surface is substantially perpendicular to the second optical surface.

5. The apparatus of claim 1, wherein the immersion medium, the sample, the first optical surface, and the second optical surface have matching indices of refraction.

6. The apparatus of claim 1, wherein the sample holder comprises an enclosed channel configured to accommodate the sample.

7. The apparatus of claim 6, wherein the enclosed channel is coupled to an inlet configured to provide fluid to the channel and an outlet configured to exhaust the fluid from the channel.

8. The apparatus of claim 1, wherein the sample holder comprises a plurality of grooves.

9. The apparatus of claim 1, wherein the sample holder comprises a plurality of wells.

10. A system, comprising:

a microscope comprising a support receptacle and an immersion fluid, wherein the microscope is configured to direct an illumination beam through the immersion fluid toward a focus region along an illumination path and to receive collected light through the immersion fluid from the focus region along a collection path; and

a modular sample holder, comprising:

a sample chamber configured to support a sample; and

a first optical surface and a second optical surface positioned along a side of the modular sample holder,

wherein the modular sample holder is removably positioned in the support receptacle such that the focal region is positionable within the sample, the first and second optical surfaces are adjacent to the immersion fluid, the first optical surface is along the illumination path and substantially perpendicular to the illumination path, and the second optical surface is along the light collection path and substantially perpendicular to the light collection path.

11. The system of claim 10, wherein the illumination beam is a light sheet.

12. The system of claim 10, wherein the sample holder comprises a groove configured to hold the sample, and wherein the first optical surface and the second optical surface form a bottom of the groove.

13. The system of claim 10, wherein the sample holder comprises a well configured to hold the sample, and wherein the first optical surface and the second optical surface form a bottom of the well.

14. The system of claim 10, wherein the sample holder comprises a channel configured to hold the sample.

15. The system of claim 10, wherein the first optical surface is substantially perpendicular to the second optical surface.

16. The system of claim 10, wherein the sample, the immersion fluid, the first optical surface, and the second optical surface have matching indices of refraction.

17. An apparatus, comprising:

illumination optics configured to provide illumination light to a sample;

collection optics configured to receive light from the sample;

an immersion chamber configured to hold an immersion fluid; and

a sample holder including an enclosed channel through material of the sample holder, the enclosed channel configured to hold the sample, the sample holder positionable such that a surface of the sample holder is adjacent to the immersion fluid, wherein the illumination light passes through the immersion medium and the sample holder before reaching the sample, and wherein the light from the sample passes through the sample holder and the immersion medium before reaching the light collection optics.

18. The apparatus of claim 17, wherein the enclosed channel has a substantially circular cross-section.

19. The apparatus of claim 17, wherein the channel is part of a microfluidic device.

20. The apparatus of claim 17, wherein the sample holder comprises a first optical surface adjacent to the immersion fluid and a second optical surface adjacent to the immersion fluid, wherein the illumination light passes through the first optical surface and the collected light passes through the second optical surface, and wherein the first optical surface is substantially perpendicular to an optical axis of the illumination light and the second optical surface is substantially perpendicular to an optical axis of the collected light.

21. The apparatus of claim 20, wherein the first optical surface is substantially perpendicular to the second optical surface.

22. The apparatus of claim 17, wherein the first optical surface and the second optical surface form a bottom surface of the enclosed channel.

23. The apparatus of claim 17, when the enclosed channel is coupled to an inlet configured to provide fluid to the channel and an outlet configured to exhaust the fluid from the channel.

24. The apparatus of claim 17, further comprising a support member configured to support the sample holder, wherein the sample holder is a modular sample holder removably positioned in the support member.

25. The apparatus of claim 17, wherein the illuminating light is a sheet of light.

Technical Field

Embodiments of the present invention relate generally to optical imaging, and in particular to microscopes.

Background

Microscopes can typically involve directing light onto a sample, and then imaging the sample based on the light received from the sample. Some microscopes place the optics of the system on the opposite (bottom) side of the sample holder from the sample. This may improve access to the sample, ease of preparing/installing the sample, etc. Thus, the illumination light and the collection light can pass through the material of the sample holder to pass between the optics and the sample.

Different types of samples may require different types of sample holders to support them. For example, some samples may rest on a plate, such as a slide, while other samples may be suspended in a liquid (e.g., in a well plate). A microscope with optics that accommodate multiple sample types and sample holders without interfering with the microscope may be useful.

Disclosure of Invention

In at least one aspect, the present disclosure relates to an apparatus that includes illumination optics, light collection optics, an immersion chamber, and a sample holder. The illumination optics direct illumination light along an illumination path to the sample. The collection optics receive light from the sample along a collection path. The immersion chamber holds an immersion medium. The sample holder supports the sample and includes a first surface and a second surface opposite the first surface. The second surface is adjacent to the immersion medium. The second surface includes: a first optical surface along the illumination path that is substantially perpendicular to the illumination path; and a second optical surface along the light collection path that is substantially perpendicular to the light collection path.

The apparatus may include a support member that may support the sample holder in an orientation relative to the illumination path and the collection path. The sample holder may be a modular sample holder removably positioned in a receptacle of the support member. The first optical surface may be substantially perpendicular to the second optical surface. The immersion medium, the sample, the first optical surface, and the second optical surface may have matching indices of refraction. The sample holder may include an enclosed channel that may contain the sample. The enclosed channel may be coupled to an inlet configured to provide a fluid to the channel and an outlet configured to exhaust the fluid from the channel. The sample holder may include a plurality of grooves. The sample holder may include a plurality of wells.

In at least one aspect, the present disclosure is directed to a system including a microscope and a modular sample holder. The microscope includes a support receptacle and an immersion fluid. The microscope directs an illumination beam through the immersion fluid along an illumination path toward a focal region and receives collected light from the focal region through the immersion fluid along a collection path. The modular sample holder includes: a sample chamber supporting a sample; and a first optical surface and a second optical surface positioned along a side of the modular sample holder. The modular sample holder is removably positioned in the support receptacle such that the focal region is positionable within the sample, the first and second optical surfaces are adjacent to the immersion fluid, the first optical surface is along the illumination path and substantially perpendicular to the illumination path, and the second optical surface is along the light collection path and substantially perpendicular to the light collection path.

The illumination beam may be a light sheet. The sample holder can include a groove configured to hold the sample, and the first optical surface and the second optical surface can form a bottom of the groove. The sample holder may include a well configured to hold the sample, and the first optical surface and the second optical surface may form a bottom of the well. The sample holder may include a channel that can hold the sample. The first optical surface may be substantially perpendicular to the second optical surface. The sample, the immersion fluid, the first optical surface, and the second optical surface may have matching indices of refraction.

In at least one aspect, the present disclosure relates to an apparatus comprising: illumination optics that provide illumination light to the sample; collection optics that receive light from the sample; an immersion chamber holding an immersion fluid; and a sample holder. The sample holder includes an enclosed channel through material of the sample holder. The closed channel holds the sample. The sample holder is positionable such that a surface of the sample holder is adjacent to the immersion fluid. The illumination light passes through the immersion medium and the sample holder before reaching the sample, and the light from the sample passes through the sample holder and the immersion medium before reaching the collection optics.

The enclosed channel may have a substantially circular cross-section. The channel may be part of a microfluidic device. The sample holder may include a first optical surface adjacent to the immersion fluid and a second optical surface adjacent to the immersion fluid. The illumination light can pass through the first optical surface and the collected light can pass through the second optical surface. The first optical surface may be substantially perpendicular to an optical axis of the illumination light and the second optical surface may be substantially perpendicular to an optical axis of the collected light. The first optical surface may be substantially perpendicular to the second optical surface.

The first optical surface and the second optical surface may form a bottom surface of the enclosed channel. The enclosed channel may be coupled to an inlet configured to provide a fluid to the channel and an outlet configured to exhaust the fluid from the channel. The apparatus may also include a support member that can support the sample holder. The sample holder may be a modular sample holder removably positioned in the support member. The illumination light may be a light sheet.

Drawings

Fig. 1 is a block diagram of an open top microscope with a solid immersion meniscus lens, according to some embodiments of the present disclosure.

Fig. 2 is a perspective view of a microscope with a modular sample holder according to some embodiments of the present disclosure.

Fig. 3 is a side view and a top view of a modular sample holder having a flat surface according to some embodiments of the present disclosure.

Fig. 4 is a side view and a top view of a modular sample holder with sample wells according to some embodiments of the present disclosure.

Fig. 5 is a side view and a top view of a modular sample holder with a sample trench according to some embodiments of the present disclosure.

Fig. 6 is a side view and a top view of a modular sample holder with a well at the bottom of a trench, according to some embodiments of the present disclosure.

Fig. 7 is a side view and a top view of a modular sample holder with a flow channel according to some embodiments of the present disclosure.

Fig. 8 is a side view and a top view of a modular sample holder with a flow channel and an optical surface according to some embodiments of the present disclosure.

Fig. 9 is a side view and a top view of a modular sample holder with a flow channel and an optical surface according to some embodiments of the present disclosure.

Fig. 10A-10B show cross-sectional side views of sample holders with open and closed flow cells, respectively, according to some embodiments of the present disclosure.

Fig. 11 is a chart of compatibility of different immersion fluids and sample holder materials according to some embodiments of the present disclosure.

Detailed Description

The following description of certain embodiments is merely exemplary in nature and is in no way intended to limit the scope of the disclosure or its application or uses. In the following detailed description of embodiments of the present systems and methods, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments in which the described systems and methods may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the presently disclosed systems and methods, and it is to be understood that other embodiments may be utilized and that structural and logical changes may be made without departing from the spirit and scope of the present disclosure. Furthermore, for the purpose of clarity, when certain features will be apparent to those skilled in the art, their detailed description will not be so discussed as to avoid obscuring the description of the embodiments of the present disclosure. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims.

Microscopes can be used in a wide range of applications to produce images of a sample that often have a field of view and/or resolution that is not typically visible to the naked eye. Illumination optics may be used to direct illumination light onto the sample. Collection optics may be used to collect light from the sample onto a detector (e.g., a CCD detector, a CMOS detector, or the user's eye). In some examples, the light reaching the detector may comprise a portion of the illumination light. In some examples, light reaching the detector may be emitted from the sample (e.g., via fluorescence) after being excited to emit light by illumination. It may be desirable to ensure that the field of view receives uniform illumination and to ensure that the illumination penetrates the depth of the sample.

The illumination and collection optics may be positioned on the opposite side of the sample holder from the sample (e.g., on the underside of the sample holder). Thus, the illuminating and collecting light may pass through the material of the sample holder. The illumination and/or collection optics may be arranged at an angle relative to the sample holder. For example, the illumination and collection optics may each be at a 45 ° angle relative to the sample holder. While this may provide the advantage of optimizing imaging parameters and imaging depth, non-normal incidence of illumination and/or collection light with respect to the sample holder may cause various aberrations. While individual sample holders may be optically optimized for a particular setup, different sample types may have different sample holder requirements. For a microscope, it may be useful to accommodate different sample holders that are compatible with the optics of the microscope.

The present disclosure relates to microscope sample holders. The microscope includes a sample holder having a first side and a second side opposite the first side. When the sample holder is positioned in the microscope, the illumination and collection optics are positioned on a second side of the sample holder. The illumination light passes through the immersion fluid and through the material of the sample holder before reaching the sample. The collected light passes through the material of the sample holder and through the immersion fluid before reaching the sample. The sample holder may be a modular component of a microscope. Different modular sample holders may have different geometries to accommodate different types of samples, different imaging modalities, different experimental conditions, and combinations thereof. Some modular sample holders may include a first optical surface along the illumination optical path between the immersion fluid and the sample and a second optical surface along the collection optical path between the sample and the immersion fluid. The first optical surface may be substantially perpendicular to an optical axis of the illumination path and the second optical surface may be substantially perpendicular to an optical axis of the collection path. The use of optical surfaces may reduce the need for index matching between the sample, the material of the sample holder, and the immersion fluid, and may also allow for objectives (e.g., illumination and/or collection objectives) with shorter working distances.

Fig. 1 is a block diagram of a microscope with a modular sample holder according to some embodiments of the present disclosure. Fig. 1 shows an optical system 100 that includes a microscope 102 and an optional controller 104 that can operate the microscope 102 and/or interpret information from the microscope 102. In some embodiments, one or more portions of the controller 104 may be omitted and the microscope 102 may be manually operated. In some embodiments, one or more portions of the controller 104 may be integrated into the microscope 102. In some embodiments, such as the example of fig. 1, the microscope 102 may be an open top microscope.

Microscope 102 includes a sample holder 108 that supports a sample 106. The bottom surface of sample holder 108 is adjacent to immersion fluid 112, which immersion fluid 112 is contained within immersion chamber 110. The microscope 102 of fig. 1 has an illumination path and a collection path that are separate from each other. The illumination path includes a source 118, illumination optics 120, and an illumination objective 122. The illumination path provides an illumination beam 124 that passes through immersion medium 112 and sample holder 108 to illuminate sample 106. The collection path includes collection objective 128, collection optics 130, and detector 132. The light collection path may collect light from a focal region 126 illuminated by the illumination beam 124.

Microscope 102 includes a modular sample holder 108 that is replaceable with various other modular sample holders 108. Examples of different types of modular sample holders 108 are discussed in more detail in fig. 3-10B. The sample holder 108 may be removably coupled to a support member 109 of the microscope 102. A support member 109 may be positioned along an upper surface of an immersion bath 110 holding an immersion fluid 112. In some embodiments, the support member 109 may form a lid or other upper housing of the immersion bath 110. The support member 109 may include an opening that exposes the immersion fluid 112. The opening may serve as a receptacle for modular sample holder 108. When sample holder 108 is positioned in the receptacle, a lower surface of sample holder 108 may be adjacent to immersion fluid 112. In some embodiments, a lower surface of sample holder 108 may contact immersion fluid 112. For example, a lower surface of sample holder 108 may extend into immersion bath 110. In some embodiments, support member 109 and/or sample holder 108 may include a gasket or other sealing member to prevent immersion fluid 112 from escaping in the middle of the seam between sample holder 108 and support member 109.

In some embodiments, the sample holder 108 may rest in a receptacle of the support member 109 without any kind of attachment (other than gravity). For example, the contour of the receptacle may be tapered and/or the receptacle may include a step that supports the sample holder 108. In some embodiments, one or more fasteners may be used to attach the sample holder 108 to the support member 109. For example, clips, screws, magnets, snaps, hook-and-loop fasteners, or a combination thereof may be used to removably couple the sample holder to the support member 109.

In some embodiments, sample holder 108 may be a unitary body that fits directly into a receptacle of support member 109. In some embodiments, an adapter plate may be used that fits in a receptacle of the support member 109 and that includes a receptacle for the sample holder 108. In some embodiments, the sample holder 108 can be attached to the adapter (e.g., using an adhesive). For example, the sample holder 108 may be bonded to the adapter along the periphery of the sample holder 108 using a UV-curable adhesive. In some embodiments, sample holder 108 can be a commercial sample holder made modular via the use of an adapter. In some embodiments, the adapter may include mounting hardware (e.g., fasteners) that mate with the support member 109.

The support member 109 may have a recessed region that holds the sample holder 108. Modular sample holder 108 is insertable into the recessed area. In some embodiments, the sample holder 108 can be shaped to limit the orientation of the sample holder 108 relative to the support member 109 (and relative to the illumination and collection paths). For example, the sample holder 108 may be square to limit placement of the sample holder 108 to one of four orientations, may be rectangular to limit placement of the sample holder 108 to one of two orientations, or may be any other shape. In some embodiments, sample holder 108 can be shaped such that there is only one orientation in which sample holder 108 fits in a receptacle of support member 109.

Source 118 provides illumination light along an illumination path to illuminate a focal region 126 of sample 106. The source 118 may be a narrow band source, such as a laser or Light Emitting Diode (LED) that can emit light in a narrow spectrum. In some embodiments, the light may be a broadband source (e.g., incandescent source, arc source) that may produce broad-spectrum (e.g., white) illumination. In some embodiments, one or more portions of the illumination light may be outside the visible range. In some embodiments, a filter (not shown) may be used as part of the illumination path to further refine the illumination light of wavelength(s). For example, a bandpass filter may receive broadband illumination from source 118 and provide illumination in a narrower spectrum. In some embodiments, the light source 103 may be a laser and may generate collimated light.

In some embodiments, the optical system 100 may be used to image fluorescence in the sample 106. The illumination beam 124 may include light of a particular excitation wavelength that may excite fluorophores in the sample 106. The illumination beam 124 may comprise a broad spectrum (which includes the excitation wavelength), or may be a narrow band centered on the excitation wavelength. In some embodiments, the light source 118 may produce a narrow spectrum centered at (or near) the excitation wavelength. In some embodiments, filter(s) (not shown) may be used in the illumination optics 120 to limit the illumination beam 124 to wavelengths near the excitation wavelength. Once excited by the illumination patch, the fluorophore in the sample 106 may emit light (which may be centered at a given emission wavelength). The collection path (e.g., collection optics 130) may include one or more filters that may be used to limit light reaching the detector 132 to wavelengths of light near the emission wavelength.

Illumination optics 120 may couple light from source 118 to illumination objective 122. For example, the illumination optics 120 may include an optical fiber that carries light from the source 118 to the back end of the illumination objective 122. In some embodiments, the illumination optics 120 may couple light between the source 118 and the objective 122 without substantially altering the light provided by the source 118. In some embodiments, the illumination optics 120 may alter the shape, wavelength, intensity, and/or other properties of the light provided by the source 118. For example, illumination optics 120 may receive broadband light from source 118 and may filter the light (e.g., using filters, diffraction gratings, acousto-optic modulators, etc.) to provide narrow-band light to objective lens 122.

In some embodiments, the illumination path may provide an illumination beam 124, the illumination beam 124 being a light sheet as part of a light sheet microscope or Light Sheet Fluorescence Microscope (LSFM). The light sheet may have a substantially elliptical cross-section with a first numerical aperture along a first axis (e.g., y-axis) and a second numerical aperture greater than the first numerical aperture along a second axis orthogonal to the first axis. Illumination optics 120 may include optics that reshape light received from source 118 into an illumination sheet. For example, the illumination optics 120 may include one or more cylindrical optics that focus light on one axis rather than on an orthogonal axis.

In some embodiments, the illumination optics 120 may include scanning optics, which may be used to scan the illumination beam 124 relative to the sample 106. For example, the area illuminated by the illumination beam may be smaller than the desired focal region 126. In this case, the illumination optics 120 may oscillate the illumination beam 124 rapidly across the desired focal region 126 to ensure illumination of the focal region 126.

Illumination objective 122 may include one or more lenses that provide an illumination beam 124. For example, the illumination objective 122 may focus the illumination beam 124 toward a focal region 126. The sample holder 108 may position the sample 106 such that the focal region 126 is substantially within the sample 106. In some embodiments, the illumination objective may be a commercial objective including one or more internal optical elements. In some embodiments, the focal region 126 may be idealized as a focal point. In some embodiments, the focal region 126 may be substantially planar in shape. For example, in an LSFM, the focal region 126 may be a plane lying along the plane of the light sheet generated as the illumination beam 124.

In some embodiments, the illumination objective 122 may be surrounded by an ambient environment (e.g., air), and the illumination objective 122 may be an air objective. The illumination objective 122 may be characterized by one or more numerical apertures, which may be based on the angle(s) at which light is concentrated at the focal region 126. In some embodiments where the illumination objective 122 is in an ambient environment outside of the illumination fluid 112, a window or lens may then be used to couple the illumination beam 124 into the immersion fluid 112. For example, the inset of fig. 1 shows an illuminating Solid Immersion Lens (SIL)114 positioned along the illumination path. In some embodiments, the illumination objective 122 may be an immersion objective in contact with the immersion fluid 112 (e.g., in a similar manner to that shown for the collection objective 128, as described in more detail herein), and the SIL 114 may be omitted.

The illumination beam 124 may be directed to pass through the illumination SIL 114 and into the immersion fluid 112. The illumination SIL 114 may be shaped to minimize refraction of light from the ambient environment including the illumination objective lens 122 (e.g., air) through the material of the illumination SIL 114 and into the immersion fluid 112. Illumination beam 124 may then pass through immersion fluid 112 toward sample 106 and sample holder 108.

Illumination beam 124 may be directed onto sample 106. The sample 106 may be supported by a sample holder 108. In some embodiments, the sample 106 may be placed directly onto the upper surface of the sample holder 108. In some embodiments, the sample 106 can be packaged in a container (e.g., on a slide, in a well plate, in a tissue culture flask, etc.) and the container can be placed on the sample holder 108. In some embodiments, the container may be integrated into the sample holder 108. In some embodiments, the sample 106 may be processed prior to imaging on the optical system 100. For example, the sample 106 may be washed, sliced, and/or labeled prior to imaging.

In some embodiments, the sample 106 may be a biological sample. For example, the sample 106 may be tissue that has been biopsied from a region of suspected disease (e.g., cancer). In some embodiments, the tissue may undergo various treatments, such as optical debridement, tissue sectioning, and/or marking, prior to inspection by the optical system 100. In some embodiments, examination of tissue using optical system 100 can be used for diagnosis, for determining treatment progress, for monitoring disease progression, and the like.

In some embodiments, the sample 106 may be non-biological. For example, the sample 106 may be a fluid and may contain one or more components for study. For example, the sample 106 may be a combustion gas, and the optical system 102 may perform Particle Image Velocimetry (PIV) measurements to characterize the composition of the gas.

In some embodiments, the sample 106 may include one or more types of fluorophores. The fluorophore can be intrinsic to the sample 106 (e.g., DNA and proteins in a biological sample) or can be a fluorescent marker applied to the sample 106 (e.g., acridine orange, eosin). Some samples 106 may contain a mixture of intrinsic types of fluorophores and fluorescent markers. Each type of fluorophore may have an excitation spectrum that may be centered at an excitation wavelength. When a fluorophore is excited by light in an excitation spectrum, it can emit light in an emission spectrum that can be centered at an emission wavelength that is different from (e.g., red-shifted from) the excitation wavelength.

Sample holder 108 may support sample 106 on a material of the sample holder that is substantially transparent to illumination beam 124 and light collected from focal region 126 of sample 106. In some embodiments, the sample holder 108 may have a window of transparent material over which the sample 106 may be positioned, and the remainder of the sample holder 108 may be formed of a non-transparent material. In some embodiments, sample holder 108 can be made of a transparent material.

The sample holder 108 can have a second surface (e.g., a lower surface) that opposes a surface of the sample holder 108 that supports the sample 106. Immersion chamber 110 holding immersion fluid 112 may be positioned below a second surface of sample holder 108. In some embodiments, immersion chamber 110 may have an open top, and immersion fluid 112 may be adjacent to a second surface of sample holder 108. In some embodiments, while the second surface of sample holder 108 may be in contact with immersion fluid 112, the first surface of sample holder 108 (which supports sample 106) may be in contact with the same environment as objective lenses 122 and 128 (e.g., air).

The support member 109 may be coupled to an actuator 107, which actuator 107 may be capable of moving the support member 109 and/or the sample holder 108 in one or more directions. In some embodiments, sample holder 108 is movable in up to three dimensions (e.g., along x, y, and z axes) relative to immersion chamber 110 and objective lenses 122 and 128. Sample holder 108 may be moved to change the position of focal region 126 within sample 106 and/or to move sample holder 108 between a loading position and an imaging position. In some embodiments, the actuator may be a manual actuator, such as a screw or a coarse/fine adjustment knob. In some embodiments, the actuator may be automated, such as an electric motor, which may be responsive to manual inputs and/or instructions from the controller 104. In some embodiments, the actuator 107 may be responsive to both manual adjustment and automatic control (e.g., a knob responsive to both manual turning and instructions from the controller 104).

Immersion chamber 110 contains immersion fluid 112. In some embodiments, the immersion chamber 110 may include a source and/or sink that may be useful for replacing the immersion fluid 112. For example, the immersion chamber 110 may be coupled to a fluid input line that provides the immersion fluid 112 (which in turn may be coupled to a pump and/or reservoir) and a drain that may be opened to remove the immersion fluid 112 from the immersion chamber 110. As described in more detail herein, the type of immersion fluid may be selected based on the index of refraction of sample 106 and/or sample holder 108.

The light collection path may receive light from the focal region 126 and direct the received light onto a detector 132, which detector 132 may image and/or otherwise measure the received light. The light from the focal region 126 may be redirected portions of the illumination beam 124 (e.g., scattered and/or reflected light), may be light emitted from the focal region 126 in response to the illumination beam 124 (e.g., via fluorescence), or a combination thereof. The collected light may pass through the sample holder 108 and immersion fluid 112 before reaching the collection objective 128. In some embodiments, the collection objective 128 may be an immersion objective having a front end positioned adjacent to the immersion fluid 112. For example, the front end of the collection objective 128 may be positioned within the immersion bath 110. In some embodiments, the collection objective 128 may be an air objective, and a lens or window may be positioned between the immersion fluid 112 and the front end of the collection objective 128.

The geometry of the focal region 126 may be defined in part by the field of view of the collection optical path, which in turn may depend in part on the numerical aperture of the collection objective 128. Similar to the illumination objective 122, the collection objective 128 may be a commercial objective including one or more lenses. In some embodiments, the collection objective 128 may be an air objective. In some embodiments, the focal region focused by the collection path and the focal region focused by the illumination path may substantially overlap at focal region 126. In some embodiments, the illumination and collection paths may have different shapes, sizes, and/or positions of their respective focal regions.

The collection path includes collection optics 130 that can redirect light from the collection objective onto a detector 132. For example, collection optics 130 may be a tube lens designed to focus light from the back end of the collection objective into an image projected on detector 132. In some embodiments, the collection optics 130 may include one or more elements that alter the light received from the collection objective 128. For example, the collection optics 130 may include filters, mirrors, descan optics, or a combination thereof.

The detector 132 may be used to image the focal region 126. In some embodiments, the detector 132 may represent an eyepiece such that the user may view the focal region 126. In some embodiments, the detector 132 may generate a signal to record an image of the focal region 126. For example, the detector 132 may include a CCD or CMOS array that may generate electronic signals based on light incident on the array.

The illumination path may direct light along a first optical axis. The light collection path may collect light along the second optical axis. In some embodiments, such as the embodiment shown in fig. 1, the first and second optical axes may be orthogonal to each other. Each of the first optical axis and the second optical axis may also be non-orthogonal to the sample holder. For example, the first optical axis may be at a 45 ° angle relative to the bottom surface of sample holder 108 and the second optical axis may also be at a 45 ° angle relative to the bottom surface of sample holder 108. Other angles between the first optical axis, the second optical axis, and/or the sample holder 108 may be used in other examples.

In some embodiments, the illumination and collection paths may be non-orthogonal to each other. For example, the illumination path may follow a first optical axis that is at a 45 ° angle relative to the bottom surface of the sample holder, while the collection path may follow a second optical axis that is at a 90 ° angle relative to the bottom surface of the holder. Thus, there may be an approximately 45 ° angle between the first optical axis and the second optical axis.

In some embodiments, one of the objectives 122 and 128 may be an air objective while the other objective may be a non-air objective (e.g., an immersion objective). In some embodiments, the non-air objective lens may be immersed with the immersion fluid 112 (or have a front surface in contact with the immersion fluid 112). In some embodiments, an air objective lens may have a SIL positioned between the objective lens and the immersion fluid, while the SIL may not be used with a non-air objective lens. For example, the illumination objective 122 may direct the illumination beam 124 through the illumination SIL 114 and into the immersion fluid 112, while the collection objective 128 may be adjacent to (e.g., in contact with) the immersion fluid 112.

The microscope 102 may be coupled to a controller 104, which controller 104 may be used to manipulate one or more portions of the microscope 102, display data from the microscope 102, interpret data from the microscope 102, or a combination thereof. In some embodiments, the controller 104 may be separate from the microscope, such as a general purpose computer. In some embodiments, one or more portions of the controller 104 may be integral with the microscope 102.

The controller 104 includes one or more input/output devices 142 that may allow a user to view feedback from the controller 104, data from the microscope 102, provide instructions to the controller 104, provide instructions to the microscope 102, or a combination thereof. For example, the input/output device 142 may include a digital display, a touch screen, a mouse, a keyboard, or a combination thereof.

The controller 104 includes a processor 140, the processor 140 may execute one or more instructions stored in a memory 144. The instructions may include control software 152, which control software 152 may include instructions on how to control the microscope 102. Based on the control software 152, the processor 140 may cause the controller 104 to send signals to various components of the microscope 102, such as the actuator 109. The instructions may include image processing software 150, which image processing software 150 may be used to process images 146 'in real time' from the detector 132 or previously stored in memory 144. Image processing software 150 may, for example, remove background noise from image 146. The instructions may include analysis software 148, which analysis software 148 may be executed by the processor 140 to determine one or more properties of the image 146. For example, the analysis software 148 may highlight nuclei in the image 146.

In some embodiments, the controller 104 may direct the microscope to collect images from several different fields of view in the sample. For example, the controller 104 may include instructions for collecting depth stacked images. The controller 104 may direct the detector 132 to collect the first image and then instruct the actuator 109 to move the sample holder 108 in a vertical direction (e.g., along the z-axis) for a set distance. This may also move the sample 106 relative to the focal region 126, which may change the height at which the focal region 126 is located within the sample. The controller 104 may then instruct the detector 132 to collect another image and then repeat the process until a set number of images in the stack and/or a set total displacement in the z-direction has been achieved. The analysis software 148 may then combine the depth-stacked images to allow 3D (or pseudo-3D) imaging of the sample 106.

Fig. 2 is a perspective view of a microscope with a modular sample holder according to some embodiments of the present disclosure. In some embodiments, microscope 200 shows an example layout of how a microscope, such as microscope 102 of fig. 1, may be assembled. In some embodiments, the components and operation of microscope 200 may be substantially similar to those of microscope 102 of fig. 1. For the sake of brevity, the components and operations described with respect to the microscope 102 of fig. 1 will not be repeated with respect to fig. 2.

The microscope 200 includes illumination optics 221, the illumination optics 221 being coupled to an illumination source 220. The illumination source 220 may be a fiber optic cable coupled to a laser (not shown in fig. 2). The illumination optics 221 may include a telescope and/or a beam expander to couple light out of the illumination source 220. Illumination optics 221 may include shaping optics to shape the illumination light into a sheet of light. The illumination optics 221 may include one or more scanning optics, which may be motorized to scan the sheet of illumination light across the back end of an illumination objective (not shown), which in turn may scan the sheet of illumination light across the sample.

The sheet of illuminating light passes through an immersion bath 212, the immersion bath 212 being positioned below the support member 209. The support member 209 may be a stage, such as a motorized stage. For example, the stage may be movable in one or more axes, such as an XY stage. In some embodiments in which the support member 209 is motorized in some axes, movement in other axes may be increased by coupling one or more portions of the microscope 200 to additional actuators (e.g., Z-axis actuators).

The view of fig. 2 shows sample holder 208 positioned over a receptacle of support member 209. Sample holder 208 is shown as a plate that can be lowered into an imaging receptacle. Different sample holder types that may be used with microscope 200 are discussed in more detail in fig. 3-10B.

The microscope 200 includes light collection optics 231 that collect light from the focal region of the microscope 200 and provide the collected light to a detector 232. The collection optics 231 may include a filter wheel that includes a number of filters that can be rotated into the collection path of the microscope 200. Each filter may be associated with a different emission spectrum (e.g., of a different type of fluorophore that may be imaged). In some embodiments, detector 232 may be a sCMOS detector.

Fig. 3-10B show various sample holders that may be used as sample holder 108 of fig. 1 and/or 208 of fig. 2 in some embodiments. Fig. 3-9 show both a top view of a sample holder and a side view of a portion of a sample holder. The side view shows a cross-section of a portion of the sample holder containing the sample. The side view shows the sample holder when placed in a microscope (e.g., microscope 102 of fig. 1 and/or microscope 200 of fig. 2) and when the microscope is aligned such that the focal region of the microscope is within the sample shown in the cross-sectional side view. Fig. 10A-10B show a pair of side views, rather than top and side views, of two variations of a sample holder.

The views of fig. 3-10B show a portion of the sample holder that does not include a means by which the sample holder is mounted to the microscope. In some embodiments, the sample holder is insertable into a receptacle of a microscope. In some embodiments, the sample holder may include mounting hardware (not shown) that is inserted into the receptacle and/or attached to a support member of the microscope (e.g., via clips, magnets, pins, screws, etc.). For example, the sample holder may be attached to an adapter plate, which in turn may fit into a receptacle and/or include accessory hardware.

Each of fig. 3-10B shows an example embodiment with an air illumination objective and an immersion collection objective. However, it should be understood that any combination of air and immersion objectives may be used for the illumination and collection paths. Each of fig. 3-10 shows a generally rectangular top view of a sample holder, however it should be understood that many features and operations between the various sample holders may be similar and for the sake of brevity such features will not be separately described for each of fig. 3-10.

For example, each of the sample holders in fig. 3-10B includes various materials, such as the material of the sample holder, the immersion fluid, the sample, the fluid surrounding the sample, and so forth. Each of these materials may have a refractive index and dispersion. For any given sample holder, two or more materials may be selected such that they have the same or similar refractive index and/or dispersion.

In some embodiments, a single sample holder may have a mixture of the geometries and features described in fig. 3-10B. For example, the sample holder may have a first region with a first geometry for holding a sample and a second region with a second geometry for holding a sample.

In some embodiments, one or more surfaces of the sample holder can include one or more coatings. For example, a hydrophobic coating may be placed on the sample holder to promote beading of the spotted sample on the sample holder.

Fig. 3 is a side view and a top view of a modular sample holder having a flat surface according to some embodiments of the present disclosure. View 300 shows a substantially planar sample holder 308. For example, sample holder 308 may be a transparent plate and sample 306 may be supported on an upper surface of the plate. When a planar sample holder 308 is used, the microscope may operate in a manner similar to a flatbed scanner, wherein a sample may be placed on the upper surface of the sample holder 308. In some embodiments, the plurality of samples 306 may be placed on a sample holder 308 and may be separated from each other by air. In some embodiments, the sample 306 may comprise a liquid phase, and the sample 306 may be a droplet deposited on a surface of the sample holder 308.

Fig. 4 is a side view and a top view of a modular sample holder with sample wells according to some embodiments of the present disclosure. Sample holder 408 may be substantially planar (e.g., similar to sample holder 308 of fig. 3), however sample holder 408 includes a surrounding medium 411 that may form a zone that contains each sample 406. For example, sample holder 408 may have defined sample regions separated from each other by surrounding medium 411. In some embodiments, surrounding medium 411 may be of the same material as the rest of sample holder 408. In some embodiments, sample holder 408 may be a flat plate of a first material, to the upper surface of which the surrounding medium 411 may be attached.

Sample holder 408 may include one or more different sample regions, each of which may have a different sample (or samples) 406 a-h placed therein. For example, the sample holder 408 may be a well plate that may hold several different samples 406 a-h.

Fig. 5 is a side view and a top view of a modular sample holder with a sample trench according to some embodiments of the present disclosure. Sample holder 508 of fig. 5 includes a plurality of sample trenches 515, each of which sample trenches 515 can hold one or more samples 506. The sample trench 515 has a bottom with a first optical surface 517 and a second optical surface 519, which can help couple to/from the illumination beam 524 and the collected light 525, respectively, of the sample 506.

The side view of fig. 5 shows a cross-section of a single one of the trenches 515. The trench may be filled with a fluid 513. Fluid 513 may be the same or different than immersion fluid 512. In some embodiments, the fluid 513 may have an index of refraction matching the index of refraction of the sample 506. In some embodiments, the fluid 513 may have a different index of refraction than the sample 506. In some embodiments, fluid 513 may have the same index of refraction as the material of sample holder 508 and/or immersion fluid 512. The fluid 513 may be used to support, stabilize and/or protect the sample 506 during imaging. For example, sample 506 may be a cell and fluid 513 may represent a cell culture medium. In some embodiments, fluid 513 may be an ambient medium such as air.

Trench 515 has a first optical surface 517 along the illumination path of illumination beam 524 between immersion fluid 512 and sample 506 (and/or between immersion fluid 512 and fluid 513). The first optical surface 517 may be positioned such that the first optical surface 517 is substantially perpendicular to an optical axis of the illumination path. This may reduce refraction of light as it passes from immersion fluid 512 into the material of sample holder 508. In some embodiments, the first optical surface 517 may be a flat surface with a uniform thickness.

The trench 515 has a second optical surface 519 between the sample 506 (and/or the fluid 513) and the immersion fluid 512 along a collection path of the collected light 525. Second optical surface 519 may be positioned such that second optical surface 519 is substantially perpendicular to an optical axis of the light collection path. This may reduce refraction of light as it passes from sample 506/fluid 513 into the material of sample holder 508 and immersion fluid 512. In some embodiments, second optical surface 519 may be a flat surface with a uniform thickness.

First optical surface 517 and second optical surface 519 may be made of the same material as the remainder of sample holder 508. For example, sample holder 508 may be a unitary body formed from a single piece of material. In some embodiments, the first and second optical surfaces 517 and 519 may be made of a different material than the remainder of the sample holder 508. For example, the first and second optical surfaces 517 and 519 can be made of a transparent material, while the remainder of the sample holder 508 is made of a non-transparent material.

The first and second optical surfaces 517 and 519 may form the 'bottom' of the trench 515. The first and second optical surfaces 517 and 519 may meet at an angle, which may be based on the angle between the illumination and collection paths. For example, the first optical surface 517 may be angled at about 90 ° relative to the second optical surface 519. In some embodiments, the first and second optical surfaces 517 and 519 may not directly intersect, and an additional piece of material may separate them. For example, an additional piece of material may be added that is substantially parallel to the bottom surface of the non-trench portion of sample holder 508, giving the bottom of trench 515 a substantially trapezoidal cross-section rather than a triangular cross-section.

Channel 515 may be elongated along the length of sample holder 508. In some embodiments, each trench 515 may be closed to prevent fluid 513 and/or sample 506 from escaping from trench 515. For example, each trench 515 may terminate at a distance from an edge of sample holder 508 such that the material of sample holder 508 encloses trench 515. In some embodiments, one or more end caps may be attached to sample holder 508 to enclose trench 515. In some embodiments, the bottom of each trench 515 can extend from the bottom of sample holder 508 (e.g., to form a series of ridges that extend along the bottom surface of sample holder 508).

Channel 515 may have a long axis that is elongated across the length of sample holder 508. The sample holder may be aligned in a microscope (e.g., in the receptacle of support member 109 of fig. 1 and/or support member 209 of fig. 2), the long axis may be substantially perpendicular to the illumination and collection paths from a 'top-down' perspective. For example, when sample holder 508 is placed in a microscope, the long axis of groove 515 may be perpendicular to the line drawn between the illumination objective and the collection objective. In some embodiments, sample holder 508 can include a plurality of grooves 515. In some embodiments, the grooves may be substantially parallel to each other.

Once the sample holder 508 is placed in the microscope, the sample holder 508 may be moved (e.g., by the actuator 107 of fig. 1) to image different regions of the sample 506 and/or different samples. Different samples may be in the same groove 515 or different grooves of sample holder 508. As sample holder 508 moves relative to immersion fluid 512, first and second optical surfaces 517 and 519 may move relative to the illumination and collection light paths. Since the first and second optical surfaces 517 and 519 are flat and perpendicular to their respective optical paths, movement of the optical surface 517/519 may result in relatively small aberrations because the illumination and collection optical paths will continue to encounter flat surfaces that are perpendicular to their respective optical axes. The imageable area of sample 506 can be based in part on the area within which imaging light beam 524 and/or collected light 525, respectively, can move relative to first optical surface 517 or second optical surface 519 without interfering with the non-optical surface portion of sample holder 508 and/or another optical surface.

In some embodiments, the first and second optical surfaces 517 and 519 may be useful for reducing the working distance of the focal region to the illumination objective (not shown in fig. 5) and/or the collection objective 528, respectively. For example, the working distance of one or both of the objective lenses may be about 5mm to 15 mm. Greater or lesser working distances may be used in other example embodiments. In some embodiments, the objective lens may be an immersion objective lens, and the objective lens may have a front surface positioned proximate to the optical surface. In some embodiments, the optical surface may be approximately parallel to a front surface (e.g., a front lens) of the objective lens. For example, in embodiments where the objective lens is an air objective lens and is separated from the immersion fluid by a window or lens (e.g., SIL), the surface of the window/lens may abut the optical surface.

Fig. 6 is a side view and a top view of a modular sample holder with a well at the bottom of a trench, according to some embodiments of the present disclosure. Fig. 6 shows a sample holder 608 that is substantially similar to sample holder 508 of fig. 5, except that sample holder 608 has a number of separate wells 615 instead of elongated grooves, each of the wells 615 having a first optical surface 617 and a second optical surface 619, the first optical surface 617 and the second optical surface 619 being substantially similar to the first and second optical surfaces of fig. 5.

Each well 615 may hold a different sample 606. Each aperture may have first and second optical surfaces 617 and 619, respectively, that are substantially perpendicular to the illumination and collection light paths. In some embodiments, optical surfaces 617 and 619 may extend along the length of sample holder 608. For example, sample holder 608 can have a similar shape as sample holder 508 of fig. 5, except that sample holder 608 can have 'walls' positioned along the groove to divide the groove into different wells 615. In some embodiments, the optical surfaces 617 and 619 may stop between each of the holes. In some embodiments, each of the holes 615 may have a rectangular cross-section from a top-down perspective. In some embodiments, other cross-sectional shapes may be used.

Fig. 7 is a side view and a top view of a modular sample holder with a flow channel according to some embodiments of the present disclosure. Sample holder 708 can include one or more channels 715 enclosed within the material of sample holder 708. Each channel 715 may be loaded with one or more samples 706, which may be surrounded by a fluid 713.

Sample holder 708 can include a channel 715 formed in the material of sample holder 708. For example, the perimeter of channel 715 may be formed by the material of sample holder 708. In some embodiments, the channel 715 may have a circular cross-section. Other shapes of cross-sections may be used in other example embodiments. In some embodiments, the channel 715 may have a shape and/or size based on the expected properties of the sample 706. For example, if the sample 706 is a cell, the channel 715 may have a diameter such that the cells of the sample 706 travel 'single file' down the length of the channel 715. In some embodiments, channel 715 may be part of a microfluidic system that extends through sample holder 708. For example, sample holder 708 may be a microfluidic chip, and channel 715 may represent a microfluidic flow channel through a transparent region of the imageable chip.

In some embodiments, sample holder 708 may include a port that may be used to load sample 706 (and/or fluid 713) into channel 715 or to drain channel 715. In some embodiments, the channels may vary in size and/or shape along their length. In some embodiments, there may be several separate channels 715. Such as a plurality of parallel channels 715 similar to the parallel grooves 515 of fig. 5. In some embodiments, there may be one long channel 715 with several parallel sections (e.g., the channel 715 may switch back on its own one or more times).

Fig. 8 is a side view and a top view of a modular sample holder with a flow channel and an optical surface according to some embodiments of the present disclosure. Sample holder 808 has a channel (similar to channel 715 of fig. 7) and first and second optical surfaces 817 and 819 (similar to optical surfaces 517 and 519 of fig. 5 and optical surface 617/619 of fig. 6).

In some embodiments, first optical surface 817 and second optical surface 819 can be facets of protrusions of material of sample holder 808 extending from a bottom surface of sample holder 808. Channel 815 may have a shape without flat surfaces corresponding to first and second optical surfaces 817 and 819. For example, as shown in FIG. 8, channel 815 may have a circular cross-section, while first and second optical surfaces 817 and 819 may form a protrusion having a substantially triangular cross-section.

Fig. 9 is a side view and a top view of a modular sample holder with a flow channel and an optical surface according to some embodiments of the present disclosure. Sample holder 908 of fig. 9 may be substantially similar to sample holder 808 of fig. 8, except that channel 915 of sample holder 908 is shaped such that first optical surface 917 and second optical surface 919 have a back surface parallel to their front surfaces. For example, the channel 915 may have the same general cross-section as the groove 515 of fig. 5 and/or the hole 615 of fig. 6, except that the channel 915 has a 'cap'. In some embodiments, the channel 915 may cut through the material of the sample holder 908, and the sample holder 908 may be a unitary body. In some embodiments, the channel 915 may be covered by a 'cap', which may be the same or different material than the rest of the sample holder 908.

Fig. 10A-10B show cross-sectional side views of sample holders with open and closed flow cells, respectively, according to some embodiments of the present disclosure. Sample holders 1000a and 1000b may be substantially similar to each other, except that sample holder 1000a is enclosed by the material of the sample holder, while sample holder 1000b is open to the ambient environment (e.g., air). For the sake of brevity, only the flow cell 1000a will be described in detail. In some embodiments, the flow cells of sample holder 1000a (or 1000b) may be incorporated into any of the geometries of sample holders 308-908 of fig. 3-9, respectively.

Sample holder 1000a includes an inlet 1021 and an outlet 1023. The inlet can provide circulating fluid 1013 to the chamber, while the outlet 1023 can remove the circulating fluid 1013. In this manner, circulating fluid 1013 can flow through the chamber. In some embodiments, one or more different circulating fluids 1013 may flow across the sample 1008 to perform automated processing of the sample. For example, the sample 1008 can be labeled in situ by flowing different reagents across the sample 1008.

In some embodiments, sample holders 1000a and 1000b can include an inlet 1021 and an outlet 1023, which inlet 1021 and outlet 1023 can mate with corresponding inlets and outlets of a microscope when sample holders 1000a and 1000b are positioned in a microscope.

Fig. 11 is a chart of compatibility of different immersion fluids and sample holder materials according to some embodiments of the present disclosure. Graph 1100 may be based on optical modeling of the interaction of different immersion fluid (e.g., 112 of fig. 1) and different sample holder (e.g., 108 of fig. 1) materials. In some embodiments in which the sample holder includes more than one type of material, the sample holder material may represent the material used in the portion(s) of the sample holder that interact with the illumination and light collection paths. For example, the sample holder material may represent the material of the first and second optical surfaces (e.g., 517 and 519 of fig. 5).

The rows represent different types of immersion fluids, each listed with their refractive index. The columns represent different types of sample holder materials. The shading of each box at the intersection of the immersion fluid and the sample holder material represents the maximum thickness of the sample holder material without significant aberrations. The maximum thickness is expressed in millimeters, or is shown to be "chemically incompatible" for combinations of structures that would cause immersion fluid to damage the sample holder.

Various example values are given throughout the specification. It should be understood that these values are approximations because perfect alignment may not be possible in real world systems. Thus, for example, a value of 'normal' or 'orthogonal' should be interpreted as "approximately 90 °", where the actual value may be within tolerance of the desired angle. For example, two things described as orthogonal may be positioned anywhere from 85 ° to 95 ° relative to each other. Other angles and measurements should be interpreted in a similar manner. Similarly, terms like 'light' are used throughout the specification to refer to electromagnetic radiation. Embodiments of the present disclosure are not limited to wavelengths within the visible spectrum. It should be understood that the various example materials listed throughout this specification may have certain optical properties over certain wavelength ranges, and that other materials may be used based on the wavelength(s) of electromagnetic radiation utilized. For example, if radiation in the far UV is used, the optical surface(s) of the sample holder may be made of a material transparent to the far UV.

Examples of the invention

Example details are given that may be used to implement, operate, and/or model one or more aspects described herein. It is understood that the specific details described herein are for specific examples only, and that the described details may be different for other example embodiments.

Multi-immersion open top light sheet microscope

Ray tracing software was used to model the optical schematic of a microscope with a modular sample holder. Illumination light is coupled into the system from a digitally controlled laser package through a single mode fiber with a numerical aperture of 0.12. The light emitted from the fiber (gaussian profile) was collimated using lens L1(f 19mm) and then spread along one axis using a 3-fold cylindrical telescope consisting of lenses C1(f 50mm) and C2(f 150mm) to provide multi-directional illumination. The resulting elliptical gaussian beam is then relayed to a scanning galvanometer GM using lenses R1(f 100mm) and R2(Thorlabs, f 50 mm). The scan mirror is driven at a frequency of 800Hz by a sinusoidal voltage from a waveform generator. A scanning lens SL (f 70mm) and a tube lens TL1(f 200mm) are used to relay the scanning beam to the back focal plane of the illumination objective. Finally, the elliptical beam travels through a plano-convex lens (R ═ 34.5mm), immersion medium, holder, and finally through the sample.

The fluorescence is collected by a multi-immersion objective lens providing in-plane resolution <1 μm for all immersion media and filtered using a motorized filter wheel with bandpass filters for 405nm, 488nm, 561nm and 638nm excitation wavelengths. The filtered fluorescence is focused by tube lens TL2(f 165mm) onto a 2048 × 2048 pixel sCMOS camera. The tube lens provides nyquist sampling of about 0.45 μm/pixel, which provides a horizontal field of view of about 0.9mm within 2048 pixels of the camera. The vertical field of view is reduced to 256 pixels to match the depth of focus (about 110 μm) of the illuminating light sheet. 256 pixels are oriented parallel to the rolling shutter readout direction of the camera, which provides an exposure time of 1.25ms and a frame rate of 800 Hz. The maximum imaging depth may be limited by the physical gap (0.5cm) of the holder and the collection objective. The illumination objective, the SIL and the collection objective are docked with the immersion chamber via an aluminum lens mount.

Image strips are collected using a motorized XY stage and Z actuator in combination with a combination of stage scanning and lateral/vertical tiling. Using stage scan firmware to send TTL trigger signals from XY stage to sCMOS camera for reproducible start positioning of each image strip (<1 μm). The spatial interval between successive frames is set to about 0.32 μm, which corresponds to a constant stage speed of about 0.25mm/sec given a 800Hz camera frame rate. For horizontal tiling, a 0.8mm offset (about 11% overlap) between adjacent image strips is used. For vertical tiling, a focal depth of 110 μm is oriented at 45 degrees, which corresponds to an image bar height of about 80 μm. Therefore, a vertical tiling offset of 70 μm (about 12% overlap) was used. According to a user-defined attenuation coefficient P ═ P₀X exp (z/μ), the laser power increases with depth to account for the attenuation of the illuminating light sheet as it penetrates deeper into the sample. The whole image acquisition is controlled by a program. The procedure consists of a series of nested loops for imaging multiple samples, collecting multiple color channels, and tiling horizontally/vertically.

Data processing and visualization

The collected data sets undergo a pre-processing routine prior to visualization in 2D and 3D. Each image strip is stored in a single DCIMG file. These DCIMG files are read by the DLL into RAM and first de-skewed by 45 degrees. By setting the interval between successive frames, de-skew is performed by shifting each plane of pixels in an image strip by an integer pixel offset. This operation may be relatively fast compared to alternative de-skewing methods that use computationally expensive affine transformations. The data is then written from RAM to disk using the hierarchical data format (HDF5), where the metadata and XML files are structured for subsequent analysis using bigstitch. A custom HDF5 compression filter (B3D) was used with default parameters to provide approximately 10 times compression, which is within the noise limits of sCMOS cameras. This pre-processing routine is applied to all DCIMG files, ultimately generating a single HDF5/XML file. Alignment of all image strips is performed and finally fused to disk in either TIFF or HDF5 file format. The resulting TIFF and HDF5 files were then visualized using an open source and commercial software package. To optionally provide a false color pseudo H & E histological image, a beer-lambert staining algorithm may be applied.

Sample holder

All holders were attached to the motorized XY stage using an aluminum adapter plate. For mouse brain sections, a 1mm thick fused quartz window with a 10 x 10cm cross section was attached to the adaptor plate using UV cured glue. Organs of mice cleared with Ce3D were imaged on custom 6-well plates. The bottom of the polystyrene 6-well plate was removed and replaced with a 0.5mm thick PMMA plate. For the expanded kidney sample, a "tympanic membrane (drumhead)" is fabricated and adapted to be mounted to a microscope. The tympanic membrane tightens a 0.1mm thick FEP film over the extrusion opening, which may be useful for imaging of the expanded sample. To overcome the hydrophobicity of the FEP film, which may cause drift of the expanded sample, the upper surface of the FEP film was treated with 0.1% (w/v) polylysine to electrically bond the sample to the FEP surface. A hiv ex lens blank was purchased and processed for human prostate biopsy. The system can also be used as a full slide scanner for conventional fluorescently labeled histological slides using commercially available slide holders.

Optical simulation

Optical simulations were performed using ray tracing software in conjunction with a "black box" model of the multi-immersion objective. For the simulation shown in fig. 11, assume that the fundamental refractive index of the immersion medium and the sample is n 1.45, and the optical path difference varies. For all scenes, the imaging depth was set to 1mm, and the PSF was measured at the center of the imaging field of view. The same relationship between strehl ratio and optical path difference was observed for other basic refractive indices and imaging depths, assuming the same optical properties of the immersion medium and the sample.

The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the various embodiments of the present invention. In this regard, no attempt is made to show structural details of the present invention in more detail than is necessary for the fundamental understanding of the present invention, the description taken with the drawings and/or the examples making apparent to those skilled in the art how the several forms of the present invention may be embodied in practice.

Of course, it should be understood that any of the examples, embodiments, or processes described herein may be combined with one or more other examples, embodiments, and/or processes, or separated and/or performed among separate devices or device portions, in accordance with the present systems, devices, and methods.

It should be understood that terms like 'top' and 'side' are used for ease of explanation and are only intended to indicate relative positioning of various components. Other embodiments may use other arrangements of components. Various components and operations may be described with respect to particular wavelengths of light. It should be understood that other wavelengths will be used (e.g., those outside the visible spectrum), and that light as used herein may represent any electromagnetic radiation. Certain materials may be described in terms of their optical properties (e.g., transparent) and it should be understood that a material having desired properties may be selected for any wavelength(s) of light used by the system.

Finally, the above-discussion is intended to be merely illustrative of the present system and should not be construed as limiting the appended claims to any particular embodiment or group of embodiments. Thus, while the present system has been described in particular detail with reference to example embodiments, it should also be appreciated that numerous modifications and alternative embodiments may be devised by those having ordinary skill in the art without departing from the broader and intended spirit and scope of the present system as set forth in the claims that follow. The specification and drawings are accordingly to be regarded in an illustrative manner and are not intended to limit the scope of the appended claims.