Open-top light-sheet microscope using non-orthogonal illumination and focusing objectives

Abstract

Apparatus, system, and method for an open-top light sheet (OTLS) microscope including an illumination objective and a focusing objective having non-orthogonal optical axes. The optical axis of the focusing objective may be orthogonal to a plane of a sample holder. The illumination objective and the focusing objective may be located below the sample holder. The OTLS microscope may optionally include a second focusing objective having an optical axis orthogonal to the optical axis of the illumination objective. The illumination objective may be an air objective, and the focusing objective may be an immersion objective.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119 of the earlier filing date of U.S. Provisional Patent Application No. 62 / 934,758, filed November 13, 2019, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] Research and development description This invention was made with government support under Grant No. K99CA240681 awarded by the National Institutes of Health and Grant No. W81XWH-18-10358 awarded by the Department of Defense. The U.S. Government has certain rights in this invention.

[0003] Background of the Invention Microscopy may generally involve directing light onto a sample and then imaging the sample based on the light received from the sample. One method of illumination is using a light sheet, in which a relatively thin plane of the sample is illuminated. This may have advantages in terms of both tissue optical properties (e.g., reduced photobleaching and phototoxicity) and increased throughput for imaging large volumes of samples. Open-top light-sheet (OTLS) microscope configurations, similar to flatbed document scanners for tissues, have been developed to enable convenient imaging of one or more tissue specimens without lateral constraints. The geometry of OTLS microscopes may impose various limitations on image resolution, imaging depth within the sample, and / or impose relatively strict refractive index tolerances on the system. It may be necessary to develop OTLS microscopes that address some of these tradeoffs while maintaining the advantageous aspects of the open-top configuration.

[0004] Summary of the Invention In at least one aspect, the present disclosure relates to an apparatus including an illumination objective and a collection objective. The illumination objective directs an illumination light sheet into a sample along an illumination axis. The collection objective receives light from an image plane of the sample along a collection axis. The illumination axis and the collection axis are non-orthogonal to each other.

[0005] The apparatus may also include a second focusing objective that can receive light from the image plane of the sample along a second focusing axis. The second focusing axis may be approximately orthogonal to the illumination axis. The apparatus may also include illumination optics that can generate an illumination light sheet. The illumination optics may be adjustable between a setting based on the focusing objective and a setting based on the second focusing objective. The focusing objective may have a first numerical aperture (NA), and the second focusing objective may have a second NA that is lower than the first NA.

[0006] The apparatus can include a third objective lens and a fourth objective lens. The third objective lens can receive light from the collection objective lens to generate a remote image. The fourth objective lens can image the remote image at an angle based on a non-orthogonal angle between the illumination axis and the collection axis.

[0007] The apparatus may include an immersion fluid. The illumination objective may not be in contact with the immersion fluid, and at least a portion of the collection objective may be in contact with the immersion fluid. The apparatus may include a sample holder configured to support a sample, and at least a portion of the sample holder may be in contact with the immersion fluid. The apparatus may include a lens positioned between the illumination objective and the immersion fluid. The lens may be a solid immersion lens (SIL) or a solid immersion meniscus lens (SIMlens).

[0008] The apparatus can include a sample holder having a first side configured to support a sample and a second side opposite the first side, and the illumination objective and the focusing objective are positioned below the second side. The focusing objective can have a depth of focus for a given field of view, and the illumination axis can be oriented such that the illumination axis does not remain within the depth of focus of the focusing objective.

[0009] In at least one aspect, the present disclosure relates to an apparatus including a sample holder, an illumination objective, and a focusing objective. The sample holder includes a first surface and a second surface opposite the first surface. The first surface supports a sample. The illumination objective directs an illumination light sheet toward the sample at a non-orthogonal angle relative to the first surface of the sample holder. The focusing objective collects light along a collection axis that is approximately orthogonal to the first surface.

[0010] The illumination objective and the collection objective can be positioned below the second surface. The apparatus can include a second collection objective that can collect light along a second collection axis that is approximately perpendicular to the illumination light sheet. The collection axis can form an acute angle with respect to the illumination light sheet. The acute angle can be between about 40° and 70°.

[0011] The apparatus may also include an immersion chamber positioned between the illumination objective and the second surface of the sample holder. The immersion chamber may hold an immersion fluid, and the illumination light sheet may pass through the immersion fluid before reaching the sample. The apparatus may include a solid immersion lens (SIL), and the illumination objective may direct the illumination light sheet into the immersion fluid through the SIL. The apparatus may include a solid immersion meniscus lens (SIMlens), and the illumination objective may direct the illumination light sheet into the immersion fluid through the SIMlens. At least a portion of the focusing objective may be positioned in the immersion fluid, and the illumination objective may not contact the immersion fluid.

[0012] In at least one embodiment, the present disclosure relates to an apparatus including first, second, and third objective lenses. The first objective lens directs an illumination sheet toward a sample in a first operating mode and a second operating mode. The first objective lens has a first optical axis. The second objective lens receives light from the sample in the first operating mode. The second objective lens has a second optical axis that is non-orthogonal to the first optical axis. The third objective lens receives light from the sample in the second operating mode. The third objective lens has a third optical axis that is approximately orthogonal to the first optical axis.

[0013] The apparatus may include a sample holder having a first surface that supports a sample. The second optical axis may be approximately perpendicular to the first surface, and the first optical axis and the third optical axis may be non-orthogonal to the first surface. The sample holder may also include a second surface opposite the first surface, and the first objective lens, the second objective lens, and the third objective lens may be positioned below the second surface. The third objective lens may also provide an illumination sheet to the sample in a third operating mode. The second objective lens may also receive light from the sample in the third operating mode.

[0014] The apparatus may also include collection optics that may generate a remote image based on light received by the second objective lens in the first mode of operation or the third mode of operation, a fourth objective lens that may collect light from the remote image at a first angle in the first mode of operation, and a fifth objective lens that may collect light from the remote image at a second angle in the third mode of operation. The apparatus may include a controller that may combine the images of the sample from the first mode of operation and the third mode of operation to generate an enhanced image of the sample.

[0015] The apparatus may include illumination optics, which may generate an illumination sheet and provide the illumination sheet to the first objective. The illumination optics may generate the illumination light sheet in a first configuration in a first mode of operation and may generate the illumination light sheet in a second configuration in a second mode of operation. The first configuration may have a first numerical aperture and a first width, and the second configuration may have a second numerical aperture smaller than the first numerical aperture and a second width larger than the first width.

[0016] In at least one aspect, the present disclosure relates to a method that includes directing an illumination light sheet to a focal region of a sample through an illumination objective, collecting light from the focal region through a collection objective, where the optical axis of the collection objective is non-orthogonal to the optical axis of the illumination objective, and imaging the collected light.

[0017] The method may also include collecting light from the focal region through a second focusing objective, where the optical axis of the second focusing objective is approximately orthogonal to the optical axis of the illumination objective, and imaging the collected light from the second focusing objective. Collecting light through the focusing objective may be part of a first mode of operation, and collecting light through the second focusing objective may be part of a second mode of operation. The method may include adjusting one or more characteristics of the illumination light sheet between the first mode of operation and the second mode of operation.

[0018] The method may also include generating a remote image based on the collected light and imaging the remote image at an angle based on a non-orthogonal angle between an optical axis of the collection objective and an optical axis of the illumination objective. The quality of the image collected by the collection objective may be diffraction limited with a Strehl ratio greater than about 0.8. The method may also include passing the illumination light sheet from the illumination light sheet through a surrounding medium, through an immersion fluid, and through a material of the sample holder to a focal region of the sample, and collecting light through the material of the sample holder and through the immersion fluid into the collection objective.

[0019] In at least one aspect, the present disclosure relates to a system including an open-top light sheet (OTLS) microscope and a controller for operating the OTLS microscope. The OTLS microscope includes an illumination objective that directs an illumination light sheet into a sample along an illumination axis, a first focusing objective, and a second focusing objective. The first focusing objective receives light from an image plane of the sample along the first focusing axis. The illumination axis and the first focusing axis are non-orthogonal to each other. The second focusing objective receives light from the image plane of the sample along a second focusing axis. The illumination axis and the second focusing axis are orthogonal to each other.

[0020] The controller images light received by the first focusing objective in a first mode of operation and images light received by the second focusing objective in a second mode of operation. The controller may combine information from the images collected in the first mode of operation and the images collected in the second mode of operation. The controller may combine the information using image processing, machine learning, deep learning, or a combination thereof.

[0021] The OTLS microscope can also include illumination optics configured to generate an illumination light sheet, and the controller can direct the illumination optics to adjust one or more characteristics of the illumination light sheet between the first and second modes of operation.

[0022] The OTLS microscope may also operate in an alternate mode, in which the second focusing objective provides an illumination light sheet and the first focusing objective receives light from an image plane of the sample. The controller may collect a first image when the illumination light sheet is provided by the first focusing objective and collect a second image when the illumination light sheet is provided by the second focusing objective, and generate an enhanced image based on the first image and the second image. The controller may generate the enhanced image based at least in part on a fusion deconvolution algorithm. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a block diagram of an open-top light sheet (OTLS) microscope according to some embodiments of the present disclosure. [Figure 2A] 2A is a schematic diagram of a portion of an OTLS microscope according to some embodiments of the present disclosure; [Figure 2B] FIG. 2B is a detailed view of a portion of the microscope of FIG. 2A. [Figure 2C] FIG. 2B is a detailed view of a portion of the microscope of FIG. 2A. [Figure 2D] FIG. 2B is a detailed view of a portion of the microscope of FIG. 2A. [Figure 3A] 3A is a schematic diagram of an OTLS microscope according to some embodiments of the present disclosure; [Figure 3B] 3B is an enlarged view showing different arrangements of illumination and collection objectives that can be used with the microscope of FIG. 3A. [Figure 3C] 3B is an enlarged view showing different arrangements of illumination and collection objectives that can be used with the microscope of FIG. 3A. [Figure 4] FIG. 1 is a diagram of a sample holder for a microscope according to some embodiments of the present disclosure. [Figure 5A] FIG. 1 is a schematic diagram of illumination and collected light in a first mode of operation of an OTLS microscope. [Figure 5B] FIG. 1 is a schematic diagram of illumination and collected light in a second mode of operation of an OTLS microscope. [Figure 6A] FIG. 1 illustrates a hybrid OTLS microscope with dual illumination modes, according to some embodiments of the present disclosure. [Figure 6B] FIG. 1 illustrates a hybrid OTLS microscope with dual illumination modes, according to some embodiments of the present disclosure. [Figure 7] FIG. 1 is a block diagram of a method for illuminating a sample using a microscope according to some embodiments of the present disclosure. [Figure 8A]8A and 8B are schematic diagrams of redirecting optics according to some embodiments of the present disclosure that can be used to redirect a non-orthogonal angle between the illumination axis and the collection axis. [Figure 8B] FIG. 8B is a schematic diagram of redirecting optics that can be used to redirect non-orthogonal angles between the illumination and collection axes. [Figure 8C] FIG. 8C is a schematic diagram of redirecting optics that can be used to redirect non-orthogonal angles between the illumination and collection axes. [Figure 8D] 8D is a schematic diagram illustrating an exemplary operation of the redirecting optics 800c of FIG. 8C in more detail.

[0024] 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 embodiments of the present system and method, reference is made to the accompanying drawings, which form a part of this specification and which show, by way of illustration, specific embodiments in which the described systems and methods may be practiced. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the presently disclosed systems and methods, and it should 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. Further, for purposes of clarity, detailed descriptions of specific features have not been provided where such details would be apparent 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.

[0025] An open-top light sheet (OTLS) microscope includes a sample holder that supports a sample to be imaged on its top surface, while illumination and collection optics are positioned below the bottom surface of the sample holder on the opposite side. Thus, light can pass from the illumination objective through the sample holder and into an illumination region of the sample. Light from the illumination region can pass through the sample holder to a collection objective, which can image the collected light onto a detector (e.g., a CCD, CMOS, etc.). It can be advantageous to utilize two separate objectives: an illumination objective for directing an illumination light sheet onto the sample, and a collection objective for collecting and directing light from the sample to the detector.

[0026] Some OTLS microscopes can use an orthogonal geometry between the optical axes of the illumination and collection objectives. For example, the illumination and collection axes may each be at 45° to the specimen and at 90° to each other. Such designs can have drawbacks. For example, the achievable imaging resolution is limited by the numerical aperture of the objective (illumination and / or collection). While higher numerical aperture objectives enable higher resolution, they also tend to have shorter working distances, thereby reducing the imaging depth into the specimen. This limitation can be exacerbated when the objectives are oriented at an oblique angle to the specimen, as the working distances of both objectives are oriented such that light must travel an additional distance to reach the imaging region. This can limit the imaging depth for relatively thick specimens, thereby limiting the thickness of the specimen that can be imaged with the desired high resolution. Another exemplary drawback is that for OTLS microscopes with high NA objectives, the quality of off-axis focused beams (i.e., beams that are not orthogonal to the sample) can be significantly degraded (caused by aberrations) by very small refractive index mismatches between transparent tissue, the specimen holder, and the immersion medium. Therefore, such systems can impose relatively stringent requirements on the refractive index matching of the immersion fluid, specimen holder, and tissue. It may be desirable to design a microscope that overcomes one or more of these challenges.

[0027] The present disclosure relates to an open-top light sheet microscope using illumination and focusing objectives in a non-orthogonal arrangement. In other words, the illumination objective can have an illumination optical axis, and the focusing objective can have a focusing optical axis, with the illumination and focusing optical axes being non-orthogonal to each other. In some embodiments, the focusing objective can be nearly orthogonal to the plane of the sample holder (e.g., the focusing axis can be perpendicular to the surface supporting the sample), while the illumination objective is non-orthogonal (e.g., at a 45° angle). This may enable an OTLS microscope in which the focusing objective can use its full imaging depth (working distance). This, in turn, may make it easier to use relatively high-NA focusing objectives (which tend to have shorter working distances), which may enable high-resolution imaging of the sample. This geometric arrangement also reduces the index-matching requirements for the collection path, since collected light can pass through the sample holder at a low angle of incidence. This geometry can also be advantageous because it places no lateral constraints on the movement of the sample.

[0028] In some embodiments, an OTLS microscope may include a second focusing objective that can be oriented orthogonal to the illumination objective (and non-orthogonal to the sample holder). The OTLS microscope may include optics that allow for swapping between two optical paths (e.g., two focusing objectives). The first focusing objective, which is non-orthogonal to the illumination (and may be orthogonal to the plane of the sample holder), may have a higher NA than the second focusing objective, which is orthogonal to the illumination objective. The use of two focusing paths may allow the microscope to operate in both high-magnification and low-magnification modes. This may be useful, for example, when screening a sample using a lower NA (and therefore larger field of view) objective to identify regions of interest and then examining those regions in more detail using a higher NA objective. The resolution of the microscope can be adjusted by adjusting the illumination and / or focusing optics.

[0029] 1 is a block diagram of an open-top light sheet (OTLS) microscope according to some embodiments of the present disclosure. FIG. 1 illustrates an optical system 100 including an open-top light sheet (OTLS) microscope 102 and an optional controller 104 capable of operating the microscope 102 and / or interpreting information from the microscope 102. In some embodiments, one or more portions of the controller 104 may be omitted, and the microscope 102 may be operated manually. In some embodiments, one or more portions of the controller 104 may be integrated into the microscope 102.

[0030] The microscope 102 includes a sample holder 108, which supports a sample 106 along its upper surface. The microscope 102 has an illumination path and a collection path that are separate from each other. The illumination path includes a light source 118, illumination optics 120, and an illumination objective 122. The illumination path provides an illumination beam 124, which passes through the sample holder 108 and illuminates the sample 106. The collection path includes a collection objective 128, collection optics 130, and a detector 132. The collection path allows light to be collected from a focal region 126 illuminated by the illumination beam 124. The optical axis of the collection objective 128 may be at an angle θ with respect to the optical axis of the illumination objective 122 (e.g., angle θ with respect to the illumination beam 124). The angle θ may be non-orthogonal (e.g., acute). Such an arrangement of illumination and optical components may generally be referred to as a non-orthogonal, dual objective (NODO) system.

[0031] The illumination objective 122 and the collection objective 128 may generally be located below the bottom surface of the sample holder 108. This may leave the top surface of the sample holder 108 relatively open, which may then facilitate placing the sample 106 on the sample holder 108. For example, the sample holder may have a top surface that is a flat plate (e.g., similar to a commercially available flatbed scanner), with different samples being placed on the flat plate. This may also reduce / eliminate lateral constraints on the sample 106.

[0032] In some embodiments, the microscope 102 may include an optional second focusing objective 160. The second focusing objective 160 may image the focal region 126 with a focusing axis that forms an angle φ with respect to the illumination axis of the illumination beam 124. The angle φ may be greater than the angle θ. In some embodiments, the angle φ may be approximately 90° (e.g., orthogonal). Imaging using the second focusing objective 160 may be referred to as orthogonal dual-objective (ODO) imaging. The second focusing objective 160 may have its own focusing optics 162, which couples collected light to a detector 164. In some embodiments, rather than using a second detector 164, the detector 132 may be shared by both focusing objectives 128 and 160. Exemplary embodiments with multiple focusing objectives are described in more detail in FIGS. 3-7.

[0033] In some embodiments, the microscope 102 may include an optional immersion fluid chamber 110 for containing an immersion fluid 112. The immersion fluid 112 may aid in coupling illumination and / or collected light into the sample. For example, the immersion fluid 112 may act as an index-matching fluid with the sample holder 108 and / or sample 106, reducing refraction of light passing through the immersion fluid 112. In some embodiments, one or both of the illumination objective 122 and the collection objective 128 may be air objectives surrounded by a surrounding medium (e.g., air). Thus, light can pass between the air and the immersion fluid 112. Optional optical elements, such as lenses or windows, may aid in coupling light across the air / immersion fluid interface. In some embodiments, one or both of the illumination objective 122 and the collection objective 128 may be immersion objectives in which at least a portion of the objective (e.g., the front lens) is in contact with the immersion fluid 112. For example, the illumination objective 122 may be an air objective, and the illumination beam 124 may pass through air and through a lens / window (not shown) into the immersion fluid 112 before reaching the sample 106. Light from the focal region 126 may be collected by the collection objective 128 through the immersion fluid 112 without passing through air.

[0034] The light source 118 provides illumination light along an illumination path to illuminate a focal region 126 of the sample 106. The light source 118 can be a narrowband light source, such as a laser or light-emitting diode (LED), which can emit light in a narrow spectrum. In some embodiments, the light can be a broadband light source (e.g., an incandescent light source, an arc light source) which can produce broadspectrum (e.g., white) illumination. In some embodiments, one or more portions of the illumination light can be outside the visible range. In some embodiments, a filter (not shown) can be used as part of the illumination path to further narrow the wavelength of the illumination light. For example, a bandpass filter can receive broadband illumination from the light source 118 and provide illumination light with a narrower spectrum. In some embodiments, the light source 118 can be a laser and can produce collimated light.

[0035] In some embodiments, the optical system 100 can be used to image fluorescence in the sample 106. The illumination beam 124 can include light at a specific excitation wavelength that can excite fluorophores in the sample 106. The illumination beam 124 can include a broad spectrum of light that includes the excitation wavelength, or it can be a narrow band centered around the excitation wavelength. In some embodiments, the light source 118 can generate a narrow spectrum of light centered around (or near) the excitation wavelength. In some embodiments, a filter (not shown) can 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 beam 124, fluorophores in the sample 106 can emit light (which can be centered around a given emission wavelength). The light collection path (e.g., the collection optics 130) can include one or more filters that can be used to limit the light reaching the detector 132 to wavelengths of light close to the emission wavelength.

[0036] 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 altering the light provided by the light 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 light source 118. For example, the illumination optics 120 can receive broadband light from the light source 118 and filter the light (e.g., using a filter, a diffraction grating, an acousto-optic modulator, etc.) to provide narrowband light to the objective 122.

[0037] In some embodiments, the illumination optics 120 can include scanning optics (e.g., scanning mirrors), and the scanning optics can be used to scan the illumination light. In some embodiments, the scanning optics can be used to generate the illumination beam 124 in the form of a light sheet (e.g., by scanning the light back and forth in one axis and not scanning in another axis). In some embodiments, the scanning optics can be used to change the position of the field of view relative to the sample 106.

[0038] In some embodiments, the illumination optics 120 may be adjustable. For example, if the microscope 102 supports more than one imaging mode (e.g., multiple focusing objectives sharing the same illumination objective), the illumination optics 120 may include one or more components that can be adjusted or tuned depending on the imaging mode. An exemplary microscope using multiple imaging modes is described in more detail in FIG. 3, and an example of adjusting the illumination optics 120 is described in more detail in FIG. 5.

[0039] The illumination path can provide illumination beam 124, which is a light sheet as part of a light-sheet microscope or light-sheet fluorescent microscopy (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. Illumination optics 120 can include optics that reshape light received from light source 118 into an illumination sheet. For example, illumination optics 120 can include one or more cylindrical optics that focus light along one axis but not along an orthogonal axis.

[0040] In some embodiments, the illumination optics 120 may include scanning optics that may be used to scan the illumination beam 124 across the sample 106. For example, the area illuminated by the illumination beam may be smaller than the desired focal region 126. In this case, the illumination optics 120 may rapidly oscillate the illumination beam 124 across the desired focal region 126 to ensure illumination of the focal region 126.

[0041] 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 so that the focal region 126 is generally within the sample 106. In some embodiments, the sample holder 108 may include one or more actuators that can position the sample 106 relative to the focal region 126. In some embodiments, the illumination objective 122 may be a commercially available objective that includes one or more internal optical elements. In some embodiments, the illumination objective 122 may be surrounded by 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, the illumination objective 122 may be an immersion objective, and at least a portion of the illumination objective 122 may be in contact with the immersion fluid 112 .

[0042] In some embodiments, the focal region 126 can ideally be treated as a focal plane. The illumination beam 124 can be directed onto the sample 106 to generate the focal region 126. The focal region 126 can ideally be treated as a flat (e.g., 2D) plane illuminated by the illumination light sheet 124. The focal plane can be aligned with the illumination light sheet 124 and can represent an area imaged by the illumination beam 124, from which the focusing objective 128 can collect light. In some embodiments, the focal region 126 can represent a single field of view of the focusing objective 128. In some embodiments, the focal region 126 can represent an area over which the field of view of the focusing objective 128 can be scanned.

[0043] The sample 106 may be supported by the top surface of the 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 on the optical system 100. For example, the sample 106 may be washed, sliced, and / or labeled before being imaged.

[0044] In some embodiments, sample 106 may be a biological sample. For example, sample 106 may be tissue biopsied from an area suspected of disease (e.g., cancer). In some embodiments, the tissue may have undergone various processes, such as optical clearance, tissue slicing, and / or labeling, before being examined by optical system 100. In some embodiments, examination of tissue using optical system 100 may be used for diagnosis, to determine progress of a treatment, to monitor disease progression, etc.

[0045] In some embodiments, the sample 106 may be a non-biological sample. For example, the sample 106 may be a fluid and may include one or more components for investigation. For example, the sample 106 may be a combustion gas, and the optical system 100 may perform particle image velocimetry (PIV) to characterize the components of the gas.

[0046] In some embodiments, the sample 106 may contain one or more types of fluorophores. The fluorophores may be inherent to the sample 106 (e.g., DNA and proteins in a biological sample) or may be fluorescent labels applied to the sample 106 (e.g., acridine orange, eosin). Some samples 106 may contain a mixture of inherent 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 by light in the excitation spectrum, it may emit light in an emission spectrum that may be centered around an emission wavelength different from (e.g., red-shifted from) the excitation wavelength.

[0047] The sample holder 108 may support the sample 106 on a material that is generally transparent to the illumination beam 124 and the light collected from the focal region 126 of the sample 106. In some embodiments, the sample holder 108 may have a window of transparent material over which the sample 106 may be positioned, and the remainder of the sample holder 108 may be formed from an opaque material. In some embodiments, the sample holder 108 may be made from a transparent material. For example, the sample holder 108 may include a glass plate that supports the sample 106.

[0048] In some embodiments, the sample holder 108 may include one or more structures for supporting the sample 106. For example, the sample holder 108 may include a clip or a well. In some embodiments, the sample holder 108 may be a modular component of the system 100, with different sample holders 108 being interchangeable depending on the type of sample, the type of imaging, the wavelength of the illumination / collected light, and combinations thereof.

[0049] The sample holder 108 can have a second surface (e.g., a bottom surface) opposite the surface of the sample holder 108 that supports the sample 106. In some embodiments, an immersion chamber 110 that holds an immersion fluid 112 can be disposed below the second surface of the sample holder 108. In some embodiments, the immersion chamber 110 can have an open top, and the immersion fluid 112 can be in contact with the second surface of the sample holder 108. In some embodiments, the second surface of the sample holder 108 can be in contact with the immersion fluid 112, while the first surface of the sample holder 108 (which supports the sample 106) can be in contact with the same environment (e.g., air) as the objective lenses 122 and 128.

[0050] The sample holder 108 can be coupled to an actuator 109, which can be capable of moving the sample holder 108 in one or more directions. In some embodiments, the sample holder 108 can be movable in one or more dimensions relative to the immersion chamber 110 and the objective lenses 122 and 128. For example, the sample holder 108 can be movable along the x-axis, y-axis, and / or z-axis and / or can be rotated (e.g., tilted, inclined, etc.). The sample holder 108 can 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 can be a manual actuator, such as a screw or coarse / fine adjustment knob. In some embodiments, the actuator can be automated, such as an electric motor that can respond to manual input and / or commands from the controller 104. In some embodiments, the actuator 109 may be responsive to both manual adjustment and automatic control (eg, it may be a knob that responds to both manual rotation and commands from the controller 104).

[0051] The optional 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 (which may be coupled to a pump and / or reservoir) that provides the immersion fluid 112 and a drain that may be opened to remove the immersion fluid 112 from the immersion chamber 110. As described in more detail herein, the type of immersion fluid may be selected based on the refractive index of the sample 106 and / or sample holder 108.

[0052] The collection path may receive light from focal region 126 and direct the received light onto detector 132, which may image and / or otherwise measure the received light. The light from focal region 126 may be a redirected portion of illumination beam 124 (e.g., scattered and / or reflected light), may be light emitted from focal region 126 in response to illumination beam 124 (e.g., via fluorescence), or a combination thereof. The collected light may pass through sample holder 108 toward collection objective lens 128.

[0053] In the NODO geometry of FIG. 1 , the collection path may have a major optical axis disposed at an angle γ with respect to the plane of the sample holder 108 (e.g., the XY plane in FIG. 1 ). In some embodiments, such as that shown in FIG. 1 , the angle γ may be approximately 90°. That is, the collection path may have a major optical axis that is approximately orthogonal to the plane of the sample holder 108. The angle γ may be sufficiently close to 90°, i.e., approximately orthogonal, provided that the quality of the image collected by the collection objective remains diffraction-limited. That is, if the Strehl ratio is used as a figure of merit, the Strehl ratio is greater than approximately 0.8. As will be apparent to those skilled in the art, the Strehl ratio may depend on many parameters potentially applicable to a given OTLS microscope system, in addition to the angle α, such as the refractive index mismatch (i.e., the optical path difference, or the product of the difference in refractive index between the holder and the immersion medium / transparent tissue sample and the thickness of the holder), the NA of the illumination and collection objectives, the field of view of the objectives, the wavelength of the illumination and / or collected light, and the particular objectives used.

[0054] The illumination path may have a principal optical axis disposed at an angle θ relative to the principal optical axis of the collection path. The angle θ may be non-orthogonal, i.e., acute. Several considerations may limit the range of acceptable values ​​for the angle θ. For example, an angle of 90° or close to 90°, i.e., nearly parallel to the plane of the specimen holder, may be impractical because it intersects the specimen holder and constrains the lateral dimensions of the specimen. Also, even if the illumination beam has a relatively low NA, index matching constraints may be very stringent. Other factors, including physical constraints imposed by the mechanical housing of the collection objective, may limit the lower end of the range of values ​​for the angle θ. Exemplary limitations imposed by the objective's geometry are described in more detail in FIG. 2.

[0055] The geometry of 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 collection objective 128. Like illumination objective 122, collection objective 128 may be a commercially available objective including one or more lenses. In some embodiments, collection objective 128 may be an air objective. In some embodiments, collection objective 128 may be an immersion objective (e.g., an oil immersion objective). In some embodiments, collection objective 128 may use an immersion medium that is different from the immersion fluid 112 used in the illumination path. In some embodiments, the focal region where the collection path is focused and the focal region where the illumination path is focused may generally overlap at focal region 126. In some embodiments, the illumination path and collection path may have different shapes, sizes, and / or locations of their respective focal regions.

[0056] The collection path includes collection optics 130, which can redirect light from the collection objective onto a detector 132. For example, collection optics 130 can 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 can include one or more elements that modify the light received from the collection objective 128. For example, collection optics 130 can include filters, mirrors, de-scanning optics, or a combination thereof.

[0057] The collection optics 130 may include optics that can redirect the view of the focal region 126. Because the axis of the collection objective lens 128 is at an angle θ relative to the focal region 126, distortion of the image may occur. The collection optics 130 may include one or more features that can redirect the image to account for the angle θ before the image is projected onto the detector 132. For example, the collection optics 130 may include a remote focus, where a first lens projects an image of the light collected by the collection objective lens 128 and a second lens forms the remote image at an angle that cancels the angle θ. This allows distortion due to the angle θ to be corrected before the light reaches the detector 132. In other exemplary embodiments, other methods of redirecting the image may be used.

[0058] Detector 132 can be used to image focal region 126. In some embodiments, detector 132 can represent an eyepiece through which a user can observe focal region 126. In some embodiments, detector 132 can generate a signal to record an image of focal region 126. For example, detector 132 can include a CCD or CMOS array that can generate an electronic signal based on light incident on the array.

[0059] The microscope 102 may be coupled to a controller 104, which may be used to operate one or more portions of the microscope 102, display data from the microscope 102, interpret data from the microscope 102, or a combination thereof. In some embodiments, the controller 104 may be separate from the microscope, such as a general-purpose computer. In some embodiments, one or more portions of the controller 104 may be integral with the microscope 102.

[0060] The controller 104 includes one or more input / output devices 142 that may allow a user to receive feedback from the controller 104, view data from the microscope 102, provide instructions to the controller 104, provide instructions to the microscope 102, or a combination thereof. For example, the input / output device 142 may include a digital display, a touch screen, a mouse, a keyboard, or a combination thereof.

[0061] The controller 104 includes a processor 140 that can execute one or more instructions stored in memory 144. The instructions may include control software 152, which may 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 109. The instructions may include image processing software 150, which can be used to process a "live" image 146 from the detector 132 or an image 146 previously stored in memory 144. The image processing software 150 can, for example, remove background noise from the image 146. The instructions may include analysis software 148, which 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.

[0062] 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 may include instructions for collecting a depth stack of images. The controller 104 can instruct the detector 132 to collect a first image and then instruct the actuator 109 to move the sample holder 108 vertically (e.g., along the z-axis) a set distance. The actuator 109 can also move the sample 106 relative to the focal region 126, thereby changing 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 the process can then be repeated until a set number of images in the stack and / or a set total displacement in the z-direction is achieved. The analysis software 148 can then combine the depth stack of images to enable 3D (or pseudo-3D) imaging of the sample 106. Similarly, various other translations can be used to collect multiple fields of view. For example, the sample may be scanned in the x-, y-, and / or z-axes. The OTLS geometry may be particularly useful for scanning in the X- or Y-directions, as the position of the objective lens (and other optics) below the sample holder 108 may allow for less constrained scanning in these directions.

[0063] In some embodiments, the controller 104 can assist in switching the microscope 102 between one or more operating (or imaging) modes. For example, the controller 104 can activate various components or start / stop one or more components. For example, in a first imaging mode, light may be collected from the focusing objective 128, while in a second imaging mode, light may be collected through a different focusing objective (not shown in FIG. 1). Exemplary embodiments having multiple imaging modes are described in more detail in FIGS. 3-6B.

[0064] 2A-2D are schematic diagrams of a portion of an OTLS microscope according to some embodiments of the present disclosure. FIG. 2A shows the layout of an OTLS microscope, and FIGS. 2B-2D show detailed views of a portion of the microscope of FIG. 2A. OTLS microscope 200, in some embodiments, may be included in microscope 102 of FIG. 1. For clarity, details and operations already described with respect to FIG. 1 will not be repeated again with respect to FIG. 2.

[0065] 2 shows a schematic diagram of the microscope, highlighting the interaction of the illumination and collection optics with the immersion chamber and sample, along with a portion of the collection optics. Microscope 200 may include additional components that are omitted from FIG. 2 for clarity of the drawing.

[0066] The microscope 200 includes an illumination objective 202 that receives illumination light from an illumination source and optional other illumination optics (not shown) and directs an illumination beam 218 through a lens 224 to an immersion fluid 222. The immersion fluid 222 is contained by an immersion chamber 220. The immersion light 218 passes through the bottom surface of a sample holder 226 and enters a sample 228. Collected light 216 from the sample 228 exits through the sample holder 226 and the immersion fluid 222 and enters the collection objective 204. The major axis of the illumination light 218 may be non-orthogonal to the plane of the sample holder 226, while the major axis of the collected light 216 may be approximately orthogonal to the plane of the sample holder 226. Thus, the illumination light 218 and the collected light 216 may be non-orthogonal to each other.

[0067] In microscope 200, exemplary redirecting optics 230 are shown that can be used to accommodate a non-orthogonal angle θ between illumination light 218 and collected light 216. Various types of redirecting optics 230 can be used. The example of FIG. 2 shows a specific implementation of redirecting optics 230 using a remote image 212. However, other embodiments may use other approaches to achieve redirecting optics 230. Various exemplary redirecting optics that can be used in place of redirecting optics 230 of FIG. 2 are described in FIGS. 8A-8D.

[0068] In the redirecting optics 230 of Figure 2, the focusing objective 204 can direct light through optional transfer optics 210 into a second focusing objective 206. The second focusing objective 206 can generate a remote image 212 that can be imaged by a third focusing objective 208. The second focusing objective 206 and the third focusing objective 208 can form a predetermined angle. This angle can be based on the angle between the collected light 216 and the illumination light 218. The third focusing objective 208 can direct the light to a detection system (not shown).

[0069] The illumination objective 202 may be an air objective, with a proximal lens (e.g., the lens emitting the illumination beam 218) positioned in air. In some embodiments, the illumination objective 202 may be positioned completely in air. However, at least a portion of the sample holder 226 may be positioned to contact the immersion fluid 222. The lens 224 may couple the illumination light 218 from the air surrounding the illumination objective 202 into the immersion fluid 222.

[0070] In some embodiments, lens 224 can be shaped to reduce refraction of light as it passes from air through the material of lens 224 and into immersion fluid 222. In some embodiments, lens 224 can be made of a material with a refractive index that matches the refractive index of immersion fluid 222. In some embodiments, lens 224 can have one or more surfaces shaped to match the wavefront of light passing through it, thereby eliminating / reducing refraction of light passing through lens 224. For example, lens 224 can be a solid immersion (SIL) lens or a solid immersion meniscus lens (SIMlens), as disclosed in U.S. Pat. No. 10,409,052 and WO 2020 / 150239, the disclosures of which are incorporated herein by reference.

[0071] After passing through the lens 224, the illumination beam 218 may pass through the immersion fluid 222 until it hits the sample holder 226. The immersion fluid 222 may have a refractive index that matches the refractive index of the sample holder 226. This may help to minimize / prevent refraction of the illumination beam 218 as it passes from the immersion fluid 222 into the material of the sample holder 226. On the other hand, the refractive index of the sample holder 226 may be selected to match the refractive index of the sample 228 (and immersion fluid 222). This may help to minimize / prevent refraction of the illumination beam 218 as it passes from the sample holder into the sample 228.

[0072] In some embodiments, the sample 228 and sample holder 226 can be immersed in the immersion fluid 222. In some embodiments, the bottom surface of the sample holder 226 can be in contact with the immersion fluid 222, and the top surface supporting the sample 228 can be in contact with air. For example, the sample holder 226 can function as a lid for the immersion chamber 220.

[0073] The collected light 216 may exit the sample 228 and pass through the sample holder 226 and the immersion fluid 222 before entering the proximal lens of the collecting objective 204. The collecting objective may be an immersion objective with a proximal lens in contact with the immersion fluid 222. The distal end of the collecting objective 204 may be positioned outside the immersion fluid 222 in the ambient environment (e.g., in air). The collected light 216 may represent a portion of the light that exits the sample 228 and passes through the collecting objective 204 and other collection optics to reach the detector. The size and geometry of the collected light 216 may be based, at least in part, on the collecting objective 204 and other collection optics.

[0074] The proximal lens of the collecting objective 204 may be located much closer to the sample 228 than the proximal lens of the illumination objective 202. This may allow the collecting objective 204 to be a higher NA objective than the illumination objective 202. Because the collecting objective 204 is nearly perpendicular to the plane of the sample holder 226, refraction (compared to the illumination beam 218) may be reduced. In some embodiments, the collecting objective 204 may be an air objective, and may be separated from the immersion fluid 222 by a window rather than being immersed in the immersion fluid 222.

[0075] The rear end of the focusing objective 204 can direct light to a second focusing objective 206 through optional transfer optics 210. Transfer optics 210 can include one or more lenses in some embodiments. For example, transfer optics 210 can be a 4f relay system.

[0076] The microscope 200 of FIG. 2 includes a feature as an optional means for redirecting the image collected by the focusing objective 204. For example, the second focusing objective 206 can project a remote image 212. The remote image 212 can be used to correct the angle between an illumination plane (e.g., illumination beam 218) within the sample 228 and the collected light 216. The remote image 212 can be imaged by the third focusing objective 208. To correct the angle between the illumination beam 218 and the collected light 216, the third focusing objective 208 can image the remote image 212 at an angle relative to the second focusing objective 206. This angle can be based on the angle between the illumination light and the collected light. For example, if the angle between the illumination beam 218 and the collected light 216 is θ, then the angle between the second objective 206 and the third objective 208 can be 90°-θ. For example, if the angle θ is approximately 45°, the angle between the second focusing objective lens 206 and the third focusing objective lens 208 may also be approximately 45°.

[0077] For example, as shown in FIG. 2B , an exemplary mechanical housing for the focusing objective allows for an illumination light path of 45° on each side, with a maximum cone angle of β (e.g., the angle between the optical axis of the illumination light 218 and the edge of the focusing objective 204). In some embodiments, the angle β may be about 7.1° (e.g., the illumination light may have an NA of about 0.12 in air). The maximum cone angle may vary based on the size / shape of the mechanical housing for the objective 204, and other angles may be used in other exemplary embodiments.

[0078] In an OTLS system with a single focusing objective with a relatively high NA, angle θ may be greater than 45°, as shown in FIG. 2C. In some embodiments, it may be desirable to have two illumination objectives for double-sided illumination and use a single focusing objective with the same relatively high NA, as shown in FIG. 2D. FIG. 2D illustrates an embodiment with a second illumination objective 240. Each of illumination objectives 202 and 240 can be positioned at a non-orthogonal angle relative to the axis of focusing objective 204. In the embodiment of FIG. 2D, both are positioned at angles greater than 45°. In other exemplary embodiments, other angles (e.g., angles equal to or less than 45°) can be used.

[0079] A focusing objective with a shallower angle and a larger housing may further constrain the range of illumination light path angles. From another perspective, the illumination light sheet may be tilted (i.e., not orthogonal) enough to not remain within the confocal parameters (depth of focus) of the focusing objective for the desired field of view. Taking this into account, a reasonable range for the angle θ may be 40° to 70°. For example, the illumination path may be along a first optical axis that is at a 45° angle relative to the bottom surface of the sample holder, while the focusing path may be along a second optical axis that is at a 90° angle relative to the plane of the sample holder 108. Thus, an angle of approximately 45° may be formed between the first and second optical axes. That is, the angle θ may be approximately 45°.

[0080] 3A-3C are schematic diagrams of an OTLS microscope according to some embodiments of the present disclosure. FIG. 3A shows microscope 300, and FIGS. 3B and 3C show close-up views of different arrangements of illumination and collection objectives that may be used with the microscope of FIG. 3A. Microscope 300, in some embodiments, may be included in microscope 102 of FIG. 1 and / or microscope 200 of FIG. 2. Microscope 300 may be generally similar to the microscopes described above, except that microscope 300 includes an additional imaging path using a collection objective that is orthogonal to the illumination sheet, in addition to the non-orthogonal imaging path described in FIGS. 1-2.

[0081] The non-orthogonal dual objective (NODO) path of microscope 300 may generally be similar in operation and components to microscope 102 of FIG. 1 and microscope 200 of FIG. 2, and therefore, for the sake of clarity, features and components already described with respect to FIGS. 1 and 2 will not be repeated with respect to FIG. 3.

[0082] The microscope 300 includes a NODO light path and an orthogonal dual objective (ODO) path. The NODO and ODO paths may share certain components, such as the illumination path. The microscope 300 includes an illumination objective 302 that directs an illumination light sheet through a lens 310 (e.g., a SIL or SIM lens) into an immersion fluid (not shown) toward a sample 308. A first focusing objective 304 can collect light from the sample 308 at a non-orthogonal angle to the illumination light sheet. The first focusing objective 304 can pass light through a first lens 314 and a second lens 316, which can form a relay. This relay can pass light to a second focusing objective 318, which can generate a remote image that is imaged at angle α by a third focusing objective 320. Angle α can be the angle between the optical axes of objectives 304 and 318 and objective 320. As shown in Figure 3, it can also be the angle between planes perpendicular to these axes. The third focusing objective 320 can pass the light through a third lens 322, which images the light onto a first detector 324.

[0083] The path from the light source (not shown) through the illumination objective 302 to the sample 308 and the path from the sample 308 through the first collecting objective 304 to the first detector 324 may form the NODO light path. The illumination light path and the collected light (e.g., the optical axes of the illumination objective 302 and the first collecting objective 304) may form a non-orthogonal angle θ with respect to each other. The microscope 300 includes redirecting optics 340 (e.g., similar to the redirecting optics 230 of FIG. 2). While a particular implementation of the redirecting optics 340 is shown in FIG. 3, other systems for redirection may be used in other exemplary embodiments. FIGS. 8A-8D include several additional exemplary redirecting optics that may be used as the redirecting optics 340.

[0084] The microscope 300 also includes a fourth focusing objective 306 having an optical axis approximately orthogonal to the illumination light sheet (e.g., the optical axis of the illumination objective 302). Like the illumination objective 302, the fourth focusing objective 306 may be an air-immersion objective or may be separated from the immersion fluid (not shown) by a second lens 312 (e.g., an SIL or SIM lens). The fourth focusing objective 306 can collect light from the sample 308 and direct the collected light through one or more ODO focusing optics to a second detector 328. For example, the ODO focusing optics may include a lens 326 that images light from the fourth focusing objective onto the second detector 328.

[0085] The path from the light source (not shown) through the illumination objective 302 to the sample 308 and the path from the sample 308 through the fourth collecting objective 306 to the second detector 328 may form an ODO light path. The illumination light path and the light collected by the fourth collecting objective 306 (e.g., the optical axis of the illumination objective 302 and the optical axis of the fourth collecting objective 306) may form an orthogonal angle with each other.

[0086] In some embodiments, the NODO and ODO light paths can share a detector rather than having separate first and second detectors 324 and 328. The microscope 300 can include additional optics (e.g., a rotating mirror, a shutter, etc.) that can switch whether light from the NODO path or the ODO path reaches the detector.

[0087] In some embodiments, the illumination path may be adjustable and may be adjusted between ODO and NODO imaging modes. For example, an illumination optics system (not shown), such as illumination optics 120 of FIG. 1, may include adjustable components that can adjust the size and shape of the illumination light sheet based on whether the first focusing objective lens 304 or the fourth focusing objective lens 306 is used. For example, the illumination optics may include a variable beam expander that can be used to adjust characteristics of the illumination light sheet, such as the NA and / or width of the light sheet. Adjustments to the illumination light sheet may be manual, automatic (e.g., managed by a controller), or a combination thereof. Adjustments to the illumination path are described in more detail in FIG. 5.

[0088] In an exemplary operation, a sample can be placed on a microscope, and the ODO optical path can be used to screen the sample. The ODO path may have lower resolution and magnification than the NODO path, but may have a larger field of view than the NODO path. Therefore, using the ODO path to screen the sample can be more efficient. In some embodiments, the sample 308 can be scanned (e.g., by movement of a focal region relative to the sample, by movement of the sample relative to the focal region, or a combination thereof). In some embodiments, multiple fields of view can be stitched together (e.g., by a controller such as 104 in FIG. 1 ). In some embodiments, the sample 308 can be scanned in three dimensions to build a volumetric image of the sample. In some embodiments, the scanning can be performed manually.

[0089] After scanning the sample (or a portion of the sample), regions of interest can be identified. In some embodiments, the regions of interest can be identified by an automated process (e.g., image processing such as segmentation, thresholding, machine learning, and / or deep learning). In some embodiments, a user (e.g., a clinician) can determine the regions of interest. Once one or more regions of interest are located, a NODO path can be used for high-resolution imaging of the regions of interest. In some embodiments, once the regions of interest are identified, the microscope 300 can be switched to NODO mode. Switching to NODO mode may include switching which detector is being used for imaging (or which light path is coupled to the detector). Switching modes may also include adjusting the illumination light sheet.

[0090] In NODO mode, microscope 300 may have higher resolution and magnification, but a smaller field of view. NODO mode may be useful for determining one or more characteristics of a region of interest. For example, a clinician may identify a region of interest and then switch to NODO mode to make a diagnosis. In some embodiments, the resolution and / or field of view in NODO and / or ODO modes may be adjustable. This may provide microscope 300 with a relatively wide operating range, each with different performance characteristics.

[0091] In some embodiments, information collected in a first operational (imaging) mode (e.g., NODO mode) can be combined with information collected in a second operational (imaging) mode (e.g., ODO mode). This process can be automated. For example, the controller (e.g., 104 in FIG. 1 ) can image an area in ODO mode and then use image processing (e.g., segmentation, thresholding, etc.), machine learning, deep learning, or a combination thereof to determine whether all or part of the imaged area is an area of ​​interest. The controller can then use NODO mode to image the area of ​​interest in more detail. In some embodiments, the process can be manual, with a user identifying the area of ​​interest. In some embodiments, a mixture of manual and automatic processes (e.g., automatic image processing but manual identification of the area of ​​interest) can also be used.

[0092] In an OTLS system having a second focusing objective, such as that shown in Figure 3A, the angle θ between the illumination objective and the first focusing objective can be optimized at 45°, as shown in Figure 3B, and the angle between the illumination objective's optical path and the second focusing objective can be approximately 90°. In an OTLS system where it is desirable to have an illumination objective with a relatively high NA, it may be desirable to have the angle between the illumination objective's optical path and the second objective's optical path greater than 90°, as shown in Figure 3C.

[0093] Figure 4 is a diagram of a microscope sample holder according to some embodiments of the present disclosure. Sample holder 400 of Figure 4 may, in some embodiments, be included in a microscope such as microscope 300 of Figure 3 using NODO and ODO optical paths. Because Figure 4 illustrates a diagram that focuses on the interaction between the objective lens and sample holder 400, various components of the microscope are omitted. For clarity, operations, features, and components previously described with respect to Figures 1-3 will not be repeated with respect to Figure 4.

[0094] The sample holder 400 supports an immersion fluid 420. A microscope including the sample holder 400 includes a NODO optical path and an ODO optical path. The illumination objective 402 provides an illumination light sheet to a focal region 410 along an illumination optical axis 403. The illumination objective 402 may be an air objective that is not immersed in the immersion fluid 420. Thus, a lens 412, such as a SIL or SIMlens, can couple the illumination light sheet into the immersion fluid 420.

[0095] The NODO collector objective 404 may receive light from a focal region 410 along a NODO collection axis 405. An angle θ may occur between the illumination axis 403 and the NODO collection axis 405. The angle θ may be non-orthogonal and, in some embodiments, may be an acute angle, such as a 45° angle. The NODO collector objective 404 may be an immersion objective, and at least a portion of the NODO collector objective 404 may be in contact with an immersion fluid 420.

[0096] The ODO collector objective 406 may receive light from the focal region 410 along an ODO optical axis 407. An angle φ may be formed between the illumination axis 403 and the ODO collection axis 407. The angle φ may be approximately perpendicular (e.g., about 90°). The ODO collector objective 406 may be an air objective. Similar to the illumination objective 402, the ODO collector objective 406 may be an air objective. Thus, a lens 414 (e.g., SIL, SIMlens) may separate the ODO collector objective 406 from the immersion fluid 420.

[0097] 4 and as described above, objectives 402, 404, and 406 can be positioned such that NODO collection objective 404 does not block light from illumination objective 402 or ODO collection objective 406. The angle of light from illumination objective 402 and light collected by ODO collection objective 406 may be based, in part, on the NA of each of these objectives. Thus, the NA of these objectives, as well as the size and shape of NODO objective 404, can be designed so that they do not interfere with one another.

[0098] 5A and 5B are schematic diagrams of illumination and collected light in a first and second operating mode of an OTLS microscope, respectively. Figures 5A and 5B represent diagrams of the interaction between the illumination light sheet and light collected by the objective lens in NODO mode (Figure 5A) and ODO mode (Figure 5B). The two diagrams in Figures 5A-5B can represent light provided by the same physical microscope in different operating modes. For example, the diagrams in Figures 5A-5B can represent the operation of a hybrid OTLS microscope, such as that described in Figures 3-4. Optical mode 500a represents NODO mode, and optical mode 500b represents ODO mode.

[0099] 5A-5B each show a respective illumination light sheet 502a / 502b and a respective cone of collected light 504a / 504b. The illumination light sheet and collected light interact to generate a field of view (FOV). The illumination light sheet can be adjusted between operational modes to accommodate different geometries of the collected light.

[0100] FIG. 5A shows an illumination light sheet 502a and a cone of collected light 504a. The illumination light sheet 502a can be at an angle θ with the collected light 504a. The angle θ can be a non-orthogonal angle, such as an acute angle, as described above with reference to FIGS. 1-4. FIG. 5B shows an illumination light sheet 502b and a cone of collected light 504b. The illumination light sheet 502b can be at an angle φ with the collected light 504b. The angle φ can be nearly orthogonal (e.g., about 90°), as described in detail above.

[0101] In NODO mode, the focusing objective may have a higher NA than the NA of the focusing objective used in ODO mode, so collected light 504a may be a wider cone than collected light 504b. Illumination light sheet 502a may have a higher NA and a smaller width W compared to illumination light sheet 502b. Thus, the FOV in optical mode 500a is smaller than the FOV in optical mode 500b. This may be due to adjustments to the illumination optics. For example, an adjustable beam expander may be used to change the W and NA of the illumination light sheet between modes.

[0102] 6A-6B illustrate a hybrid OTLS microscope with dual illumination modes, according to some embodiments of the present disclosure. The diagrams shown in FIGS. 6A-6B represent a portion of a hybrid OTLS microscope having both ODO and NODO modes. For example, the diagrams in FIGS. 6A-6B may represent a portion of microscope 300 of FIG. 3 in some embodiments. For clarity, features, components, and operations described above with respect to one or more previous figures will not be repeated with respect to FIGS. 6A-6B.

[0103] 6A-6B illustrate additional imaging modes that can be used to utilize three objectives (e.g., an illumination objective, a NODO collection objective, and an ODO collection objective) that can direct and receive light from a sample. Rather than providing illumination to the NODO path through a single illumination objective, the system can also be configured for a mode that can provide illumination through the ODO collection objective. FIG. 6A illustrates a microscope operating in a first mode in which illumination light is provided by a first objective 602 and received by a collection objective 604 at a non-orthogonal angle. FIG. 6B illustrates the same microscope operating in a second mode in which illumination light is provided by a second objective 606 and received by the collection objective 604 at a non-orthogonal angle. In some embodiments, images captured using both modes can be combined to improve imaging performance, for example, compared to a single image.

[0104] The microscope of FIGS. 6A-6B includes a first objective lens 602, a second objective lens 606, and a third focusing objective lens 604. Light collected by the focusing objective lens 604 can pass through one or more transfer optics (not shown) to reach optics that can redirect the collected image. Because there are two illumination paths, each with a different angle (e.g., +45° and −45°) relative to the focusing objective lens 604, the redirecting optics may need to correct for the two different angles. The exemplary microscope of FIGS. 6A-6B includes a remote imaging system. The remote imaging system includes a fourth objective lens 608, a fifth objective lens 610, and a sixth objective lens 612. The fourth objective lens 608 can generate a remote image that can be imaged by either the fifth objective lens 610 or the sixth objective lens 612, depending on the imaging mode.

[0105] In ODO imaging mode (not shown in FIGS. 6A-6B), illumination can pass between objectives 602 and 606. For example, objective 602 can function as an illumination objective to provide an illumination light sheet at the sample, and objective 606 can image the sample at a nearly orthogonal angle to the illumination light sheet. The opposite arrangement can also be used, where objective 606 provides the illumination light sheet and objective 602 collects at a nearly orthogonal angle.

[0106] In some embodiments, various optical components along the illumination path can be shared between the modes represented in Figures 6A and 6B. For example, there can be a single light source that can be coupled to either of the two objective lenses 602 or 606 depending on the mode. Similarly, there can be shared components in the collection path. For example, one or more detectors can be coupled to objective lenses 602, 604, or 606 depending on which imaging mode is being used.

[0107] In NODO imaging mode, depending on whether objective 602 or 606 is used to provide the illumination light sheet, the light sheet may have a different angle relative to the collected light. For example, if the angle between the axis of objective 602 and the axis of objective 604 is θ, then the angle between the axis of objective 606 and the axis of objective 604 may be −θ (90°−θ), where the negative sign indicates that the angle is in the opposite direction (relative to the axis of objective 604) from angle θ. In some embodiments, angle θ may be approximately 45°, and the two objectives 602 and 606 may have axes that are each approximately 45° from (but in opposite directions from) the axis of collecting objective 604.

[0108] To account for different angles and / or directions when different objective lenses are used for illumination, the distant focus generated by objective lens 608 can also be imaged from different angles. For example, in the imaging mode of FIG. 6A where objective lens 602 provides a light sheet, objective lens 610 may be used to image the distant focus. In the imaging mode of FIG. 6B where objective lens 606 provides a light sheet, objective lens 612 may image the distant focus. In some embodiments, these objective lenses can be coupled to different detectors or the same detector.

[0109] The use of two different observation modes with different illumination angles can be useful to help correct distortions due to the angle between the illumination and collected light (in NODO mode). For example, the point-spread function (PSF) of an image can be deconvolved by combining two elliptical PSFs for each individual viewing angle. The microscope controller (e.g., 104 in Figure 1) can combine the two images using various computational techniques. For example, a processor (e.g., 140 in Figure 1) can execute instructions stored in memory (e.g., 144 in Figure 1) to combine information from images collected under different illumination modes. For example, a fusion deconvolution algorithm can be used.

[0110] The microscope of Figures 6A-6B is shown as using a particular set of redirecting optics, particularly the remote focus system described in more detail in, for example, redirecting optics 230 of Figure 2 and / or redirecting optics 340 of Figure 3. However, other exemplary embodiments that use different viewing modes at different viewing angles may use one or more redirecting optics in addition to (or instead of) the remote focus system shown in Figures 6A-6B. For example, one of the remote focus systems described in Figures 8A-8D could be used instead.

[0111] 7 is a block diagram of a method for illuminating a sample using a microscope according to some embodiments of the present disclosure. Method 700 may generally be performed by one or more of the optical systems described in FIGS.

[0112] Method 700 may generally begin at block 710, which describes directing an illumination light sheet to a focal region of a sample through an illumination objective. For example, illumination optics (e.g., 120 in FIG. 1 ) may generate the illumination light sheet and direct it to the back end of the illumination objective. In some embodiments, the characteristics (e.g., width, NA) of the illumination light sheet may be adjusted based on the operating mode of the microscope. The illumination objective (e.g., 122 in FIG. 1 ) may direct the illumination light sheet to the sample. In some embodiments, the illumination light sheet may pass through material of a sample holder on its way to the sample. In some embodiments, the illumination light sheet may pass through an immersion fluid between the illumination objective and the sample. In some embodiments, the illumination objective may be an air objective, and the illumination light sheet may pass through a lens or window (e.g., SIMlens, SIL) between the illumination objective and the immersion fluid.

[0113] Block 710 may be generally followed by block 720, which describes collecting light from the focal region through a collection objective, where the optical axis of the collection objective is not orthogonal to the optical axis of the illumination objective. In some embodiments, the angle between the illumination axis and the collection axis may be an acute angle, e.g., a 45° angle. In some embodiments, the angle may be larger or smaller (e.g., 10°-80°). In some embodiments, the collection objective may be an immersion objective, and the proximal end of the collection objective may be in contact with an immersion fluid. Thus, light may be collected through the sample holder and the immersion fluid into the collection objective.

[0114] Block 720 may be followed by block 730, which generally describes imaging the collected light. Light from the collecting objective may be directed onto a detector (and / or eyepiece) that may be used to present an image to a user.

[0115] In some embodiments, the collection optics can direct the collected light to a remote image, and an additional objective lens can focus the remote image at an angle based on the angle between the illumination axis and the collection axis.

[0116] In some embodiments, the method 700 may also include imaging the sample through a second focusing objective having a second focusing axis that is orthogonal to the illumination axis. The focusing objective and the second focusing objective may be used as part of different imaging modes.

[0117] In some embodiments, the above-described embodiments can be combined with conventional (e.g., orthogonal) open-top light sheet microscopes. For example, a non-orthogonal focusing objective (e.g., the first focusing objective in FIG. 1 ) and an orthogonal focusing objective can be provided, each using the same illumination objective. For example, FIG. 2 shows an illumination objective oriented at 45° to the non-orthogonal focusing objective and a second orthogonal focusing objective, also oriented at 45° (forming a 90° angle to the excitation objective), opposite the non-orthogonal focusing objective. In this combined multi-modality system, the non-orthogonal and orthogonal focusing paths can be handled separately using independent optical paths or combined into a single optical path ( FIG. 3 ). In some embodiments, the combined system provides multiscale imaging capabilities, with the non-orthogonal arrangement providing high-resolution imaging and the orthogonal arrangement providing low- to medium-resolution imaging ( FIG. 4 ).

[0118] 8A-8D are schematic diagrams of different redirecting optics according to some embodiments of the present disclosure. FIGS. 8A-8C are schematic diagrams of redirecting optics that may be used to redirect a non-orthogonal angle between the illumination axis and the collection axis, and FIG. 8D is a schematic diagram illustrating an exemplary operation of redirecting optics 800c of FIG. 8C in more detail. Any of the redirecting optics of FIGS. 8A-8D may be used in any of the microscopes described herein. For example, any of redirecting optics 800a-800c may be included in redirecting optics 230 of FIG. 2 and / or redirecting optics 340 of FIG. 3 in some embodiments. FIGS. 8A-8D illustrate many features already described with respect to previous figures, and therefore, for the sake of clarity, some components and features will not be described again.

[0119] 8A shows redirecting optics 800a that uses a detector 810 tilted relative to the optical path of the collected light. A collecting objective 802 (e.g., 204 in FIG. 2) collects light at a non-orthogonal angle relative to the illuminated focal plane 801. Tube lenses 804 and 806 direct the light to an additional collecting objective 808, which images the light onto detector 810. Unlike the redirecting optics shown in 230 in FIG. 2 and 340 in FIG. 3, redirecting optics 800a directly projects the remote focus onto detector 810 at a predetermined angle rather than using additional optics to image the remote focus at a predetermined angle.

[0120] The redirecting optics 800a can offer certain advantages. For example, the far focus should be approximately equal to the refractive index of the specimen, ideally in the range of 1.33 to 1.56. Therefore, the image plane at the far focus is approximately the same size as the image plane at the specimen. For higher-resolution imaging, the size of the image plane is typically on the order of 0.5 to 1.0 mm. This means that the detector must also have very small pixels of this same size to achieve Nyquist sampling. One potential advantage of placing the tilted detector at this location rather than at an intermediate image plane is that the image plane remains at 45° and does not tilt any further.

[0121] FIG. 8B shows redirecting optics 800b, where the second tube lens 806 and objective lens 808 are omitted; instead, tube lens 804 images directly onto detector 810, which is at an angle relative to the optical axis of tube lens 804 (and objective lens 802). Tube lens 804 can provide Nyquist sampling of the focal region 801 within the sample. The magnification for this image plane can be in the range of about 10x. This change in magnification causes the tilted image plane to be further tilted at the detector, which may not be ideal. However, this can be mitigated by using a detector with a particularly small pixel size.

[0122] 8C and 8D show a redirecting optical system 800c in which an adjustable lens 804 is used to change the focus of an oblique image of a focal region 801 onto a detector 810 that is not tilted relative to the optical axis of the collected light. The use of an adjustable lens 804 can allow the image to be redirected by synchronizing the adjustment of the lens to the operation of the detector 810 (e.g., to the rolling shutter of the camera 810). Figure 8D shows a schematic diagram illustrating an exemplary operation of using an adjustable lens 820 (e.g., adjustable lens 804 of optical system 800c) to change which portion of the obliquely focused light is focused on the detector 810 as the detector rolling shutter moves across the surface of the detector 810.

[0123] Of course, it should be understood that any of the examples, embodiments, or processes described herein may be combined with one or more other examples, embodiments, and / or processes, or may be performed among separable and / or distinct devices or device portions in accordance with the present systems, devices, and methods.

[0124] Finally, the foregoing description is intended to be merely illustrative of the present system and should not be construed as limiting the appended claims to any particular embodiment or group of embodiments. Accordingly, 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 skilled in the art without departing from the broader intended spirit and scope of the present system as set forth in the following claims. Accordingly, the specification and drawings are to be regarded as illustrative and are not intended to limit the scope of the appended claims.

Claims

1. a sample holder having a first side configured to support a sample and a second side opposite the first side; an illumination objective configured to direct an illumination light sheet into the sample along an illumination axis in a first mode and a second mode of operation; a collecting objective configured to receive light from an image plane of the sample along a collection axis in the first and third modes of operation; a second objective configured to receive light from an image plane of the sample along a second optical axis in the second mode of operation and to direct an illumination light sheet into the sample along the second optical axis in the third mode of operation; Equipped with the illumination axis and the collection axis are non-orthogonal to each other; the second optical axis is substantially perpendicular to the illumination axis; the illumination objective and the collection objective are positioned below the second side of the sample holder; Device.

2. The apparatus of claim 1 , further comprising an illumination optical system configured to generate the illumination light sheet, the illumination optical system being adjustable between a setting based on the focusing objective lens and a setting based on the second objective lens.

3. 3. The apparatus of claim 1, wherein the focusing objective lens has a first numerical aperture (NA) and the second objective lens has a second NA that is lower than the first NA.

4. The device comprises: a third objective configured to receive light from the collecting objective and generate a remote image; a fourth objective lens configured to image the remote image at an angle based on a non-orthogonal angle between the illumination axis and the collection axis; The apparatus of claim 1 , further comprising:

5. 5. The apparatus according to claim 1, further comprising an immersion fluid, wherein the illumination objective is not in contact with the immersion fluid and at least a portion of the collection objective is in contact with the immersion fluid.

6. The apparatus of claim 5 , wherein at least a portion of the sample holder is in contact with the immersion fluid.

7. 7. The apparatus according to claim 5, further comprising a lens positioned between the illumination objective and the immersion fluid.

8. The apparatus of claim 7 , wherein the lens is a solid immersion lens (SIL), a solid immersion meniscus lens (SIMlens).

9. 9. The apparatus of claim 1, wherein the focusing objective lens has a depth of focus for a given field of view, and the illumination axis is tilted at an angle of 40° to 70° so that the illumination light sheet does not remain within the depth of focus of the focusing objective lens for the given field of view.

10. a sample holder including a first surface and a second surface opposite the first surface, the first surface configured to support a sample; an illumination objective configured to direct an illumination light sheet toward the sample at a non-orthogonal angle relative to the first surface of the sample holder in a first mode and a second mode of operation; a collecting objective configured to collect light along a collection axis substantially perpendicular to the first surface in the first and third modes of operation; a second objective configured to receive light from an image plane of the sample along a second optical axis in the second mode of operation and to direct an illumination light sheet into the sample along the second optical axis in the third mode of operation; Equipped with the second optical axis is substantially perpendicular to the illumination light sheet; the illumination objective and the collection objective are positioned below the second surface of the sample holder. Device.

11. 11. The apparatus of claim 10, wherein the angle between the light collection axis and the illumination light sheet is between about 40° and 70°.

12. 12. The apparatus of claim 10 or 11, further comprising an immersion chamber positioned between the illumination objective and the second surface of the sample holder, the immersion chamber configured to hold an immersion fluid, and the illumination light sheet passes through the immersion fluid before reaching the sample.

13. The apparatus of claim 12 , further comprising a solid immersion lens (SIL), the illumination objective configured to direct the illumination light sheet through the SIL and into the immersion fluid.

14. 13. The apparatus of claim 12, further comprising a solid immersion meniscus lens (SIMlens), wherein the illumination objective lens is configured to direct the illumination light sheet through the SIMlens into the immersion fluid.

15. 15. Apparatus according to any one of claims 12 to 14, wherein at least a part of the collection objective is positioned in the immersion fluid, and the illumination objective is not in contact with the immersion fluid.

16. a sample holder having a first surface configured to support a sample and a second surface opposite the first surface; a first objective configured to direct an illumination sheet at the sample in a first mode of operation and a second mode of operation, the first objective having a first optical axis; a second objective configured to receive light from the sample in the first mode of operation, the second objective having a second optical axis that is non-orthogonal to the first optical axis; a third objective configured to receive light from the sample in the second mode of operation, the third objective having a third optical axis substantially orthogonal to the first optical axis; Equipped with the third objective lens is further configured to provide an illumination sheet at the sample in a third mode of operation; the second objective lens is further configured to receive light from the sample in the third mode of operation; the first objective lens, the second objective lens, and the third objective lens are positioned below the second surface of the sample holder. Device.

17. 17. The apparatus of claim 16, wherein the second optical axis is substantially perpendicular to the first surface, and the first optical axis and the third optical axis are non-orthogonal to the first surface.

18. The device comprises: a collection optical system configured to generate a remote image based on light received by the second objective lens in the first mode of operation or the third mode of operation; a fourth objective lens configured to collect light from the remote image at a first angle in the first mode of operation; a fifth objective lens configured to collect light from the remote image at a second angle in the third mode of operation; and 18. The apparatus of claim 16 or 17, further comprising:

19. 20. The apparatus of claim 18, further comprising a controller configured to combine images of the specimen from the first mode of operation and the third mode of operation to generate a highlighted image of the specimen.

20. 20. The apparatus of claim 16, further comprising an illumination optical system configured to generate the illumination sheet and provide the illumination sheet to the first objective lens, the illumination optical system configured to generate the illumination sheet in a first configuration in the first operating mode and configured to generate the illumination sheet in a second configuration in the second operating mode.

21. 21. The apparatus of claim 20, wherein the first arrangement has a first numerical aperture and a first width, and the second arrangement has a second numerical aperture smaller than the first numerical aperture and a second width larger than the first width.

22. in a first mode of operation and a second mode of operation, directing an illumination light sheet through an illumination objective to a focal region of a sample, the sample being supported on a first side of a sample holder, the sample holder further having a second side opposite the first side; in the first and third modes of operation, collecting light from the focal region through a collection objective, the optical axis of the collection objective being non-orthogonal to the optical axis of the illumination objective; receiving light from an image plane of the sample along a second optical axis through a second objective lens in the second mode of operation; directing an illumination light sheet through the second objective lens along the second optical axis to the focal region of the sample in the third mode of operation; imaging the collected light; Including, the second optical axis is substantially perpendicular to the optical axis of the illumination objective lens; the illumination objective and the collection objective are positioned below the second side of the sample holder; method.

23. 23. The method of claim 22, further comprising adjusting one or more characteristics of the illumination light sheet between the first and second modes of operation.

24. The method comprises: generating a remote image based on the collected light; imaging the remote image at an angle based on the non-orthogonal angle between the optical axis of the collection objective and the optical axis of the illumination objective; 24. The method of claim 22 or 23, further comprising:

25. 25. The method of any one of claims 22 to 24, wherein the quality of the image collected by the focusing objective is diffraction limited with a Strehl ratio greater than about 0.

8.

26. The method comprises: passing the illumination light sheet from the illumination light sheet through the surrounding medium, through an immersion fluid, and through the material of the sample holder to the focal region of the sample; collecting light through the material of the sample holder and through the immersion fluid onto the collecting objective; 26. The method of any one of claims 22 to 25, further comprising:

27. 1. A system comprising: an open-top light sheet (OTLS) microscope; a controller configured to operate the OTLS microscope; Equipped with The OTLS microscope comprises: a sample holder having a first side configured to support a sample and a second side opposite the first side; an illumination objective configured to direct an illumination light sheet into the sample along an illumination axis in a first mode and a second mode of operation; a first focusing objective configured to receive light from an image plane of the sample along a first focusing axis in the first and third modes of operation, wherein the illumination axis and the first focusing axis are non-orthogonal to one another; a second objective configured to receive light from an image plane of the sample along a second optical axis in the second mode of operation and to direct an illumination light sheet into the sample along the second optical axis in the third mode of operation, wherein the illumination axis and the second optical axis are substantially orthogonal to one another; Equipped with the illumination objective and the collection objective are positioned below the second side of the sample holder; system.

28. 28. The system of claim 27, wherein the controller is configured to combine information from images collected in the first mode of operation and images collected in the second mode of operation.

29. 30. The system of claim 28, wherein the controller is configured to combine the information using image processing, machine learning, deep learning, or a combination thereof.

30. the OTLS microscope further comprises illumination optics configured to generate the illumination light sheet; the controller is configured to instruct the illumination optics to adjust one or more characteristics of the illumination light sheet between the first mode of operation and the second mode of operation.

30. A system according to any one of claims 27 to 29.

31. The OTLS microscope is further configured to operate in an alternate mode, wherein the second objective is configured to provide an illumination light sheet and the first focusing objective is configured to receive light from the image plane of the sample; the controller is further configured to collect a first image when the illumination light sheet is provided by the illumination objective lens, collect a second image when the illumination light sheet is provided by the second objective lens, and generate an enhanced image based on the first image and the second image.

31. A system according to any one of claims 27 to 30.

32. 32. The system of claim 31, wherein the controller is configured to generate the enhanced image based at least in part on a fusion deconvolution algorithm.

Images