Apparatuses, systems and methods for microscope sample holders

Abstract

To provide a microscope capable of accommodating a variety of sample types and sample holders without interfering with the optics of the microscope.SOLUTION: The invention provides apparatuses, systems and methods for modular sample holders. A microscope may direct illumination light along an illumination path towards a sample, and collect light from the sample along a collection path. Light along the illumination path may pass through an immersion fluid and a material of a sample holder to reach the sample. Light along the collection path may pass through the material of the sample holder and the immersion fluid. The sample holder may have a first optical surface along the illumination path which is generally perpendicular to an optical axis of the illumination path. The sample holder may have a second optical surface along the collection path which is generally perpendicular to an optical axis of the collection path. The sample holder may be modular. The sample holder may contain the sample in an enclosed channel.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119 of the earlier filing date of U.S. Provisional Patent Application No. 62 / 804,606, filed February 12, 2019, the entire contents of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Research and Development Statement This invention was made with government support under Grant No. W81XWH-14-2-0183 awarded by the Department of Defense and Grant No. F32CA213615 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] FIELD OF THE DISCLOSURE Embodiments of the present invention relate generally to optical imaging, and in particular to microscopy. [Background technology]

[0004] Microscopes may generally involve directing light at a sample and then imaging the sample based on the light received from the sample. In some microscopes, the optics of the system may be located on the opposite side (bottom) of the sample holder from the sample. This may allow for better access to the sample, easier sample preparation / mounting, etc. Thus, the illumination light and collected light may pass through the material of the sample holder to pass between the optics and the sample. Summary of the Invention [Problem to be solved by the invention]

[0005] Different types of samples may require different types of sample holders to support them. For example, some samples may be mounted on a flat plate such as a glass slide, while other samples may be suspended in a liquid (e.g., in a well plate). It may be useful to have a microscope that can accommodate a variety of sample types and sample holders without interfering with the microscope's optics. [Means for solving the problem]

[0006] overview In at least one aspect, the present disclosure relates to an apparatus including an illumination optics arrangement, a collection optics arrangement, an immersion chamber, and a sample holder. The illumination optics arrangement directs illumination light along an illumination path to a sample. The collection optics arrangement receives 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 generally perpendicular to the illumination path, and a second optical surface along the collection path that is generally perpendicular to the collection path.

[0007] The apparatus may include a support member capable of supporting a sample holder in an orientation relative to the illumination path and the collection path. The sample holder may be a modular sample holder removably positionable in a receptacle of the support member. The first optical surface may be generally perpendicular to the second optical surface. The immersion medium, the sample, the first optical surface, and the second optical surface may have matched refractive indices. The sample holder may include an enclosed channel capable of containing the sample. The enclosed channel may be coupled to an inlet configured to provide fluid to the channel and an outlet configured to drain fluid from the channel. The sample holder may include a plurality of troughs. The sample holder may include a plurality of wells.

[0008] In at least one aspect, the disclosure relates 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 of light along an illumination path through the immersion fluid 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 that supports 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 positionable 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 generally perpendicular to the illumination path, and the second optical surface is along the collection path and generally perpendicular to the collection path.

[0009] The illumination beam may be a light sheet. The sample holder may include a trough configured to hold the sample. In that case, the first optical surface and the second optical surface may form a bottom of the trough. The sample holder may include a well configured to hold the sample. In that case, the first optical surface and the second optical surface may form a bottom of the well. The sample holder may include a channel capable of holding the sample. The first optical surface may be approximately perpendicular to the second optical surface. The sample, the immersion fluid, the first optical surface and the second optical surface may have matched refractive indices.

[0010] In at least one aspect, the present disclosure relates to an apparatus including illumination optics for providing illumination light to a sample, collection optics for receiving light from the sample, an immersion chamber for holding an immersion fluid, and a sample holder. The sample holder includes a sealed channel through a material of the sample holder. The sealed channel holds a sample. The sample holder is positionable such that a surface of the sample holder is adjacent to the immersion fluid. Illumination light passes through the immersion medium and the sample holder before reaching the sample, and light from the sample passes through the sample holder and the immersion medium before reaching the collection optics.

[0011] The enclosed channel may have a generally 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 may pass through the first optical surface and the collected light may pass through the second optical surface. The first optical surface may be generally perpendicular to an optical axis of the illumination light and the second optical surface may be generally perpendicular to an optical axis of the collected light. The first optical surface may be generally perpendicular to the second optical surface.

[0012] The first optical surface and the second optical surface may form a bottom surface of a confined channel. The confined channel may be coupled to an inlet configured to provide a fluid to the channel and an outlet configured to drain the fluid from the channel. The apparatus may also include a support member capable of supporting a sample holder. The sample holder may be a modular sample holder removably positionable on the support member. The illumination light may be a light sheet. [Brief description of the drawings]

[0013] [Figure 1] 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. [Diagram 2] FIG. 1 is a perspective view of a microscope having a modular sample holder according to some embodiments of the present disclosure. [Diagram 3] 1A and 1B are side and top views of a modular sample holder having a flat surface according to some embodiments of the present disclosure. [Figure 4] 1A and 1B are side and top views of a modular sample holder having a sample well according to some embodiments of the present disclosure. [Diagram 5] 1A and 1B are side and top views of a modular sample holder having a sample trough according to some embodiments of the present disclosure. [Figure 6]1A-1D are side and top views of a modular sample holder having a well with a trough bottom according to some embodiments of the present disclosure. [Figure 7] 1A and 1B are side and top views of a modular sample holder having a flow channel according to some embodiments of the present disclosure. [Figure 8] 1A-1D are side and top views of a modular sample holder having flow channels and optical surfaces according to some embodiments of the present disclosure. [Figure 9] 1A-1D are side and top views of a modular sample holder having flow channels and optical surfaces according to some embodiments of the present disclosure. [Figure 10A] FIG. 1 is a side cross-sectional view of a sample holder having an open flow cell according to some embodiments of the present disclosure. [Figure 10B] FIG. 1 is a side cross-sectional view of a sample holder having a closed flow cell according to some embodiments of the present disclosure. [Figure 11] 1 is a chart of compatibility of different immersion fluids and sample holder materials, according to some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description The following description of specific embodiments is merely exemplary in nature and is not intended to limit the scope of the disclosure or its application or uses. In the following detailed description of the embodiments of the system and method according to the present invention, reference is made to the accompanying drawings, which form a part of this specification and which are shown for illustration of specific embodiments in which the described system and method may be practiced. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the presently disclosed system and method, and it should be understood that other embodiments may be utilized and structural and logical changes may be made without departing from the spirit and scope of the present disclosure. Moreover, for the purposes of clarity, detailed descriptions of specific features will not be discussed if they are obvious to those skilled in the art so as not to obscure the description of the embodiments of the present disclosure. Therefore, the following detailed description is not to be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims.

[0015] Microscopy may be used in a wide range of applications to generate images of a sample with a field of view and / or resolution that is typically invisible to the naked eye. Illumination light may be directed to the sample using illumination optics. Collection optics may be used to focus light from the sample onto a detector (e.g., a CCD detector, a CMOS detector, or the user's eye). In some cases, the light that reaches the detector may include a portion of the illumination light. In some cases, the light that reaches the detector may be emitted from the sample (e.g., via fluorescence) after being excited to emit by the illumination light. It may be desirable to ensure that the field of view is uniformly illuminated and that the illumination penetrates to the depth of the sample.

[0016] The illumination optics and collection optics may be positioned on the opposite side of the sample holder from the sample (e.g., underneath the sample holder). Thus, the illumination and collection light may pass through the material of the sample holder. The illumination optics and / or collection optics may also be positioned at an angle to the sample holder. For example, the illumination optics and collection optics may each be positioned at a 45° angle to the sample holder. This may provide advantages for optimizing imaging parameters and imaging depth, but non-normal incidence of the illumination and / or collection light on the sample holder may lead to various aberrations. Each individual sample holder may be optically optimized for a particular setup, but different sample types may have different sample holder requirements. It may be useful for a microscope to accommodate different sample holders that are compatible with the microscope's optics.

[0017] The present disclosure is directed to a microscope sample holder. The microscope includes a sample holder having a first side and a second side opposite the first side. The illumination optics and collection optics are positioned on the second side of the sample holder when the sample holder is positioned in the microscope. The illumination light passes through an immersion fluid and a material of the sample holder before reaching the sample. The collection light passes through a material of the sample holder and through the immersion fluid before reaching the sample. The sample holder may be a modular component of the 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 an illumination optical path between the immersion fluid and the sample, and a second optical surface along a collection optical path between the sample and the immersion fluid. The first optical surface may be approximately perpendicular to the optical axis of the illumination path, and the second optical surface may be approximately perpendicular to the optical axis of the collection path. The use of optical surfaces may reduce the need for refractive index matching between the sample and sample holder materials and the immersion fluid, and may also allow the use of objectives (e.g., illumination and / or collection objectives) with shorter working distances.

[0018] 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 including a microscope 102 and an optional controller 104 that may operate the microscope 102 and / or interpret information from the microscope 102. In some embodiments, one or more components of the controller 104 may be omitted and the microscope 102 may be manually operated. In some embodiments, one or more components of the controller 104 may be integrated into the microscope 102. In some embodiments, as in the example of FIG. 1, the microscope 102 may be an open-top microscope.

[0019] The microscope 102 includes a sample holder 108 that supports a sample 106. A bottom surface of the sample holder 108 is adjacent to an immersion fluid 112 contained within an immersion chamber 110. The microscope 102 of FIG. 1 has an illumination path and a collection path that are separated from each other. The illumination path includes a light source 118, an illumination optics 120, and an illumination objective 122. The illumination path provides an illumination beam 124 that passes through the immersion medium 112 and the sample holder 108 to illuminate the sample 106. The collection path includes a collection objective 128, a collection optics 130, and a detector 132. The collection path can collect light from a focal region 126 illuminated by the illumination beam 124.

[0020] The microscope 102 includes a modular sample holder 108, which may be interchangeable with various other modular sample holders 108. Examples of different types of modular sample holders 108 are described in more detail in FIGS. 3-10B. The sample holder 108 may be removably coupleable to a support member 109 of the microscope 102. The support member 109 may be disposed along a top surface of an immersion tank 110 that holds an immersion fluid 112. In some embodiments, the support member 109 may form a lid or other upper housing for the immersion tank 110. The support member 109 may include an opening that exposes the immersion fluid 112. The opening may act as a receptacle for the modular sample holder 108. When the sample holder 108 is positioned in the receptacle, a bottom surface of the sample holder 108 may be adjacent to the immersion fluid 112. In some embodiments, the underside of the sample holder 108 can contact the immersion fluid 112. For example, the underside of the sample holder 108 can extend into the immersion bath 110. In some embodiments, the support member 109 and / or the sample holder 108 can include a gasket or other sealing member to prevent the immersion fluid 112 from leaking between the seams between the sample holder 108 and the support member 109.

[0021] In some embodiments, the sample holder 108 can rest within the receptacle of the support member 109 without any type of attachment (other than gravity). For example, the receptacle may have a tapered profile and / or 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 combinations thereof may be used to removably couple the sample holder to the support member 109.

[0022] In some embodiments, the sample holder 108 may be a one-piece body that fits directly into a receptacle in the support member 109. In some embodiments, an adapter plate may be used that fits into the receptacle in the support member 109 and includes a receptacle for the sample holder 108. In some embodiments, the sample holder 108 may be attached (e.g., with an adhesive) to the adapter. For example, the sample holder 108 may be glued to the adapter with a UV-curable adhesive along the perimeter of the sample holder 108. In some embodiments, the sample holder 108 may be a commercially available sample holder that is modularized through the use of an adapter. In some embodiments, the adapter may include mounting hardware (e.g., fasteners) that mate with the support member 109.

[0023] The support member 109 may have a recessed area that holds the sample holder 108. The modular sample holder 108 may be inserted into this recessed area. In some embodiments, the sample holder 108 may 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 the placement of the sample holder 108 to one of four orientations, rectangular to limit the placement of the sample holder 108 to one of two orientations, or any other shape. In some embodiments, the sample holder 108 may be shaped such that there is only one orientation in which the sample holder 108 fits into the receptacle of the support member 109.

[0024] The light source 118 provides illumination light along the illumination path to illuminate the focal region 126 of the sample 106. The light source 118 may be a narrowband light source, such as a laser or light emitting diode (LED), capable of emitting light in a narrow spectrum. In some embodiments, the light source may be a broadband light source (e.g., incandescent light source, arc light source) capable of producing 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 subdivide the wavelengths of the illumination light. For example, a bandpass filter may receive the broadband illumination from the light source 118 and provide a narrower spectrum of illumination light. In some embodiments, the light source 103 may be a laser and may produce collimated light.

[0025] In some embodiments, the optical system 100 may be used to image fluorescence in the sample 106. The illumination beam 124 may include light at a specific excitation wavelength that may excite fluorophores in the sample 106. The illumination beam 124 may include a broad spectrum of light that includes the excitation wavelength, or may be narrowband centered on the excitation wavelength. In some embodiments, the light source 118 may generate a narrow spectrum of light centered on (or close to) the excitation (wavelength). In some embodiments, a filter (not shown) may be used in the illumination optics 120 to limit the illumination beam 124 to wavelengths close to the excitation wavelength. When excited by the illumination sheet, fluorophores in the sample 106 may emit light (which may be centered on a predetermined emission wavelength). The collection path (e.g., collection optics 130) may include one or more filters that may be used to limit the light reaching the detector 132 to wavelengths of light close to the emission wavelength.

[0026] The illumination optics 120 can couple light from the light source 118 to the illumination objective 122. For example, the illumination optics 120 can include an optical fiber that transmits light from the light source 118 to the back end of the illumination objective 122. In some embodiments, the illumination optics 120 can couple light between the light source 118 and the objective 122 without substantially changing the light provided by the light source 118. In some embodiments, the illumination optics 120 can change the shape, wavelength, intensity, and / or other characteristics of the light provided by the light source 118. For example, the illumination optics 120 can receive broadband light from the light source 118 and filter the light (e.g., with a filter, a diffraction grating, an acousto-optic modulator, etc.) to provide narrowband light to the objective 122.

[0027] In some embodiments, the illumination path can provide illumination beam 124, which is a light sheet microscope or a light sheet as part of a light sheet fluorescence microscope (LSFM). The light sheet can have a generally elliptical cross-section with a first numerical aperture along a first axis (e.g., the Y axis) and a second numerical aperture along a second axis orthogonal to the first axis, the second numerical aperture being greater than the first numerical aperture. The illumination optics 120 can include optical devices that reshape the light received from the light source 118 into an illumination sheet. For example, the illumination optics 120 can include one or more cylindrical optical devices that focus the light in one axis but not in an orthogonal axis.

[0028] In some embodiments, the illumination optics 120 may include scanning optics that 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 intended focal region 126. In this case, the illumination optics 120 may rapidly oscillate the illumination beam 124 across the intended focal region 126 to ensure illumination of the focal region 126.

[0029] The illumination objective 122 may include one or more lenses that provide the 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 generally within the sample 106. The illumination objective may, in some embodiments, be a commercially available objective lens that includes 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 generally flat in shape. For example, in a LSFM, the focal region 126 may be a plane that lies along the plane of a light sheet generated as the illumination beam 124.

[0030] 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 at which light converges at the focal region 126. In some embodiments where the illumination objective 122 is in the ambient environment outside the immersion fluid 112, a window or lens may then be used to couple the illumination beam 124 into the immersion fluid 112. For example, in the illustration of FIG. 1, an illumination solid immersion lens (SIL) 114 is shown 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., similar to how the focusing objective 128 is shown, as described in more detail herein), and the SIL 114 may be omitted.

[0031] 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 surrounding environment (e.g., air), including the illumination objective 122, through the material of the illumination SIL 114 and into the immersion fluid 112. The illumination beam 124 may then pass through the immersion fluid 112 towards the sample 106 and sample holder 108.

[0032] The illumination beam 124 may be directed at the sample 106. The sample 106 may be supported by a sample holder 108. In some embodiments, the sample 106 may be placed directly on the top surface of the sample holder 108. In some embodiments, the sample 106 may be packaged in a container (e.g., on a glass slide, in a well plate, in a tissue culture flask, etc.) and the container may 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 before being imaged with the optical system 100. For example, the sample 106 may be washed, sliced, and / or labeled prior to imaging.

[0033] In some embodiments, the sample 106 may be a biological sample. For example, the sample 106 may be tissue biopsied from an area suspected of disease (e.g., cancer). In some embodiments, the tissue may undergo various processes, such as optical distance, tissue slicing, and / or labeling, before being examined by the optical system 100. In some embodiments, examination of the tissue by the optical system 100 may be used for diagnosis, to determine progress of a treatment, to monitor disease progression, etc.

[0034] In some embodiments, the sample 106 may be non-living. For example, the sample 106 may be a fluid and may contain one or more components for investigation. For example, the sample 106 may be a combustion gas and the optical system 102 may perform particle image velocimetry (PIV) to characterize the components of the gas.

[0035] In some embodiments, the sample 106 may include one or more types of fluorophores. The fluorophores may be endogenous to the sample 106 (e.g., DNA and proteins in a biological sample) or may be fluorescent labels (e.g., acridine orange, eosin) that are deposited on the sample 106. Some samples 106 may include a mixture of endogenous types of fluorophores and fluorescent labels. Each type of fluorophore may have an excitation spectrum that may be centered around an excitation wavelength. When a fluorophore is excited with light of the excitation spectrum, it may emit light of an emission spectrum that may be centered around an emission wavelength different from (e.g., red-shifted from) the excitation wavelength.

[0036] The sample holder 108 can support the sample 106 on a material of the sample holder that is generally transparent to the light collected from the illumination beam 124 and the focal region 126 of the sample 106. In some embodiments, the sample holder 108 can have a window of transparent material over which the sample 106 can be positioned, and the remainder of the sample holder 108 can be formed from a non-transparent material. In some embodiments, the sample holder 108 can be made from a transparent material.

[0037] The sample holder 108 may have a second surface (e.g., a bottom surface) opposite the surface of the sample holder 108 that supports the sample 106. An immersion chamber 110 that holds an immersion fluid 112 may be positioned below the second surface of the sample holder 108. In some embodiments, the immersion chamber 110 may have an open top. In that case, the immersion fluid 112 may be adjacent to the second surface of the sample holder 108. In some embodiments, the second surface of the sample holder 108 may be in contact with the immersion fluid 112, while the first surface of the sample holder 108 (which supports the sample 106) may be in contact with the environment (e.g., air) that is equivalent to the objective lenses 122 and 128.

[0038] The support member 109 may be coupled to an actuator 107 that may be capable of moving the support member 109 and / or the sample holder 108 in one or more directions. In some embodiments, the sample holder 108 may be capable of moving in up to three dimensions (e.g., along the x-axis, y-axis, and z-axis) relative to the immersion chamber 110 and the objective lenses 122, 128. The sample holder 108 may be moved to change the position of the focal region 126 within the sample 106 and / or to move the 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 that may respond to manual input and / or commands from the controller 104. In some embodiments, the actuator 107 may accommodate both manual adjustment and automatic control (e.g., a knob that responds to both manual rotation and commands from the controller 104).

[0039] The immersion chamber 110 contains the immersion fluid 112. In some embodiments, the immersion chamber 110 may include a source and / or sink that may be useful for exchanging the immersion fluid 112. For example, the immersion chamber 110 may be coupled to a fluid input line that supplies the immersion fluid 112 (which may in turn be coupled to a pump and / or reservoir) and a drain that may be opened to drain 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 refractive index of the sample 106 and / or sample holder 108.

[0040] The collection path can receive light from the focal region 126 and direct the received light to a detector 132 that can image and / or otherwise measure the received light. The light from the focal region 126 can be a redirected portion of the illumination beam 124 (e.g., scattered light and / or reflected light), can be light emitted from the focal region 126 in response to the illumination beam 124 (e.g., via fluorescence), or can be a combination thereof. The collected light can pass through the sample holder 108 and the immersion fluid 112 before reaching the collection objective 128. In some embodiments, the collection objective 128 can 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 can be positioned inside the immersion bath 110. In some embodiments, the focusing 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 focusing objective 128.

[0041] The geometry of the focal region 126 may be defined in part by the field of view of the collection path, which in turn may depend in part on the numerical aperture of the collection objective 128. Like the illumination objective 122, the collection objective 128 may be a commercially available objective including one or more lenses. In some embodiments, the collection objective 128 may be an air objective. In some embodiments, the focal region where the collection path is focused and the focal region where the illumination path is focused may substantially overlap at the focal region 126. In some embodiments, the illumination and collection paths may have different shapes, sizes, and / or locations of their respective focal regions.

[0042] 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 that is projected onto the detector 132. In some embodiments, collection optics 130 may include one or more elements that modify the light received from collection objective 128. For example, collection optics 130 may include filters, mirrors, descanning optics, or combinations thereof.

[0043] Detector 132 may be used to image focal region 126. In some embodiments, detector 132 may represent an eyepiece so that a user can view focal region 126. In some embodiments, detector 132 may generate a signal to record an image of focal region 126. For example, detector 132 may include a CCD array or a CMOS array capable of generating an electronic signal based on light incident on the array.

[0044] The illumination path can direct light along a first optical axis. The collection path can collect light along a second optical axis. In some embodiments, such as that shown in FIG. 1, the first optical axis and the second optical axis can be mutually orthogonal. Each of the first optical axis and the second optical axis can be non-orthogonal to the sample holder. For example, the first optical axis can be at a 45° angle with respect to the bottom surface of the sample holder 108, and the second optical axis can also be at a 45° angle with respect to the bottom surface of the sample holder 108. Other angles between the first optical axis, the second optical axis, and / or the sample holder 108 can be used in other examples.

[0045] 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 to the bottom surface of the sample holder, while the collection path may follow a second optical axis that is at a 90° angle to the bottom surface of the holder. Thus, there may be an angle of about 45° between the first and second optical axes.

[0046] 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), which in some embodiments may be immersed in (or have a front surface in contact with) the immersion fluid 112. In some embodiments, the air objective may have a SIL positioned between the objective and the immersion fluid, although a SIL may not be used with a non-air objective. For example, the illumination objective 122 may direct the illumination beam 124 through the illumination SIL 114 into the immersion fluid 112, while the collection objective 128 may be adjacent to (e.g., in contact with) the immersion fluid 112.

[0047] The microscope 102 may be coupled to a controller 104 that may be used to operate one or more components of the microscope 102, display data from the microscope 102, interpret data from the microscope 102, or combinations 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 components of the controller 104 may be integral to the microscope 102.

[0048] The controller 104 includes one or more input / output devices 142 that enable a user to display feedback from the controller 104, data from the microscope 102, provide commands to the controller 104, provide commands to the microscope 102, or combinations thereof. For example, the input / output devices 142 may include a digital display, a touch screen, a mouse, a keyboard, or combinations thereof.

[0049] The controller 104 includes a processor 140 that can execute one or more instructions stored in the memory 144. These instructions can include control software 152. The control software 152 can then include instructions on how to control the microscope 102. Based on the control software 152, the processor 140 can cause the controller 104 to send signals to various components of the microscope 102, such as the actuator 107. The instructions can include image processing software 150 that can be used to process a "live" image from the detector 132 or an image 146 that was stored in the memory 144 at a previous time. The image processing software 150 can, for example, eliminate background noise from the image 146. The instructions can include analysis software 148 that can be executed by the processor 140 to determine one or more characteristics of the image 146. For example, the analysis software 148 can highlight cell nuclei in the image 146.

[0050] In some embodiments, the controller 104 can instruct the microscope to collect images from multiple different fields of view within the sample. For example, the controller 104 can include instructions to collect a stack depth of images. The controller 104 can instruct the detector 132 to collect a first image, and then instruct the actuator 107 to move the sample holder 108 vertically (e.g., along the z-axis) a set distance. This can also move the sample 106 relative to the focal region 126, which can change the height within the sample at which the focal region 126 is located. The controller 104 can then instruct the detector 132 to collect another image, and then repeat the process, up to a set number of images in the stack and / or until a set total displacement in the z-direction is achieved. The analysis software 148 can then combine the stack depth of images to enable 3D (or pseudo-3D) imaging of the sample 106.

[0051] 2 is a perspective view of a microscope having a modular sample holder according to some embodiments of the present disclosure. In some embodiments, microscope 200 illustrates an example layout of how a microscope, such as microscope 102 of FIG. 1, may be assembled. The components and operation of microscope 200 may, in some embodiments, be generally similar to the components and operation of microscope 102 of FIG. 1. For brevity, the components and operations described with respect to microscope 102 of FIG. 1 are not repeated again with respect to FIG. 2.

[0052] The microscope 200 includes an illumination optics 221 coupled to an illumination source 220, which 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 for combining the light output from the illumination source 220. The illumination optics 221 may include forming optics that form the illumination light into a light sheet. The illumination optics 221 may include one or more scanning optics, which may then be motorized to scan the illumination light sheet across a back end of an illumination objective (not shown), which can then be scanned across the sample.

[0053] The illumination light sheet passes through an immersion bath 212 positioned below the support member 209. The support member 209 may be a stage, such as a motorized stage. For example, the stage may move in one or more axes, such as an XY stage. In some embodiments where the support member 209 is motorized in several axes, movement in other axes may be added by coupling one or more components of the microscope 200 to additional actuators (such as a Z-axis actuator).

[0054] The view of Figure 2 shows a sample holder 208 positioned over a receptacle of a support member 209. The sample holder 208 is shown as a flat plate that may be lowered into an imaging receptacle. Different sample holder types that may be used with the microscope 200 are discussed in more detail in Figures 3-10B.

[0055] Microscope 200 includes collection optics 231 that collects light from a focal region of microscope 200 and provides the collected light to detector 232. The collection optics 231 may include a filter wheel that includes multiple filters that may be rotated into the collection path of microscope 200. Each filter may be associated with a different emission spectrum (e.g., a different type of fluorophore that may be imaged). Detector 232 may be an sCMOS detector in some embodiments.

[0056] 3-10B show various sample holders that may be used in some embodiments as sample holder 108 of FIG. 1 and / or sample holder 208 of FIG. 2. FIGS. 3-9 show both a top view of the sample holder and a side view of a portion of the sample holder. The side view shows a cross section of a portion of the sample holder containing a sample. The side view shows the sample holder when it is placed in a microscope (e.g., microscope 102 of FIG. 1 and / or microscope 200 of FIG. 2) and the microscope is aligned such that the focal region of the microscope is within the sample shown in the side cross section view. FIGS. 10A-10B show a pair of side views of two variations of the sample holder, rather than top and side views.

[0057] The drawings in Figures 3-10B show a portion of the sample holder that does not include a means for attaching the sample holder to a microscope. In some embodiments, the sample holder can be plugged into a receptacle on a microscope. In some embodiments, the sample holder can include a mounting hardware (not shown) that plugs into the receptacle and / or attaches to a support member of the microscope (e.g., via a clip, magnet, pin, screw, etc.). For example, the sample holder can be attached to an adapter plate that can subsequently fit into the receptacle and / or can include a mounting hardware.

[0058] Each of Figures 3-10B shows an exemplary embodiment having an air illumination objective and an immersion collection objective, although it should be understood that any combination of air and immersion objectives may be used for the illumination and collection paths. Each of Figures 3-10 shows a generally rectangular top view of a sample holder, however it should be understood that many features and operations may be similar between the various sample holders, and for brevity such features will not be separately described in each of Figures 3-10.

[0059] For example, each of the sample holders of Figures 3-10B includes various materials, such as the material of the sample holder, the immersion fluid, the sample, the fluid surrounding the sample, etc. 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.

[0060] In some embodiments, a single sample holder can have a mixture of the geometries and features described in Figures 3-10B. For example, a sample holder can have a first region having a first geometry for holding a sample and a second region having a second geometry for holding a sample.

[0061] In some embodiments, one or more surfaces of the sample holder may include one or more coatings, for example, a hydrophobic coating may be disposed on the sample holder to promote bead-up of samples spotted onto the sample holder.

[0062] 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. The diagram 300 shows a sample holder 308 that is generally flat. For example, the sample holder 308 may be a transparent plate and the sample 306 may be supported on the top surface of the plate. When a flat sample holder 308 is used, the microscope may operate similarly to a flatbed scanner, where the sample may be placed on the top surface of the sample holder 308. In some embodiments, multiple samples 306 may be placed on the sample holder 308 and may be separated from each other by air. In some embodiments, the sample 306 may include a liquid phase. In that case, the sample 306 may be droplet deposited on the surface of the sample holder 308.

[0063] 4 illustrates a side view and a top view of a modular sample holder with sample wells, according to some embodiments of the present disclosure. The sample holder 408 may be generally flat (e.g., similar to the sample holder 308 of FIG. 3), however, the sample holder 408 includes a surrounding medium 411 that may form an area containing each sample 406. For example, the sample holder 408 may have defined sample areas separated from each other by the surrounding medium 411. In some embodiments, the surrounding medium 411 may be the same material as the remainder of the sample holder 408. In some embodiments, the sample holder 408 may be a flat plate of a first material on whose top surface the surrounding medium 411 may be attached.

[0064] The sample holder 408 may include one or more different sample regions, each of which may have a different sample (or samples) 406a-h disposed therein. For example, the sample holder 408 may be a well plate capable of holding a number of different samples 406a-h.

[0065] 5 illustrates a side view and a top view of a modular sample holder with a sample trough, according to some embodiments of the present disclosure. The sample holder 508 of FIG. 5 includes multiple sample troughs 515, each of which can hold one or more samples 506. The sample troughs 515 have a bottom with a first optical surface 517 and a second optical surface 519, which can assist in coupling an illumination beam 524 to the sample 506 and coupling collected light 525 from the sample 506, respectively.

[0066] The side view of FIG. 5 shows a cross section of one of these troughs 515. The trough may be filled with a fluid 513. This fluid 513 may be the same as the immersion fluid 512 or may be different. In some embodiments, the fluid 513 may have a refractive index that matches the refractive index of the sample 506. In some embodiments, the fluid 513 may have a different refractive index than the sample 506. In some embodiments, the fluid 513 may have the same refractive index as the material of the sample holder 508 and / or the immersion fluid 512. The fluid 513 may be used to support, stabilize and / or protect the sample 506 during imaging. For example, the sample 506 may be cells and the fluid 513 may represent cell culture medium. In some embodiments, the fluid 513 may be an ambient medium such as air.

[0067] The trough 515 has a first optical surface 517 along the illumination path of the illumination beam 524 between the immersion fluid 512 and the sample 506 (and / or between the immersion fluid 512 and the fluid 513). The first optical surface 517 may be positionable such that the first optical surface 517 is approximately perpendicular to the optical axis of the illumination path. This may reduce refraction of light as it passes from the immersion fluid 512 into the material of the sample holder 508. In some embodiments, the first optical surface 517 may be a flat surface with a uniform thickness.

[0068] The trough 515 has a second optical surface 519 along the collection path of the collected light 525 between the sample 506 (and / or fluid 513) and the immersion fluid 512. The second optical surface 519 may be positionable such that the second optical surface 519 is approximately perpendicular to the optical axis of the collection path, thereby reducing refraction of light as it passes from the sample 506 / fluid 513 through the material of the sample holder 508 and the immersion fluid 512. In some embodiments, the second optical surface 519 may be a flat surface with a uniform thickness.

[0069] The first optical surface 517 and the second optical surface 519 may be made of the same material as the remainder of the sample holder 508. For example, the sample holder 508 may be a one-piece body formed from a single piece of material. In some embodiments, the first optical surface 517 and the second optical surface 519 may be made of a different material than the remainder of the sample holder 508. For example, the first optical surface 517 and the second optical surface 519 may be made of a transparent material, while the remainder of the sample holder 508 is made of a non-transparent material.

[0070] The first optical surface 517 and the second optical surface 519 can form the "bottom" of the trough 515. The first optical surface 517 and the second optical surface 519 can be at an angle, which may be based on the angle between the illumination path and the collection path. For example, the first optical surface 517 may be at an angle of about 90° with respect to the second optical surface 519. In some embodiments, the first optical surface 517 and the second optical surface 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 generally parallel to the bottom surface of the non-trough portion of the sample holder 508, giving the bottom surface of the trough 515 a generally trapezoidal cross-section rather than a triangular cross-section.

[0071] The troughs 515 may extend along the length of the sample holder 508. In some embodiments, each trough 515 may be closed to prevent leakage of the fluid 513 and / or sample 506 from the trough 515. For example, each trough 515 may terminate a distance from the edge of the sample holder 508 such that the material of the sample holder 508 seals the trough 515. In some embodiments, one or more end caps may be attached to the sample holder 508 to seal the trough 515. In some embodiments, the bottom of each trough 515 may extend from the bottom of the sample holder 508 (e.g., to form a series of ridges extending along the bottom surface of the sample holder 508).

[0072] The trough 515 may have a long axis that extends across the length of the sample holder 508. The sample holder may be positionable within the microscope (e.g., within a receptacle of the support member 109 of FIG. 1 and / or the support member 209 of FIG. 2) and the long axis may be generally perpendicular to the illumination and collection paths from a "top-down" perspective. For example, the long axis of the trough 515 may be perpendicular to a line drawn between the illumination and collection objectives when the sample holder 508 is placed within the microscope. In some embodiments, the sample holder 508 may include multiple troughs 515. In some embodiments, the troughs may be generally parallel to one another.

[0073] Once the sample holder 508 is placed in the microscope, it may be moved (e.g., by actuator 107 in FIG. 1 ) to image different areas of the sample 506 and / or different samples. The different samples may be in the same trough 515 or different troughs of the sample holder 508. As the sample holder 508 moves relative to the immersion fluid 512, the first optical surface 517 and the second optical surface 519 may move relative to the illumination and collection paths. Because the first optical surface 517 and the second optical surface 519 are flat and perpendicular to their respective optical paths, the movement of the optical surfaces 517 / 519 may introduce relatively small aberrations because the illumination and collection paths continue to encounter flat surfaces perpendicular to their respective optical axes. The imageable area of ​​the sample 506 may be based in part on the area over which the imaging beam 524 and / or the collected light 525 can move relative to the first optical surface 517 or the second optical surface 519, respectively, without interfering with non-optical surface portions and / or other optical surfaces of the sample holder 508.

[0074] In some embodiments, the first optical surface 517 and the second optical surface 519 may be useful to reduce the working distance of the focal region for 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 objectives may be about 5-15 mm. In other exemplary embodiments, longer or shorter working distances may be used. In some embodiments, the objective may be an immersion objective, and the objective may have a front surface positioned near the optical surface. In some embodiments, the optical surface may be approximately parallel to the front surface of the objective (e.g., the front lens). For example, in embodiments where the objective is an air objective and is separated from the immersion fluid by a window or lens (e.g., SIL), the window / lens surface may be near the optical surface.

[0075] Figure 6 shows a side view and a top view of a modular sample holder having a trough bottom and a well, according to some embodiments of the present disclosure. Figure 6 shows a sample holder 608 that is generally similar to sample holder 508 of Figure 5 except that rather than being an elongated trough, the sample holder 608 has a number of separate wells 615, each of which has a first optical surface 617 and a second optical surface 619 generally similar to the first and second optical surfaces of Figure 5.

[0076] Each well 615 can hold a different sample 606. Each well can have first and second optical surfaces 617 and 619 that are generally perpendicular to the illumination and collection paths, respectively. In some embodiments, the optical surfaces 617 and 619 can extend along the length of the sample holder 608. For example, the sample holder 608 can have a shape similar to the sample holder 508 of FIG. 5, except that the sample holder 608 can have a "wall" positioned along the trough to separate the trough into different wells 615. In some embodiments, the optical surfaces 617 and 619 can stop between each well. In some embodiments, each of the wells 615 can have a rectangular cross-section when viewed from above and below. In some embodiments, other cross-sectional shapes may be used.

[0077] 7 illustrates a side and top view of a modular sample holder with flow channels according to some embodiments of the present disclosure. The sample holder 708 can include one or more channels 715 that are sealed within the material of the sample holder 708. Each channel 715 can be loaded with one or more samples 706, which can be surrounded by a fluid 713.

[0078] The sample holder 708 may include a channel 715 formed in the material of the sample holder 708. For example, the periphery of the channel 715 may be formed from the material of the sample holder 708. In some embodiments, the channel 715 may have a circular cross-section. In other exemplary embodiments, other cross-section shapes may be used. 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 cellular, the channel 715 may have a diameter that allows the cells of the sample 706 to pass "single file" along the length of the channel 715. In some embodiments, the channel 715 may be part of a microfluidic system that extends through the sample holder 708. For example, the sample holder 708 may be a microfluidic chip, and the channel 715 may represent a microfluidic flow channel through a transparent region of the chip that may be imaged.

[0079] In some embodiments, the sample holder 708 may include ports that can be used to fill or empty the channel 715 with the sample 706 (and / or fluid 713). In some embodiments, the channel may vary in size and / or shape along its length. In some embodiments, they may be multiple separate channels 715, such as multiple parallel channels 715 similar to the parallel troughs 515 of FIG. 5. In some embodiments, they may be one long channel 715 with multiple parallel segments (e.g., the channel 715 may switch back on itself one or more times).

[0080] 8 illustrates a side view and a top view of a modular sample holder having a flow channel and optical surfaces according to some embodiments of the present disclosure. Sample holder 808 has a channel (similar to channel 715 in FIG. 7) and first and second optical surfaces 817 and 819 (similar to optical surfaces 517 and 519 in FIG. 5 and 617 / 619 in FIG. 6).

[0081] The first optical surface 817 and the second optical surface 819 may in some embodiments be facets of a protrusion of the material of the sample holder 808 extending from a bottom surface of the sample holder 808. The channel 815 may have a shape that does not have flat surfaces corresponding to the first optical surface 817 and the second optical surface 819. For example, as shown in FIG. 8, the channel 815 may have a circular cross-section, while the first and second optical surfaces 817 and 819 may form a protrusion having a generally triangular cross-section.

[0082] 9 is a side view and a top view of a modular sample holder having a flow channel and optical surfaces according to some embodiments of the present disclosure. The sample holder 908 of FIG. 9 may be generally similar to the sample holder 808 of FIG. 8, except that the channel 915 of the sample holder 908 is shaped such that the first optical surface 917 and the second optical surface 919 have a back surface that is parallel to their front surfaces. For example, the channel 915 may have a general cross-section similar to the trough 515 of FIG. 5 and / or the well 615 of FIG. 6, except that the channel 915 has a "lid". In some embodiments, the channel 915 may be 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 "lid" that may be the same or different from the material of the remainder of the sample holder 908.

[0083] 10A-10B are side cross-sectional views of sample holders having an open flow cell and a closed flow cell, respectively, according to some embodiments of the present disclosure. Sample holders 1000a and 1000b may be generally similar to each other, except that sample holder 1000a is closed by the material of the sample holder, whereas sample holder 1000b is open to the surrounding environment (e.g., air). For brevity, only flow cell 1000a will be described in detail. In some embodiments, the flow cell of sample holder 1000a (or 1000b) may be incorporated into any of the geometries of sample holders 308-908 of FIGS. 3-9, respectively.

[0084] The sample holder 1000a includes an inlet 1021 and an outlet 1023. The inlet can supply a circulating fluid 1013 to the chamber, while the outlet 1023 can drain the circulating fluid 1013. The circulating fluid 1013 can thus flow through the chamber. In some embodiments, one or more different circulating fluids 1013 can be flowed across a 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.

[0085] In some embodiments, the sample holders 1000a and 1000b may include an inlet port 1021 and an outlet port 1023 that may mate with corresponding inlet and outlet ports of a microscope when the sample holders 1000a and 1000b are positioned within the microscope.

[0086] 11 is a compatibility chart of different immersion fluids and sample holder materials according to some embodiments of the present disclosure. This chart 1100 may be based on optical modeling of the interaction of different immersion fluids (e.g., 112 in FIG. 1) with different sample holder materials (e.g., 108 in FIG. 1). In some embodiments where the sample holder includes more than one material, the sample holder material may represent the material used in the portion of the sample holder that interacts with the illumination and collection paths. For example, the sample holder material may represent the material of the first and second optical surfaces (e.g., 517 and 519 in FIG. 5).

[0087] The rows represent different types of immersion fluids, each listed with its 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. This maximum thickness is expressed in millimeters, and is marked "chemically incompatible" for combinations where the immersion fluid would cause damage to the sample holder structures.

[0088] Various example values ​​are provided throughout this specification. It should be understood that these values ​​are approximate since perfect alignment may not be possible in a practical system. Thus, values ​​such as "perpendicular" or "orthogonal" should be interpreted as representing "near 90°" where the actual value may be within the intended angular tolerance. For example, two things described as orthogonal may be positioned anywhere between 85° and 95° from each other. Other angles and measurements should be interpreted similarly. Similarly, terms such as "light" are used throughout this specification to refer to electromagnetic radiation. The 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 specific optical properties over a particular wavelength range, and other materials may be used based on the wavelength of electromagnetic radiation utilized. For example, when using deep UV light, the optical surfaces of the sample holder may be made of a material transparent to deep UV light.

[0089] Working Example In the following, exemplary details are provided that can be used to implement, operate, and / or model one or more aspects described herein. It should be understood that the specific details described herein are for specific examples only, and that the details described may differ in other exemplary embodiments.

[0090] Multi-immersion open-top light sheet microscope The optical schematic of the microscope with a modular sample holder is modeled using ray tracing software. Illumination light is coupled into the system by a single-mode fiber with a numerical aperture of 0.12 from a digitally controlled laser package. The light (Gaussian profile) emerging from the fiber is collimated with lens L1 (f=19mm) and then expanded along one axis using a 3x cylindrical telescope composed of lenses C1 (f=50mm) and C2 (f=150mm) to provide multi-directional illumination. The resulting elliptical Gaussian beam is then relayed to the scanning galvanometer GM using lenses R1 (f=100mm) and R2 (Thorlabs, f=50mm). The scanning mirror is driven by a sinusoidal voltage with a frequency of 800Hz from a waveform generator. The scanning beam is relayed to the back focal plane of the illumination objective using a scan lens SL (f=70mm) and a cylindrical lens TL1 (f=200mm). Finally, the elliptical beam passes through a plano-convex lens (R = 34.5 mm), an immersion medium, a holder and finally reaches the specimen.

[0091] The fluorescence is collected by a multi-immersion objective lens providing an in-plane resolution of less than 1 μm for all immersion media and filtered using a motorized filter wheel with bandpass filters for excitation wavelengths of 405 nm, 488 nm, 561 nm and 638 nm. The filtered fluorescence is focused by a cylindrical lens TL2 (f=165 mm) onto a 2048×2048 pixel sCMOS camera. The cylindrical lens provides Nyquist sampling to 0.45 μm / pixel, which provides a horizontal field of view of 0.9 mm over the 2048 pixels of the camera. The vertical field of view is reduced to 256 pixels to match the focal depth of the illumination light sheet (to 110 μm). The 256 pixels are oriented parallel to the rolling shutter read direction of the camera providing an exposure time of 1.25 ms and a frame rate of 800 Hz. The maximum imaging depth may be limited by the physical separation between the holder and the collecting objective lens (0.5 cm). The illumination objective, SIL and collection objective interface with the immersion chamber via aluminum mounts.

[0092] Image strips are collected by a combination of stage scanning and horizontal / vertical tiling using a motorized XY stage and Z actuator. The stage scanning firmware is used to send a TTL trigger signal from the XY stage to the sCMOS camera for reproducible positioning start (<1 μm) of each image strip. The spatial interval between consecutive frames is set to 0.32 μm, which corresponds to a constant stage speed of 0.25 mm / s given a camera frame rate of 800 Hz. For horizontal tiling, an offset of 0.8 mm between adjacent image strips is used (overlap of 11%). For vertical tiling, a focal depth of 110 μm is oriented at 45°, which corresponds to an image strip height of 80 μm. Hence, a vertical tiling offset of 70 μm is used (overlap of 12%). The laser power increases with depth by a user-defined attenuation coefficient P=P0×exp(z / μ) to account for attenuation as the illumination light sheet penetrates deeper into the specimen. The entire image acquisition is controlled by a program that consists of a series of nested loops for imaging multiple specimens, collecting multiple color channels, and horizontal / vertical tiling.

[0093] Data Processing and Visualization The collected dataset undergoes a pre-processing routine before being visualized in 2D and 3D. Each image stripe is stored in a single DCIMG file. These DCIMG files are read into RAM by a DLL and initially skewed at 45 degrees. By setting the interval between successive frames, this skew is done by shifting each plane of pixels in the image strip by an integer pixel offset. This operation may be relatively fast compared to alternative skew removal means using computationally expensive affine transformations. The data is then written from RAM to disk using a hierarchical data format (HDF5) with metadata and a structured XML file for later analysis using BigStitche. A custom HDF5 compression filter (B3D) is used with default parameters to provide up to 10x compression within the noise limits of the sCMOS camera. This pre-processing routine is applied to all DCIMG files, resulting in a single HDF5 / XML file at the end. All image strips are aligned and finally fused to disk in TIFF or HDF5 file formats. The resulting TIFF and HDF5 files are then visualized using open source and commercial packages, and optionally a Beer-Lambert staining algorithm may be applied to provide false-colored pseudo-H&E tissue images.

[0094] Sample Holder All holders are mounted on a motorized XY stage using an aluminum adapter plate. For mouse brain slices, a 1 mm thick fused silica window with a 10 × 10 cm cross section was attached to the adapter plate using UV-curable adhesive. Mouse organs cleared using Ce3D are imaged on customized 6-well plates. The bottom surface of the polystyrene 6-well plate is removed and replaced with a 0.5 mm thick PMMA plate. For the spread kidney specimens, a "drumhead" was made and adapted for mounting on the microscope. This drumhead holds a 0.1 mm thick FEP film taut over the extruded opening, which may help with imaging the spread specimens. To overcome the hydrophobicity of the FEP film (which may cause drift of the spread specimen), the top surface of the FEP film is treated with 0.1% (w / v) polylysine for charge-based adhesion of the specimen to the FEP surface. For human prostate biopsies, HIVEX lens blanks were purchased and machined. The system can also be used as a whole slide scanner for conventional fluorescently labeled histology slides using commercially available slide holders.

[0095] Optical Simulation Optical simulations were performed using ray tracing software with a "black box" model of the multi-immersion objective. In the simulations shown in Fig. 11, the base refractive index of the immersion medium and the specimen was assumed to be n = 1.45, and the optical path difference was varied. For all scenarios, the imaging depth was set to 1 mm, and the PSF was measured at the center of the imaging field. Under the assumption that the optical properties of the immersion medium and the specimen were identical, a similar relationship between the Strehl ratio and the optical path difference was observed for other base refractive indices and imaging depths.

[0096] The details shown in this specification are for the purpose of illustrative discussion of preferred embodiments of the present invention only, by way of example, and are presented for the reason of providing what is believed to be the most useful and easily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt has been made to show structural details of the present invention beyond those deemed necessary for a fundamental understanding of the present invention, and the description taken together with the figures and / or examples will make clear to those skilled in the art how some forms of the present invention may be embodied in practice.

[0097] Of course, it should be understood that any one of the examples, embodiments, or processes described herein may be combined with one or more other examples, embodiments and / or processes, and may be separated and / or performed among separate devices or device portions in accordance with the present systems, devices, and methods.

[0098] It should be understood that terms such as "top" and "side" are used for ease of description and are intended only to indicate the relative positioning of the various components. In other embodiments, other arrangements of components may be used. The various components and operations may be described in terms of specific wavelengths of light. As such, it should also be understood that other wavelengths (such as wavelengths outside the visible spectrum) may be used, and that light as used herein may refer to any electromagnetic radiation. It should also be understood that certain materials may be described in terms of their optical properties (e.g., transmissivity), and thus a material having the desired properties may be selected for any wavelength of light used by the system.

[0099] Finally, the above discussion is intended to be merely illustrative of the inventive system and should not be construed as limiting the appended claims to any particular embodiment or group of embodiments. Thus, while the inventive system has been described in particular detail with reference to exemplary embodiments, it should be understood that many modifications and alternative embodiments may be devised by those skilled in the art without departing from the broadly intended spirit and scope of the inventive system as set forth in the following claims. Thus, the specification and drawings are to be regarded as illustrative and not intended as a limitation on the scope of the appended claims.

Claims

1. an illumination optics arrangement configured to direct illumination light along an illumination path to the sample; collection optics configured to receive light from the sample along a collection path; an immersion chamber configured to hold an immersion medium; a sample holder configured to support the sample; An apparatus comprising: the sample holder includes a first surface and a second surface opposite the first surface; the second surface is adjacent to an immersion medium; The apparatus, wherein the second surface includes a first optical surface along the illumination path that is generally perpendicular to the illumination path, and a second optical surface along the collection path that is generally perpendicular to the 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 positionable in a receptacle of the support member.

4. The apparatus of claim 1 , wherein the first optical surface is generally 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 matched refractive indices.

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

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

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

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

10. a microscope including a support receptacle and an immersion fluid, the microscope being configured to direct an illumination beam along an illumination path through the immersion fluid towards a focal region and to receive collected light from the focal region along a collection path through the immersion fluid; a modular sample holder; A system comprising: The modular sample holder comprises: a sample chamber configured to support a sample; a first optical surface and a second optical surface positioned along a side of the modular sample holder; Equipped with The modular sample holder is removably positionable in the support receptacle as follows: 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 generally perpendicular to the illumination path; The system, wherein the modular sample holder is removably positionable in the support receptacle such that the second optical surface is along the collection path and generally perpendicular to the collection path.

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

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

13. 11. The system of claim 10, wherein the sample holder comprises a well configured to hold the sample, and 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 generally 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 matched refractive indices.

17. an illumination optics arrangement configured to provide illumination light to the specimen; collection optics configured to receive light from the sample; an immersion chamber configured to hold an immersion fluid; a sample holder including a sealed channel extending through a material of the sample holder; An apparatus comprising: the enclosed channel is configured to hold the sample; the sample holder is positionable such that a surface of the sample holder is adjacent the immersion fluid; the illumination light passes through an immersion medium and the sample holder before reaching the sample; An apparatus wherein light from the sample passes through the sample holder and the immersion medium before reaching the collection optics.

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

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

20. 18. 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, the illumination light passes through the first optical surface and the collected light passes through the second optical surface, the first optical surface being generally perpendicular to an optical axis of the illumination light and the second optical surface being generally perpendicular to an optical axis of the collected light.

21. 21. The apparatus of claim 20, wherein the first optical surface is generally 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. 20. The device of claim 17, wherein the enclosed channel is coupled to an inlet configured to supply a fluid to the channel and an outlet configured to exhaust a fluid from the channel.

24. 20. The apparatus of claim 17, further comprising a support member configured to support the sample holder, the sample holder being a modular sample holder removably positionable on the support member.

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

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