Apparatus, system and method for microscope sample holder

Abstract

The invention relates to an apparatus, system and method for a microscope sample holder. A microscope may direct illumination light along an illumination path toward a sample and collect light from the sample along a light collection path. Light along the illumination path may pass through an immersion fluid and a 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 may have a first optical surface along the illumination path substantially perpendicular to an optical axis of the illumination path. The sample holder may have a second optical surface along the light collection path substantially perpendicular to the optical axis of the light collection path. The sample holder may be modular. The sample holder may receive the sample in a closed channel.

Description

[0001] Divisional Application

[0002] This application is a divisional application of a patent application for an invention titled "Devices, Systems, and Methods for a Microscope Sample Holder" with an application date of February 12, 2020, an application number of 202080013952.0.

[0003] Cross - Reference to Related Applications

[0004] This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 62 / 804,606, filed on February 12, 2019, and the entire disclosure of said application is incorporated herein by reference for any purpose.

[0005] Statement Regarding Research and Development

[0006] This invention was made with government support under Award No. W81XWH-14-2-0183 awarded by the Department of Defense and Award No. F32 CA213615 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0007] Embodiments of the present invention generally relate to optical imaging, and more particularly to microscopes. Background Art

[0008] Microscopes typically may 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 relative to the sample. This can improve access to the sample, ease of preparing / mounting the sample, etc. Thus, illumination light and collected light can pass through the material of the sample holder to transfer between the optics and the sample.

[0009] Different types of samples may require different types of sample holders to support them. For example, some samples may rest on a flat plate such as a slide, while other samples may be suspended in a liquid (e.g., in a well plate). A microscope that can accommodate multiple sample types and sample holders without disturbing the optics of the microscope may be useful. Summary of the Invention

[0010] 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 a sample. The light collection optics receive light from the sample along a light 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 light collection path that is generally perpendicular to the light collection path.

[0011] The apparatus may include a support member that supports the sample holder in an orientation relative to the illumination path and the light 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 generally perpendicular to the second optical surface. The immersion medium, the sample, the first optical surface, and the second optical surface may have a matched refractive index. The sample holder may include a closed channel that can accommodate the sample. The closed channel may be coupled to an inlet configured to supply fluid to the channel and an outlet configured to discharge the fluid from the channel. The sample holder may include a plurality of grooves. The sample holder may include a plurality of holes.

[0012] In at least one aspect, the present disclosure relates to a system that includes a microscope and a modular sample holder. The microscope includes a support receptacle and an immersion fluid. The microscope directs an illumination beam along an illumination path through the immersion fluid toward a focus region and receives collected light from the focus region through the immersion fluid along a light 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 positioned in the support receptacle such that the focus region can be positioned 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 light collection path and generally perpendicular to the light collection path.

[0013] The illumination beam can be a light sheet. The sample holder can include a trench configured to hold the sample, and the first and second optical surfaces can form the bottom of the trench. The sample holder can include a hole configured to hold the sample, and the first and second optical surfaces can form the bottom of the hole. The sample holder can include a channel that can hold the sample. The first optical surface can be substantially perpendicular to the second optical surface. The sample, the immersion fluid, the first optical surface, and the second optical surface can have a matching refractive index.

[0014] In at least one aspect, the present disclosure relates to an apparatus that includes: illumination optics that provide illumination light to a sample; collection optics that receive light from the sample; an immersion chamber that holds an immersion fluid; and a sample holder. The sample holder includes an enclosed channel that passes through the material of the sample holder. The enclosed channel holds the sample. The sample holder can be positioned 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.

[0015] The enclosed channel can have a generally circular cross-section. The channel can be part of a microfluidic device. The sample holder can 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 can be substantially perpendicular to the optical axis of the illumination light and the second optical surface can be substantially perpendicular to the optical axis of the collected light. The first optical surface can be substantially perpendicular to the second optical surface.

[0016] The first and second optical surfaces can form the bottom surface of the enclosed channel. The enclosed channel can be coupled to an inlet configured to provide fluid to the channel and an outlet configured to drain the fluid from the channel. The apparatus can also include a support member that can support the sample holder. The sample holder can be a modular sample holder removably positioned in the support member. The illumination light can be a light sheet. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a block diagram of an open-top microscope with a solid immersion meniscus lens according to some embodiments of the present disclosure.

[0018] Figure 2 is a perspective view of a microscope with a modular sample holder according to some embodiments of the present disclosure.

[0019] Figure 3 Side and top views of a modular sample holder with a flat surface, according to some embodiments of the present disclosure.

[0020] Figure 4 Side and top views of a modular sample holder with sample wells, according to some embodiments of the present disclosure.

[0021] Figure 5 Side and top views of a modular sample holder with sample grooves, according to some embodiments of the present disclosure.

[0022] Figure 6 Side and top views of a modular sample holder with holes in the groove bottom, according to some embodiments of the present disclosure.

[0023] Figure 7 Side and top views of a modular sample holder with flow channels, according to some embodiments of the present disclosure.

[0024] Figure 8 Side and top views of a modular sample holder with flow channels and optical surfaces, according to some embodiments of the present disclosure.

[0025] Figure 9 Side and top views of a modular sample holder with flow channels and optical surfaces, according to some embodiments of the present disclosure.

[0026] Figures 10A to 10B Cross-sectional side view showing a sample holder with flow cells that are open and closed, respectively, according to some embodiments of the present disclosure.

[0027] Figure 11 Graph showing the compatibility of different immersion fluids and sample holder materials, according to some embodiments of the present disclosure. Detailed Description

[0028] The following description of certain embodiments is merely exemplary in nature and is in no way intended to limit the scope of the present disclosure or its application or use. In the following detailed description of embodiments of the present system and method, reference is made to the accompanying drawings which form a part hereof and which illustrate, by way of example, specific embodiments in which the described system and method may be practiced. The embodiments are described in sufficient detail to enable those skilled in the art to practice the presently disclosed system and method, 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. Additionally, for the sake of clarity, when certain features will be apparent to those skilled in the art, their detailed description will not be recited so as not to obscure the description of the embodiments of the present disclosure. Accordingly, the following detailed description should not be taken in a limiting sense, and the scope of the present disclosure is defined only by the appended claims.

[0029] Microscopes can be used in a wide range of applications to produce sample images that often have a field of view and / or resolution that is not normally visible to the naked eye. Illumination optics can be used to direct illumination light onto the sample. Condensing optics can 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 include a portion of the illumination light. In some examples, the light reaching the detector may be emitted from the sample after being excited to emit by the illumination light (e.g., via fluorescence). It may be desirable to ensure that the field of view receives uniform illumination and that the illumination penetrates the depth of the sample.

[0030] The illumination and condensing optics can be positioned on the side of the sample holder opposite the sample (e.g., on the underside of the sample holder). Thus, the illumination and the collected light can pass through the material of the sample holder. The illumination and / or condensing optics can be arranged at an angle relative to the sample holder. For example, the illumination and condensing optics can each be at a 45° angle relative to the sample holder. While this can provide the advantage of optimizing the imaging parameters and the imaging depth, the non-normal incidence of the illumination and / or the collected light relative to the sample holder may result in various aberrations. While individual sample holders can be optically optimized for specific setups, 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.

[0031] The present disclosure relates to a microscope sample holder. 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 light collection optics are positioned on the second side of the sample holder. The illumination light passes through an 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 can be a modular component of the microscope. Different modular sample holders can have different geometries to accommodate different types of samples, different imaging modalities, different experimental conditions, and combinations thereof. Some modular sample holders can include a first optical surface along the illumination optical path between the immersion fluid and the sample and a second optical surface along the light collection optical path between the sample and the immersion fluid. The first optical surface can be generally perpendicular to the optical axis of the illumination path, and the second optical surface can be generally perpendicular to the optical axis of the light collection path. The use of the optical surfaces can reduce the need for refractive index matching between the sample, the material of the sample holder, and the immersion fluid, and can also allow for an objective lens (e.g., an illumination and / or light collection objective lens) with a shorter working distance.

[0032] Figure 1 is a block diagram of a microscope having a modular sample holder according to some embodiments of the present disclosure. Figure 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 can be omitted, and the microscope 102 can be manually operated. In some embodiments, one or more portions of the controller 104 can be integrated into the microscope 102. In some embodiments, for example Figure 1 as an example, the microscope 102 can be an open-top microscope.

[0033] The microscope 102 includes a sample holder 108 that supports a sample 106. The bottom surface of the sample holder 108 is adjacent to an immersion fluid 112 that is contained within an immersion chamber 110. Figure 1 The microscope 102 has separate illumination and light collection paths. The illumination path includes a source 118, illumination optics 120, and an illumination objective lens 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 light collection path includes a light collection objective lens 128, light collection optics 130, and a detector 132. The light collection path can collect light from a focused region 126 illuminated by the illumination beam 124.

[0034] The microscope 102 includes a modular sample holder 108 that can be replaced with various other modular sample holders 108. In Figures 3 to 10BInstances of different types of modular sample holders 108 are discussed in greater detail below. The sample holder 108 can be removably coupled to a support member 109 of the microscope 102. The support member 109 can be placed along the upper surface of the immersion bath 110 that holds the immersion fluid 112. In some embodiments, the support member 109 can form a lid or other upper housing of the immersion bath 110. The support member 109 can include an opening that exposes the immersion fluid 112. The opening can serve as a receptacle for the modular sample holder 108. When the sample holder 108 is positioned in the receptacle, the lower surface of the sample holder 108 can be adjacent to the immersion fluid 112. In some embodiments, the lower surface of the sample holder 108 can contact the immersion fluid 112. For example, the lower surface 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 component to prevent the immersion fluid 112 from escaping through the seam between the sample holder 108 and the support member 109.

[0035] In some embodiments, the sample holder 108 can rest in the receptacle of the support member 109 without any kind of attachment (other than gravity). For example, the profile of the receptacle can be tapered and / or the receptacle can include a step for supporting the sample holder 108. In some embodiments, one or more fasteners can be used to attach the sample holder 108 to the support member 109. For example, clips, screws, magnets, snap buttons, hook-and-loop fasteners, or combinations thereof can be used to removably couple the sample holder to the support member 109.

[0036] In some embodiments, the sample holder 108 can be a single body that fits directly into the receptacle of the support member 109. In some embodiments, an adapter plate can be used that fits into the 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 can be bonded to the adapter along the outer perimeter of the sample holder 108 using a UV-curing adhesive. In some embodiments, the sample holder 108 can be a commercial sample holder made modular via the use of an adapter. In some embodiments, the adapter can include mounting hardware (e.g., fasteners) that mates with the support member 109.

[0037] The support member 109 may have a recessed area for holding the sample holder 108. The modular sample holder 108 may be inserted into the 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 light 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.

[0038] The source 118 provides illumination light along the illumination path to illuminate the focused area 126 of the sample 106. The source 118 may be a narrowband source, such as a laser or a light emitting diode (LED) that emits light in a narrow spectrum. In some embodiments, the light may be a broadband source (e.g., an incandescent light source, an arc light source) that produces broad-spectrum (e.g., white) illumination. In some embodiments, one or more portions of the illumination light may be outside the visible light range. In some embodiments, a filter (not shown) may be used as part of the illumination path to further refine the illumination light of (a) certain wavelength(s). For example, a bandpass filter may receive broadband illumination from the source 118 and provide illumination light in a narrower spectrum. In some embodiments, the light source 118 may be a laser and may generate collimated light.

[0039] 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 specific excitation wavelength that can excite fluorophores in the sample 106. The illumination beam 124 may include a broad spectrum (which includes the excitation wavelength) or may be narrowband centered at 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, (a) filter(s) (not shown) may be used in the illumination optics 120 to limit the illumination beam 124 to wavelengths close to the excitation wavelength. Once excited by the illumination beam, the fluorophores in the sample 106 may emit light (which may be centered at a given emission wavelength). The light collection path (e.g., the light collection optics 130) may include one or more filters that can be used to limit the light reaching the detector 132 to wavelengths close to the emission wavelength.

[0040] The illumination optics 120 can couple light from the source 118 into the illumination objective 122. For example, the illumination optics 120 can include an optical fiber that carriers light from the source 118 to the rear end of the illumination objective 122. In some embodiments, the illumination optics 120 can couple light between the source 118 and the objective 122 with substantially no change to the light provided by the source 118. In some embodiments, the illumination optics 120 can alter the shape, wavelength, intensity, and / or other properties of the light provided by the source 118. For example, the illumination optics 120 can receive broadband light from the source 118 and can filter the light (e.g., using a filter, diffraction grating, acousto-optic modulator, etc.) to provide narrowband light to the objective 122.

[0041] In some embodiments, the illumination path can provide an illumination beam 124 that is a light sheet as part of a light sheet microscope or 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 greater than the first numerical aperture along a second axis orthogonal to the first axis. The illumination optics 120 can include optics that reshape the light received from the source 118 into an illumination sheet. For example, the illumination optics 120 can include one or more cylindrical optics that focus the light on one axis rather than on an orthogonal axis.

[0042] In some embodiments, the illumination optics 120 can include scanning optics that can be used to scan the illumination beam 124 relative to the sample 106. For example, the area illuminated by the illumination beam can be smaller than the desired focus area 126. In such a case, the illumination optics 120 can cause the illumination beam 124 to rapidly oscillate across the desired focus area 126 to ensure illumination of the focus area 126.

[0043] The illumination objective 122 can include one or more lenses that provide the illumination beam 124. For example, the illumination objective 122 can focus the illumination beam 124 toward the focus area 126. The sample holder 108 can position the sample 106 such that the focus area 126 is generally within the sample 106. In some embodiments, the illumination objective can be a commercial objective that includes one or more internal optical elements. In some embodiments, the focus area 126 can be idealized as a focus. In some embodiments, the focus area 126 can be generally planar in shape. For example, in LSFM, the focus area 126 can be a plane that lies along the plane of the light sheet generated as the illumination beam 124.

[0044] In some embodiments, the illumination objective lens 122 may be surrounded by the surrounding environment (e.g., air), and the illumination objective lens 122 may be an air objective lens. The illumination objective lens 122 may be characterized by one or more numerical apertures, which may be based on the (several) angles at which light converges at the focal region 126. In some embodiments where the illumination objective lens 122 is in the surrounding environment outside 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, Figure 1 The illustration of Figure 1 shows an illumination solid immersion lens (SIL) 114 positioned along the illumination path. In some embodiments, the illumination objective lens 122 may be an immersion objective lens in contact with the immersion fluid 112 (e.g., similar to the way the condenser objective lens 128 is shown, as described in more detail herein), and the SIL 114 may be omitted.

[0045] The illumination beam 124 may be guided to pass through the illumination SIL 114 and into the immersion fluid 112. The illumination SIL 114 may be shaped to minimize the refraction of light passing from the surrounding environment containing the illumination objective lens 122 (e.g., air) 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 the sample holder 108.

[0046] The illumination beam 124 may be guided onto the sample 106. The sample 106 may be supported by the sample holder 108. In some embodiments, the sample 106 may be directly placed on the upper surface of the sample holder 108. In some embodiments, the sample 106 may be encapsulated in a container (e.g., on a 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 by the optical system 100. For example, the sample 106 may be washed, sectioned, and / or labeled before imaging.

[0047] In some embodiments, the sample 106 may be a biological sample. For example, the sample 106 may be tissue biopsied from a suspected disease (e.g., cancer) area. In some embodiments, the tissue may undergo various processes before being examined by the optical system 100, such as optical clearing, tissue sectioning, and / or labeling. In some embodiments, the examination of the tissue using the optical system 100 may be used for diagnosis, for determining treatment progress, for monitoring disease progression, etc.

[0048] 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 components of the gas.

[0049] In some embodiments, sample 106 may include one or more types of fluorophores. The fluorophores may be inherent to sample 106 (e.g., DNA and proteins in a biological sample) or may be fluorescent labels applied to sample 106 (e.g., acridine orange, eosin). Some samples 106 may include a mixture of inherent types of fluorophores and fluorescent labels. Each type of fluorophore may have an excitation spectrum centered at an excitation wavelength. When the fluorophore is excited by light in the excitation spectrum, it may emit light in an emission spectrum centered at an emission wavelength that may be different from the excitation wavelength (e.g., red-shifted from the excitation wavelength).

[0050] 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 the focal region 126 of sample 106. In some embodiments, sample holder 108 may have a window of transparent material, sample 106 may be positioned above the window, and the remainder of sample holder 108 may be formed of non-transparent material. In some embodiments, sample holder 108 may be made of transparent material.

[0051] Sample holder 108 may have a second surface (e.g., a lower surface) that is opposite to the surface of sample holder 108 that supports sample 106. Immersion chamber 110 holding immersion fluid 112 may be positioned below the 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 the second surface of sample holder 108. In some embodiments, although the second surface of sample holder 108 may contact immersion fluid 112, the first surface of sample holder 108 (which supports sample 106) may contact the same environment as objectives 122 and 128 (e.g., air).

[0052] Support member 109 may be coupled to actuator 107 that may be capable of moving support member 109 and / or sample holder 108 in one or more directions. In some embodiments, sample holder 108 may be moved relative to immersion chamber 110 and objectives 122 and 128 in up to three dimensions (e.g., along the x, y, and z axes). 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 a motor, that may respond to a manual input and / or instructions from controller 104. In some embodiments, actuator 107 may respond to both manual adjustment and automatic control (e.g., a knob that responds to both manual rotation and instructions from controller 104).

[0053] The immersion chamber 110 holds an immersion fluid 112. In some embodiments, the immersion chamber 110 may include a source and / or a 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 may in turn be coupled to a pump and / or a reservoir) and may be open to a drain port for removing 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 the sample holder 108.

[0054] The light collection path may receive light from the focus region 126 and direct the received light onto the detector 132, which may image and / or otherwise measure the received light. The light from the focus region 126 may be a redirected portion of the illumination beam 124 (e.g., scattered and / or reflected light), light emitted from the focus 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 the immersion fluid 112 before reaching the light collection objective 128. In some embodiments, the light 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 light collection objective 128 may be positioned within the immersion bath 110. In some embodiments, the light 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 light collection objective 128.

[0055] The geometry of the focus region 126 may be partially defined by the field of view of the light collection path, which in turn may depend in part on the numerical aperture of the light collection objective 128. Similar to the illumination objective 122, the light collection objective 128 may be a commercial objective including one or more lenses. In some embodiments, the light collection objective 128 may be an air objective. In some embodiments, the focus region focused by the light collection path and the focus region focused by the illumination path may substantially overlap at the focus region 126. In some embodiments, the illumination path and the light collection path may have different shapes, sizes, and / or positions of their respective focus regions.

[0056] The light collection path includes light collection optics 130 that may redirect light from the light collection objective onto the detector 132. For example, the light collection optics 130 may be a tube lens designed to focus light from the rear end of the light collection objective onto an image projected on the detector 132. In some embodiments, the light collection optics 130 may include one or more elements that modify the light received from the light collection objective 128. For example, the light collection optics 130 may include filters, mirrors, de-scanning optics, or combinations thereof.

[0057] The detector 132 can be used to image the focal region 126. In some embodiments, the detector 132 can represent an eyepiece such that a user can observe the focal region 126. In some embodiments, the detector 132 can generate a signal to record an image of the focal region 126. For example, the detector 132 can include a CCD or CMOS array that can generate an electrical signal based on light incident on the array.

[0058] The illumination path can direct light along a first optical axis. The light collection path can collect light along a second optical axis. In some embodiments (such as Figure 1 the embodiment shown in), the first optical axis and the second optical axis can be orthogonal to each other. Each of the first optical axis and the second optical axis can also be non-orthogonal to the sample holder. For example, the first optical axis can be at a 45° angle relative to the bottom surface of the sample holder 108 and the second optical axis can also be at a 45° angle relative 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 instances.

[0059] In some embodiments, the illumination path and the light collection path can be non-orthogonal to each other. For example, the illumination path can follow a first optical axis that is at a 45° angle relative to the bottom surface of the sample holder, while the light collection path can follow a second optical axis that is at a 90° angle relative to the bottom surface of the holder. Thus, there can be an angle of approximately 45° between the first optical axis and the second optical axis.

[0060] In some embodiments, one of the objective lenses 122 and 128 can be an air objective lens, while the other objective lens can be a non-air objective lens (e.g., an immersion objective lens). In some embodiments, the non-air objective lens can be immersed in (or the front surface can be in contact with) the immersion fluid 112. In some embodiments, the air objective lens can have a SIL positioned between the objective lens and the immersion fluid, and the SIL may not be used with the non-air objective lens. For example, the illumination objective lens 122 can direct the illumination beam 124 through the illumination SIL 114 and into the immersion fluid 112, while the light collection objective lens 128 can be adjacent to (e.g., in contact with) the immersion fluid 112.

[0061] The microscope 102 can be coupled to a controller 104 that can be used to operate one or more parts 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 can be separate from the microscope, such as a general-purpose computer. In some embodiments, one or more parts of the controller 104 can be integrated with the microscope 102.

[0062] 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 devices 142 may include a digital display, a touch screen, a mouse, a keyboard, or a combination thereof.

[0063] The controller 104 includes a processor 140 that may execute one or more instructions stored in the memory 144. The instructions may include control software 152 that 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 that may be used to process 'live' images from the detector 132 or images 146 previously stored in the memory 144. The image processing software 150 may, for example, remove background noise from the image 146. The instructions may include analysis software 148 that 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 the cell nuclei in the image 146.

[0064] In some embodiments, the controller 104 may direct the microscope to collect images from several different fields of view in a sample. For example, the controller 104 may include instructions for collecting a depth stack of images. The controller 104 may direct the detector 132 to collect a first image and then instruct the actuator 109 to move the sample holder 108 a set distance in a vertical direction (e.g., along the z-axis). 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 have been achieved. The analysis software 148 may then combine the depth stack of images to allow 3D (or pseudo-3D) imaging of the sample 106.

[0065] Figure 2 is a perspective view of a microscope with a modular sample holder according to some embodiments of the present disclosure. In some embodiments, the microscope 200 shows an example layout of a microscope such as Figure 1 of the microscope 102. In some embodiments, the components and operations of the microscope 200 may be generally similar to the components and operations of Figure 1 of the microscope 102. For the sake of brevity, the components and operations described with respect to Figure 1 of the microscope 102 will not be repeated with respect to Figure 2

[0066] The microscope 200 includes illumination optics 221 that are coupled to an illumination source 220. The illumination source 220 can be an optical fiber cable coupled to a laser ( Figure 2 not shown). The illumination optics 221 can include a telescope and / or a beam expander to couple light out of the illumination source 220. The illumination optics 221 can include shaping optics to shape the illumination light into a light sheet. The illumination optics 221 can include one or more scanning optics that can be motorized to scan the illumination light sheet across the back end of an illumination objective (not shown), which in turn can scan the illumination light sheet across the sample.

[0067] The illumination light sheet passes through an immersion bath 212 that is positioned below a support member 209. The support member 209 can be a stage, such as a motorized stage. For example, the stage can be movable in one or more axes, such as an XY stage. In some embodiments where the support member 209 is motorized in some axes, movement in other axes can be added by coupling one or more parts of the microscope 200 to additional actuators (such as a Z-axis actuator).

[0068] Figure 2 The view shows a sample holder 208 positioned above a receptacle of the support member 209. The sample holder 208 is shown as a flat plate that can be lowered into an imaging receptacle. In Figures 3 to 10B different types of sample holders that can be used with the microscope 200 are discussed in more detail.

[0069] The microscope 200 includes condenser optics 231 that collect light from a focused region of the microscope 200 and provide the collected light to a detector 232. The condenser optics 231 can include a filter wheel that includes several filters that can be rotated into the collection path of the microscope 200. Each filter can be associated with a different emission spectrum (e.g., of different types of fluorophores that can be imaged). In some embodiments, the detector 232 can be an sCMOS detector.

[0070] Figures 3 to 10B Shows various sample holders that can be used as Figure 1 the sample holder 108 and / or Figure 2 208 in some embodiments. Figures 3 to 9 Shows 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 that includes the sample. The side view shows when the sample holder is placed in a microscope (e.g., Figure 1 the microscope 102 and / or Figure 2A sample holder when in a microscope 200 and when the microscope is aligned such that the focal region of the microscope is within the sample as shown in a cross-sectional side view. Figures 10A to 10B A pair of side views showing two variants of the sample holder, rather than a top view and a side view.

[0071] Figures 3 to 10B The view shows a portion of the sample holder that does not include the member by which the sample holder is mounted to the microscope. In some embodiments, the sample holder can be inserted into a receptacle of the microscope. In some embodiments, the sample holder can include mounting hardware (not shown) that is inserted into the receptacle and / or attached 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, which in turn can be fitted into the receptacle and / or include attachment hardware.

[0072] Figures 3 to 10B Each of those in [reference number] shows an exemplary embodiment with an air illumination objective and an immersion condenser objective. However, it should be understood that any combination of air and immersion objectives can be used for the illumination and collection paths. Figures 3 to 1 Each of those in [reference number] shows a generally rectangular top view of the sample holder. However, it should be understood that many features and operations among the various sample holders may be similar, and for the sake of brevity, such features will not be described individually for each of those in [reference number]. Figures 3 to 1 Each of those in [reference number].

[0073] For example, Figures 3 to 10B Each of the sample holders in [reference number] 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 can have a refractive index and dispersion. For any given sample holder, two or more materials can be selected such that they have the same or similar refractive index and / or dispersion.

[0074] In some embodiments, a single sample holder can have Figures 3 to 10B a mixture of the geometries and features described in [reference number]. For example, the sample holder can have a first region with a first geometry for holding the sample and a second region with a second geometry for holding the sample.

[0075] In some embodiments, one or more surfaces of the sample holder can include one or more coatings. For example, a hydrophobic coating can be placed on the sample holder to promote beading of the sample that dots the sample holder.

[0076] Figure 3Side and top views of a modular sample holder with a flat surface, according to some embodiments of the present disclosure. View 300 shows a generally planar sample holder 308. For example, the sample holder 308 can be a transparent plate and the sample 306 can be supported on the upper surface of the plate. When using the planar sample holder 308, the microscope can operate in a manner similar to a flatbed scanner, where the sample can be placed on the upper surface of the sample holder 308. In some embodiments, multiple samples 306 can be placed on the sample holder 308 and can be separated from each other by air. In some embodiments, the sample 306 can include a liquid phase, and the sample 306 can be a droplet deposited on the surface of the sample holder 308.

[0077] Figure 4 Side and top views of a modular sample holder with sample wells, according to some embodiments of the present disclosure. The sample holder 408 can be generally planar (e.g., similar to Figure 3 the sample holder 308), however the sample holder 408 includes a surrounding medium 411 that can form regions for accommodating each sample 406. For example, the sample holder 408 can have defined sample regions that are separated from each other by the surrounding medium 411. In some embodiments, the surrounding medium 411 can have the same material as the rest of the sample holder 408. In some embodiments, the sample holder 408 can be a flat plate of a first material, and the surrounding medium 411 can be attached to the upper surface of the flat plate.

[0078] The sample holder 408 can include one or more different sample regions, each of which can have a different sample (or samples) 406a to h placed therein. For example, the sample holder 408 can be a well plate that can hold several different samples 406a to h.

[0079] Figure 5 Side and top views of a modular sample holder with sample trenches, according to some embodiments of the present disclosure. Figure 5 The sample holder 508 includes a plurality of sample trenches 515, each of which can hold one or more samples 506. The sample trenches 515 have bottoms that have a first optical surface 517 and a second optical surface 519, which can help couple the illumination beam 524 to / from the sample 506 and the collected light 525, respectively.

[0080] Figure 5The side view shows the cross-section of a single groove in groove 515. The groove may be filled with fluid 513. Fluid 513 may be the same as or different from immersion fluid 512. In some embodiments, fluid 513 may have a refractive index that matches the refractive index of sample 506. In some embodiments, fluid 513 may have a refractive index different from that of sample 506. In some embodiments, fluid 513 may have the same refractive index as the material of sample holder 508 and / or immersion fluid 512. Fluid 513 may be used to support, stabilize, and / or protect sample 506 during imaging. For example, sample 506 may be a cell, and fluid 513 may represent cell culture medium. In some embodiments, fluid 513 may be an ambient medium such as air.

[0081] Groove 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). First optical surface 517 may be positionable such that first optical surface 517 is generally perpendicular to the optical axis of the illumination path. This may reduce the refraction of light as it passes from immersion fluid 512 into the material of sample holder 508. In some embodiments, first optical surface 517 may be a flat surface with a uniform thickness.

[0082] Groove 515 has a second optical surface 519 along the light collection path of collected light 525 between sample 506 (and / or fluid 513) and immersion fluid 512. Second optical surface 519 may be positionable such that second optical surface 519 is generally perpendicular to the optical axis of the light collection path. This may reduce the 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.

[0083] 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 single body formed from a single piece of material. In some embodiments, first and second optical surfaces 517 and 519 may be made of a material different from the remainder of sample holder 508. For example, first and second optical surfaces 517 and 519 may be made of a transparent material while the remainder of sample holder 508 is made of a non-transparent material.

[0084] The first and second optical surfaces 517 and 519 may form the 'bottom' of the groove 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 light collection paths. For example, the first optical surface 517 may be at an angle of approximately 90° relative to the second optical surface 519. In some embodiments, the first and second optical surfaces 517 and 519 may not intersect directly, 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-grooved portion of the sample holder 508, thereby giving the bottom of the groove 515 a generally trapezoidal cross-section rather than a triangular cross-section.

[0085] The groove 515 may extend along the length of the sample holder 508. In some embodiments, each groove 515 may be closed to prevent the fluid 513 and / or the sample 506 from escaping from the groove 515. For example, each groove 515 may terminate at a distance from the edge of the sample holder 508 such that the material of the sample holder 508 encloses the groove 515. In some embodiments, one or more end caps may be attached to the sample holder 508 to close the groove 515. In some embodiments, the bottom of each groove 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).

[0086] The groove 515 may have a major axis that extends across the length of the sample holder 508. The sample holder may be aligned in a microscope (e.g., in the receptacle of the support member 109 of Figure 1 and / or Figure 2 the support member 209 of ), and from a 'top-down' perspective, the major axis may be generally perpendicular to the illumination and light collection paths. For example, when the sample holder 508 is placed in a microscope, the major axis of the groove 515 may be perpendicular to the line drawn between the illumination objective and the light collection objective. In some embodiments, the sample holder 508 may include a plurality of grooves 515. In some embodiments, the grooves may be generally parallel to each other.

[0087] Once the sample holder 508 is placed in the microscope, the sample holder 508 may be moved (e.g., by Figure 1The actuator 107) is used to image different regions of the sample 506 and / or different samples. Different samples can be in the same groove 515 or different grooves of the sample holder 508. As the sample holder 508 moves relative to the immersion fluid 512, the first and second optical surfaces 517 and 519 can move relative to the illumination and light collection paths. Since the first and second optical surfaces 517 and 519 are flat and perpendicular to their respective optical paths, the movement of the optical surfaces 517 / 519 can result in relatively small aberrations because the illumination and light collection paths will continue to encounter flat surfaces perpendicular to their respective optical axes. The imaged area of the sample 506 can be partially based on the area where 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 disturbing the non-optical surface portion of the sample holder 508 and / or another optical surface.

[0088] In some embodiments, the first and second optical surfaces 517 and 519 can be useful for respectively reducing the working distance from the focusing zone to the illumination objective ( Figure 5 not shown) and / or the light collection objective 528. For example, the working distance of one or both of the objectives can be about 5 mm to 15 mm. Larger or smaller working distances can be used in other example embodiments. In some embodiments, the objective can be an immersion objective, and the objective can have a front surface positioned close to the optical surface. In some embodiments, the optical surface can 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 a lens (e.g., SIL), the surface of the window / lens can be close to the optical surface.

[0089] Figure 6 is a side view and a top view of a modular sample holder with holes having groove bottoms according to some embodiments of the present disclosure. Figure 6 Shows Figure 5 a sample holder 608 that is generally similar to the sample holder 508 of Figure 5 , except that the sample holder 608 has several individual holes 615 instead of elongated grooves, and each of the holes 615 has a first optical surface 617 and a second optical surface 619, and the first optical surface 617 and the second optical surface 619 are generally similar to the first and second optical surfaces of

[0090] Each hole 615 can hold a different sample 606. Each hole can have a first and a second optical surface 617 and 619 that are respectively generally perpendicular to the illumination and light collection paths. 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 Figure 5The sample holder 508 has a similar shape, except that the sample holder 608 may have 'walls' positioned along the grooves to divide the grooves into different holes 615. In some embodiments, the optical surfaces 617 and 619 may stop between each of the holes. In some embodiments, when viewed from a top-down perspective, each of the holes 615 may have a rectangular cross-section. In some embodiments, other cross-sectional shapes may be used.

[0091] Figure 7 are side and top views of a modular sample holder with flow channels according to some embodiments of the present disclosure. The sample holder 708 may include one or more channels 715 enclosed within the material of the sample holder 708. Each channel 715 may be loaded with one or more samples 706, and the one or more samples 706 may be surrounded by a fluid 713.

[0092] The sample holder 708 may include channels 715 formed in the material of the sample holder 708. For example, the perimeter of the channel 715 may be formed by the material of the sample holder 708. In some embodiments, the channel 715 may have a circular cross-section. Other cross-sectional shapes 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 pass 'in single file' down 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 can be imaged.

[0093] In some embodiments, the sample holder 708 may include ports that can be used to load the sample 706 (and / or the fluid 713) into the channel 715 or to empty the channel 715. In some embodiments, the channels may change size and / or shape along their length. In some embodiments, there may be several separate channels 715. For example, several parallel channels 715 similar to the Figure 5 parallel grooves 515. In some embodiments, there may be one long channel 715 with several parallel segments (e.g., the channel 715 may switch back on itself one or more times).

[0094] Figure 8 are side and top views of a modular sample holder with flow channels and optical surfaces according to some embodiments of the present disclosure. The sample holder 808 has channels (similar to the Figure 7 channels 715) and first and second optical surfaces 817 and 819 (similar to the Figure 5 optical surfaces 517 and 519 andFigure 6 are similar to the optical surfaces 617 / 619).

[0095] In some embodiments, the first optical surface 817 and the second optical surface 819 may be facets of protrusions that extend from the bottom surface of the sample holder 808 by the material of the sample holder 808. The channel 815 may have a shape that does not have flat surfaces corresponding to the first and second optical surfaces 817 and 819. For example, as Figure 8 shown, the channel 815 may have a circular cross-section, while the first and second optical surfaces 817 and 819 may form protrusions having a generally triangular cross-section.

[0096] Figure 9 are a side view and a top view of a modular sample holder having a flow channel and an optical surface according to some embodiments of the present disclosure. Figure 9 The sample holder 908 of Figure 8 may be generally similar to the sample holder 808 of Figure 5 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 rear surface parallel to their front surface. For example, the channel 915 may have a substantially the same cross-section as the groove 515 of Figure 6 and / or the hole 615 of

[0097] Figures 10A to 10B shows a cross-sectional side view of a sample holder having flow units that are respectively open and closed according to some embodiments of the present disclosure. The sample holders 1000a and 1000b may be generally similar to each other, except that the sample holder 1000a is enclosed by the material of the sample holder, while the sample holder 1000b is open to the surrounding environment (e.g., air). For the sake of brevity, only the flow unit 1000a will be described in detail. In some embodiments, the flow units of the sample holder 1000a (or 1000b) may be respectively incorporated into Figures 3 to 9 any one of the geometries of the sample holders 308 to 908 of

[0098] The sample holder 1000a includes an inlet 1021 and an outlet 1023. The inlet can supply the circulating fluid 1013 to the chamber, and the outlet 1023 can remove the circulating fluid 1013. In this way, the circulating fluid 1013 can flow through the chamber. In some embodiments, one or more different circulating fluids 1013 can 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.

[0099] In some embodiments, the sample holders 1000a and 1000b can include an inlet 1021 and an outlet 1023 that can mate with corresponding inlets and outlets of a microscope when the sample holders 1000a and 1000b are positioned in the microscope.

[0100] Figure 11 is a chart of the compatibility of different immersion fluids and sample holder materials according to some embodiments of the present disclosure. Chart 1100 can be based on optical modeling of the interaction of different immersion fluids (e.g., Figure 1 of 112) and different sample holder (e.g., Figure 1 of 108) materials. In some embodiments where the sample holder includes more than one type of material, the sample holder material can represent the material in the (some) portions of the sample holder that interact with the illumination and light collection paths. For example, the sample holder material can represent the material of the first and second optical surfaces (e.g., Figure 5 of 517 and 519).

[0101] Rows represent different types of immersion fluids, each immersion fluid listed with its refractive index. Columns represent different types of sample holder materials. The shading of each box at the intersection of an immersion fluid and a sample holder material represents the maximum thickness of the sample holder material with no significant aberration. The maximum thickness is shown in millimeters or as "chemically incompatible" for combinations that will cause the immersion fluid to damage the structure of the sample holder.

[0102] Throughout the specification, various example values are given. It should be understood that these values are approximate, as perfect alignment may not be possible in a real-world system. Thus, for example, values of 'normal' or 'orthogonal' should be interpreted as "approximately 90°", where the actual value may be within the tolerance of the desired angle. For example, two things described as orthogonal may be positioned at any position from 85° to 95° relative to each other. Other angles and measurements should be interpreted in a similar manner. Similarly, throughout the specification, terms such as 'light' are used 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 the specification may have certain optical properties within certain wavelength ranges, and other materials may be used based on the (several) wavelengths of the electromagnetic radiation utilized. For example, if radiation in the far UV is used, then the (several) optical surfaces of the sample holder may be made of a material that is transparent to far UV.

[0103] Example

[0104] Example details are given 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 only for specific examples, and for other example embodiments, the described details may be different.

[0105] Multi-immersion open-top light-sheet microscope

[0106] Ray tracing software is used to model the optical schematic of a microscope with a modular sample holder. Illumination light is coupled into the system from a numerically controlled laser package through a single-mode fiber with a numerical aperture of 0.12. A lens L1 (f = 19 mm) is used to collimate the light (Gaussian profile) emitted from the fiber, and then a 3x cylindrical telescope consisting of lenses C1 (f = 50 mm) and C2 (f = 150 mm) is used to expand the light along one axis to provide multi-directional illumination. Then, lenses R1 (f = 100 mm) and R2 (Thorlabs, f = 50 mm) are used to relay the resulting elliptical Gaussian beam to a scanning galvanometer GM. The scanning mirror is driven at a frequency of 800 Hz by a sinusoidal voltage from a waveform generator. A scanning lens SL (f = 70 mm) and a tube lens TL1 (f = 200 mm) 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 = 34.5 mm), an immersion medium, a holder, and finally through the sample.

[0107] Collect fluorescence with a multi-immersion objective that provides in-plane resolution of <1 μm for all immersion media and filter the fluorescence using a motorized filter wheel with bandpass filters for excitation wavelengths of 405 nm, 488 nm, 561 nm, and 638 nm. Focus the filtered fluorescence onto a 2048×2048 pixel sCMOS camera via the tube lens TL2 (f = 165 mm). The tube lens provides Nyquist sampling of approximately 0.45 μm / pixel, which provides a horizontal field of view of approximately 0.9 mm within the 2048 pixels of the camera. Reduce the vertical field of view to 256 pixels to match the focal depth of the illumination light sheet (approximately 110 μm). The 256 pixels are oriented parallel to the rolling shutter readout direction of the camera, which provides an exposure time of 1.25 ms and a frame rate of 800 Hz. The maximum imaging depth may be limited by the physical gap (0.5 cm) between the holder and the condenser objective. The illumination objective, SIL, and condenser objective are docked to the immersion chamber via aluminum lens mounts.

[0108] Collect image strips using a motorized XY stage and Z actuator in combination with a combination of stage scanning and lateral / vertical tiling. Use stage scanning firmware to send a TTL trigger signal from the XY stage to the sCMOS camera for reproducible starting positioning (<1 μm) of each image strip. The spatial interval between consecutive frames is set to approximately 0.32 μm, which corresponds to a constant stage speed of approximately 0.25 mm / sec at a camera frame rate of 800 Hz. For lateral tiling, use an offset of 0.8 mm (approximately 11% overlap) between adjacent image strips. For vertical tiling, the 110-μm focal depth is oriented at 45 degrees, which corresponds to an image strip height of approximately 80 μm. Therefore, use a vertical tiling offset of 70 μm (approximately 12% overlap). The laser power increases with depth according to the user-defined attenuation coefficient P = P0×exp(z / μ) to account for the attenuation of the illumination light sheet as it penetrates deeper into the sample. The entire image acquisition is controlled by a program. The program consists of a series of nested loops for imaging multiple samples, collecting multiple color channels, and lateral / vertical tiling.

[0109] Data Processing and Visualization

[0110] The collected dataset undergoes a preprocessing routine before 2D and 3D visualization. Each image strip is stored in a single DCIMG file. These DCIMG files are read by a DLL into RAM and first de-skewed by 45 degrees. The de-skewing is performed by setting the interval between consecutive frames and shifting each plane of the pixels in the image strip by an integer pixel offset. This operation can 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 BigStitche. A custom HDF5 compression filter (B3D) is used with default parameters to provide approximately 10-fold compression, which is within the noise limit of the sCMOS camera. This preprocessing 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 TIFF or HDF5 file format. Open-source and commercial software packages are then used to visualize the resulting TIFF and HDF5 files. To optionally provide false-color pseudo-H&E histological images, the Beer-Lambert coloring algorithm can be applied.

[0111] Sample holder

[0112] All holders are attached to a motorized XY stage using aluminum adapter plates. For mouse brain slices, a 1-mm thick fused silica window with a 10×10 cm cross-section is attached to the adapter plate using UV-curing glue. Mouse organs imaged using Ce3D are placed on a custom 6-well plate. The bottom of the polystyrene 6-well plate is removed and replaced with a 0.5-mm thick PMMA plate. For inflated kidney samples, a “drumhead” is fabricated and adapted to fit onto the microscope. The drumhead tightens a 0.1-mm thick FEP membrane over an extruded opening, which can be useful for imaging inflated samples. To overcome the hydrophobicity of the FEP membrane, which can cause drift of the inflated sample, the upper surface of the FEP membrane is treated with 0.1% (w / v) polylysine to electrostatically bond the sample to the FEP surface. For human prostate biopsies, HIVEX lens blanks are purchased and processed. The system can also be used as a whole-slide scanner for conventional fluorescence-labeled histological slides using commercially available slide holders.

[0113] Optical simulation

[0114] Optical simulations are performed using ray-tracing software in combination with a “black box” model of a multi-immersion objective lens. For Figure 11The simulations presented herein assume a base refractive index of n = 1.45 for the immersion medium and the sample, and the optical path difference varies. For all scenarios, the imaging depth is set to 1 mm, and the PSF is measured at the center of the imaging field of view. The same relationship between the Strehl ratio and the optical path difference is observed for other base refractive indices and imaging depths, assuming the same optical properties for the immersion medium and the sample.

[0115] The details presented herein are for purposes of example only and for an illustrative discussion of the preferred embodiments of the present invention and are presented in order to provide 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 has been made to show the structural details of the present invention in more detail than is necessary for a fundamental understanding of the present invention, and the description, in conjunction with the drawings and / or examples, enables those skilled in the art to understand how the present invention may be embodied in several forms.

[0116] 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 according to the present system, apparatus, and method, or separated and / or performed in separate devices or device parts.

[0117] It should be understood that terms such as 'top' and'side' are used for ease of explanation and are only intended to indicate the relative positioning of the various components. Other embodiments may use other arrangements of the components. The various components and operations may be described with respect to light of a particular wavelength. It should be understood that other wavelengths (e.g., those outside the visible spectrum) will be used, and 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 materials with the desired properties may be selected for any wavelength(s) of light used by the system.

[0118] Finally, the foregoing discussion is only intended to illustrate 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 exemplary embodiments, it should also be understood that numerous modifications and alternative embodiments may be devised by those of ordinary skill in the art without departing from the broader and intended spirit and scope of the present system as set forth in the appended claims. Accordingly, the specification and drawings are to be regarded as illustrative and not intended to limit the scope of the appended claims.

Claims

1. A microscope including an open-top microscope, comprising: Illumination optics configured to direct illumination light along an illumination path to a sample; Condensing optics configured to receive light from the sample along a condensing path separate from the illumination path; An immersion chamber configured to hold an immersion fluid; And A sample holder containing a material, the sample holder having a first surface and a second surface opposite the first surface, the first surface and the second surface forming a flat bottom of the sample holder, wherein the first surface is configured to support the sample and the second surface is in contact with the immersion fluid, such that the first surface is fluidly isolated from the immersion fluid and thereby the sample is fluidly isolated from the immersion fluid, and Wherein at least one of the illumination path or the condensing path is non-orthogonal to the flat bottom of the sample holder, the flat bottom having a thickness in the range of 0.1 mm to 1 mm; Wherein the refractive index of the immersion fluid is selected to match the refractive index of the sample, and wherein the material of the sample holder is based on a combination of the sample refractive indices selected from the following list: Wherein the sample refractive index is approximately 1.56, and the sample holder material is COC; Wherein the sample refractive index is approximately 1.45, and the sample holder material is fused silica; or Wherein the sample refractive index is approximately 1.49, and the sample holder material is PMMA.

2. The microscope according to claim 1, wherein the material of the sample holder has a thickness based on a combination of the sample refractive index and the sample holder material.

3. The microscope according to claim 1, wherein the sample is treated with a chemical that matches the combination of the sample refractive indices selected from the list in claim 1, and wherein the chemical is selected from the following list: Wherein the sample refractive index is approximately 1.56, and the chemical is DISCO or ECI; Wherein the sample refractive index is approximately 1.45, and the chemical is CLARITY; Wherein the sample refractive index is approximately 1.49, and the chemical is Ce3D; or Wherein the sample refractive index is approximately 1.33, and the chemical is FxM.

4. The microscope according to claim 3, wherein the chemical and the immersion fluid are the same.

5. The microscope according to claim 1, wherein the sample holder includes: a first optical surface and a second optical surface, wherein the first optical surface is along the illumination path and is substantially perpendicular to the illumination path, and wherein the second optical surface is along the condensing path and is substantially perpendicular to the condensing path.

6. The microscope according to claim 5, wherein the first optical surface is substantially perpendicular to the second optical surface.

7. The microscope according to claim 1, wherein the sample holder is a modular component configured to be assembled in a receptacle of the immersion chamber.

8. The microscope according to claim 7, wherein the sample holder forms a lid of the immersion chamber.

9. The microscope according to claim 1, wherein the illumination optics are configured to produce a light sheet as the illumination light.

10. A method of using the microscope according to claim 1, comprising: processing the sample such that the sample has the refractive index; selecting the material of the immersion fluid and the sample holder to match the refractive index; and imaging the sample by guiding the illumination light through the immersion fluid to a focal region within the sample using the illumination optics, and collecting the light received through the immersion fluid using the condenser optics.

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