Open-top light-sheet microscope with non-orthogonal arrangement of illumination and focusing objective lenses.

Abstract

The geometric configuration of an OTLS microscope imposes various limitations on image resolution, depth of field within the sample, and / or may impose relatively tight refractive index tolerances on the system. [Solution] Apparatus, system, and method for an open-top light-sheet (OTLS) microscope 100, including an illumination objective lens and a condensing objective lens having mutually non-orthogonal optical axes. The optical axis of the condensing objective lens 128 may be orthogonal to the plane of the sample holder 108. The illumination objective lens 122 and the condensing objective lens may be located below the sample holder. The OTLS microscope may optionally include a second condensing objective lens having an optical axis orthogonal to the optical axis of the illumination objective lens. The illumination objective lens may be an air objective lens, and the condensing objective lens may be an immersion objective lens.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Patent Application No. 62 / 934,758, filed on November 13, 2019, the entire content of which is incorporated herein by reference in its entirety for all purposes.

[0002] Description of Research and Development 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 United States government has certain rights in this invention.

[0003] Background of the Invention Microscopy generally can involve directing light onto a sample and then imaging the sample based on the light received from the sample. One method of illumination is to use a light sheet where a relatively thin plane of the sample is illuminated. This can have advantages in terms of both the optical properties of the tissue (e.g., reduction of photobleaching and phototoxicity) and improvement of throughput for imaging large numbers of samples. An open - top light - sheet (OTLS) microscope configuration, similar to a flat - bed document scanner for tissues, has been developed to enable convenient imaging of one or more tissue specimens without lateral constraints. The geometric arrangement of the OTLS microscope can impose various limitations on the resolution of the image, the depth of imaging within the sample, and / or can impose relatively stringent refractive index tolerances on the system. There may be a need to develop an OTLS microscope that addresses some of these trade - offs while maintaining the advantageous aspects of the open - top configuration.

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

[0005] The apparatus may also include a second focusing objective lens capable of receiving light from the imaging plane of the sample along a second focusing axis. The second focusing axis may be substantially perpendicular to the illumination axis. The apparatus may also include an illumination optical system capable of generating an illumination light sheet. The illumination optical system may be adjustable between a setting based on the first focusing objective lens and a setting based on the second focusing objective lens. The first focusing objective lens may have a first numerical aperture (NA), and the second focusing objective lens may have a second NA lower than the first NA.

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

[0007] The apparatus may include an immersion fluid. The illumination objective lens does not need to be in contact with the immersion fluid, but at least a portion of the focusing objective lens 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 lens and the immersion fluid. The lens may be a solid immersion lens (SIL) or a solid immersion meniscus lens (SIMlens).

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

[0009] In at least one aspect, the present disclosure relates to an apparatus comprising a sample holder, an illumination objective lens, and a focusing objective lens. The sample holder includes a first surface and a second surface opposite the first surface. The first surface supports the sample. The illumination objective lens orients an illumination light sheet toward the sample at an angle not perpendicular to the first surface of the sample holder. The focusing objective lens collects light along a focusing axis substantially perpendicular to the first surface.

[0010] The illumination objective lens and the focusing objective lens may be positioned below the second surface. The apparatus may include a second focusing objective lens capable of collecting light along a second focusing axis that is substantially perpendicular to the illumination light sheet. The focusing axis may be at an acute angle with respect to the illumination light sheet. The acute angle may be approximately 40° to 70°.

[0011] The apparatus may also include an immersion chamber positioned between the illumination objective lens and a 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), through which the illumination objective lens may orient the illumination light sheet into the immersion fluid. The apparatus may also include a solid immersion meniscus lens (SIMlens), through which the illumination objective lens may orient the illumination light sheet into the immersion fluid. At least a portion of the focusing objective lens may be positioned in the immersion fluid, and the illumination objective lens may not be in contact with the immersion fluid.

[0012] In at least one embodiment, the present disclosure relates to an apparatus comprising first, second, and third objective lenses. The first objective lens orients an illumination sheet to a sample in a first and 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 not perpendicular 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 substantially perpendicular to the first optical axis.

[0013] The apparatus may include a sample holder having a first surface for supporting a sample. The second optical axis may be substantially perpendicular to the first surface, and the first and third optical axes may be non-perpendicular to the first surface. The sample holder may also include a second surface opposite to the first surface, and the first, second, and third objective lenses 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 a third operating mode.

[0014] The apparatus may also include a focusing optical system capable of generating a remote image based on light received by a second objective lens in a first or third operating mode, a fourth objective lens capable of collecting light from the remote image at a first angle in the first operating mode, and a fifth objective lens capable of collecting light from the remote image at a second angle in the third operating mode. The apparatus may also include a controller capable of combining images of the sample from the first and third operating modes to generate a enhanced image of the sample.

[0015] The apparatus may include an illumination optical system that generates and provides an illumination sheet to a first objective lens. The illumination optical system may generate an illumination light sheet in a first configuration in a first operating mode, and may generate an illumination light sheet in a second configuration in a second operating mode. 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 comprising orienting an illumination light sheet to a focal region of a sample through an illumination objective lens, collecting light from the focal region through a condensing objective lens, wherein the optical axis of the condensing objective lens is non-orthogonal to the optical axis of the illumination objective lens, and imaging the collected light.

[0017] The method may also include collecting light from a focal region through a second condensing objective lens, wherein the optical axis of the second condensing objective lens is substantially perpendicular to the optical axis of the illumination objective lens, and imaging the light collected from the second condensing objective lens. Collecting light through the condensing objective lens may be part of a first operating mode, and collecting light through the second condensing objective lens may be part of a second operating mode. The method may also include adjusting one or more characteristics of the illumination light sheet between the first operating mode and the second operating mode.

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

[0019] In at least one aspect, the 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 lens that orients an illumination light sheet into a sample along an illumination axis, a first condensing objective lens, and a second condensing objective lens. The first condensing objective lens receives light from the imaging plane of the sample along a first condensing axis. The illumination axis and the first condensing axis are non-orthogonal to each other. The second condensing objective lens receives light from the imaging plane of the sample along a second condensing axis. The illumination axis and the second condensing axis are orthogonal to each other.

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

[0021] An OTLS microscope may also include an illumination optics configured to produce an illumination light sheet. A controller may instruct the illumination optics to adjust one or more characteristics of the illumination light sheet between a first operating mode and a second operating mode.

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

[0023] [Figure 1] Block diagram of an open-top light sheet (OTLS) microscope according to some embodiments of the present disclosure. [Figure 2A] Schematic diagram of a part of an OTLS microscope according to some embodiments of the present disclosure. FIG. 2A is a diagram showing the layout of the OTLS microscope. [Figure 2B] Detailed view showing a part of the microscope of FIG. 2A. [Figure 2C] Detailed view showing a part of the microscope of FIG. 2A. [Figure 2D] Detailed view showing a part of the microscope of FIG. 2A. [Figure 3A] Schematic diagram of an OTLS microscope according to some embodiments of the present disclosure. FIG. 3A is a diagram showing the microscope 300. [Figure 3B] Enlarged view showing different arrangements of an illumination objective lens and a condenser objective lens that can be used with the microscope of FIG. 3A. [Figure 3C] Enlarged view showing different arrangements of an illumination objective lens and a condenser objective lens that can be used with the microscope of FIG. 3A. [Figure 4] Diagram of a sample holder of a microscope according to some embodiments of the present disclosure. [Figure 5A] Schematic diagram of illumination and collected light in the first operating mode of an OTLS microscope. [Figure 5B] Schematic diagram of illumination and collected light in the second operating mode of an OTLS microscope [Figure 6A] Diagram showing a hybrid OTLS microscope with a dual illumination mode according to some embodiments of the present disclosure. [Figure 6B] Diagram showing a hybrid OTLS microscope with a dual illumination mode according to some embodiments of the present disclosure. [Figure 7] Block diagram of a method of illuminating a sample using a microscope according to some embodiments of the present disclosure. [Figure 8A]Figure 8A is a schematic diagram of a redirection optical system according to some embodiments of the present disclosure. [Figure 8B] Figure 8B is a schematic diagram of a redirecting optical system that can be used to redirect non-orthogonal angles between the illumination axis and the focusing axis. [Figure 8C] Figure 8C is a schematic diagram of a redirecting optical system that can be used to redirect non-orthogonal angles between the illumination axis and the focusing axis. [Figure 8D] This is a schematic diagram illustrating the exemplary operation of the redirection setting optical system 800c in Figure 8C in more detail.

[0024] Detailed explanation The following descriptions of specific embodiments are substantially illustrative and are not intended to limit the scope of this disclosure or its use or application. The following detailed descriptions of embodiments of the systems and methods refer to the accompanying drawings, which form part of this specification and illustrate specific embodiments in which the systems and methods described may be put into practice. These embodiments are described in sufficient detail to enable those skilled in the art to implement the systems and methods currently disclosed, and it should be understood that other embodiments may be used, and that structural and logical modifications may be made without departing from the spirit and scope of this disclosure. Furthermore, for clarity, detailed descriptions of certain features are omitted where they would be obvious to those skilled in the art, so as not to obscure the descriptions of embodiments in this disclosure. Therefore, the following detailed descriptions should not be constrained, and the scope of this disclosure is defined solely by the accompanying claims.

[0025] An open-top light-sheet (OTLS) microscope includes a sample holder that supports the sample to be imaged on the top surface of the sample holder, while the illumination and focusing optics are positioned on the opposite side, below the bottom surface of the sample holder. Thus, light can enter the illumination area of ​​the sample from the illumination objective lens, through the sample holder. Light from the illumination area can pass through the sample holder to the focusing objective lens, which can then image the collected light onto a detector (e.g., CCD, CMOS, etc.). It may be advantageous to use two separate objective lenses: an illumination objective lens for orienting the illumination light sheet onto the sample, and a focusing objective lens for collecting light from the sample and orienting it towards the detector.

[0026] Some OTLS microscopes can use a geometric arrangement where the optical axes of the illumination and focusing objectives are orthogonal. For example, the illumination and focusing axes may be 45° to the specimen and 90° to each other. Such a design can have drawbacks. For example, the achievable imaging resolution is limited by the numerical aperture (NA) of the objectives (illumination and / or focusing). Objectives with higher NAs allow for higher resolution, but they also tend to have shorter working distances, thus resulting in a shorter depth of field into the specimen. If the objectives are oriented obliquely to the specimen, this limitation can be more severe because the working distances of both objectives are oriented so that light needs to travel a further distance to reach the imaging area. This can limit the depth of field in relatively thick specimens, thus limiting the thickness of specimens that can be imaged at the desired high resolution. Another exemplary drawback is that, in the case of OTLS microscopes with high NA objective lenses, the quality of the off-axis focused beam (i.e., a beam not orthogonal to the sample) can be significantly degraded (aberrations occur) due to very small refractive index mismatches between the transparent tissue, the sample holder, and the immersion medium. Therefore, such systems may impose relatively stringent requirements for refractive index matching between the immersion solution, the sample holder, and the tissue. It may be desirable to design a microscope that overcomes one or more of these challenges.

[0027] This disclosure relates to an open-top light-sheet microscope using a non-orthogonal arrangement of illumination and focusing objective lenses. In other words, the illumination objective lens may have an illumination axis, and the focusing objective lens may have a focusing axis, and the illumination and focusing axes are non-orthogonal to each other. In some embodiments, the focusing objective lens may be substantially orthogonal to the plane of the sample holder (e.g., the focusing axis may be perpendicular to the surface supporting the sample), while the illumination objective lens is non-orthogonal (e.g., at an angle of 45°). This can enable an OTLS microscope in which the focusing objective lens can utilize its entire imaging depth (working distance). This, in turn, can make it easier to use focusing objective lenses with relatively high NAs (which tend to have shorter working distances), thereby enabling high-resolution imaging of the sample. Furthermore, using this geometric arrangement allows the collected light to pass through the sample holder at a low angle of incidence, thus reducing the refractive index matching requirement for the focusing path. This geometric arrangement can also be advantageous because it does not impose lateral constraints on the movement of the sample.

[0028] In some embodiments, an OTLS microscope may include a second focusing objective lens that can be oriented perpendicular to the illumination objective lens (and not perpendicular to the sample holder). The OTLS microscope may include an optical system that allows for the exchange of two optical paths (e.g., two focusing objective lenses). The first focusing objective lens, which is not perpendicular to the illumination (and may be perpendicular to the plane of the sample holder), may have a higher NA than the second focusing objective lens, which is perpendicular to the illumination objective lens. Using 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 with an objective lens with a lower NA (and therefore a larger field of view) to identify areas of interest, and then examining these areas in more detail with an objective lens with a higher NA. The resolution of the microscope can be adjusted by adjusting the illumination and / or focusing optical system.

[0029] Figure 1 is a block diagram of an open-top light-sheet (OTLS) microscope according to several embodiments of the present disclosure. Figure 1 shows an optical system 100, which includes an open-top light-sheet (OTLS) microscope 102 and a controller 104 as an optional means capable of operating the microscope 102 and / or interpreting information from the microscope 102. In some embodiments, one or more parts of the controller 104 may be omitted, and the microscope 102 can be operated manually. In some embodiments, one or more parts of the controller 104 may be integrated with 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 a light path and a focusing path that are separated from each other. The light path includes a light source 118, a light optics system 120, and a light objective lens 122. The light path provides a light beam 124, which passes through the sample holder 108 to illuminate the sample 106. The focusing path includes a focusing objective lens 128, a focusing optics system 130, and a detector 132. The focusing path can collect light from a focal region 126 illuminated by the light beam 124. The optical axis of the focusing objective lens 128 may be at an angle θ with respect to the optical axis of the light objective lens 122 (e.g., angle θ with respect to the light beam 124). The angle θ may be non-orthogonal (e.g., acute). Such arrangements of illumination and optical components can generally be referred to as a non-orthogonal, dual objective (NODO) system.

[0031] The illumination objective lens 122 and the focusing objective lens 128 can generally be located below the bottom surface of the sample holder 108. This allows the top surface of the sample holder 108 to be relatively open, and then makes it easier to place the sample 106 on the sample holder 108. For example, the sample holder may have a top surface that is a flat plate (similar to, for example, a commercially available flatbed scanner), and different samples may be placed on the flat plate. This also reduces / eliminates lateral constraints on the sample 106.

[0032] In some embodiments, the microscope 102 may include a second focusing objective lens 160 as an additional optional means. The second focusing objective lens 160 can image a focal region 126 at a focusing axis that makes 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 about 90° (e.g., orthogonal). Imaging using the second focusing objective lens 160 may be referred to as orthogonal dual-objective (ODO) imaging. The second focusing objective lens 160 may have its own focusing optical system 162, which couples the collected light to a detector 164. In some embodiments, instead of using a second detector 164, a detector 132 may be shared by both the focusing objective lenses 128 and 160. Exemplary embodiments having multiple focusing objective lenses are described in more detail in Figures 3 to 7.

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

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

[0035] In some embodiments, an optical system 100 can be used to image fluorescence within the sample 106. The illumination beam 124 may contain light of a specific excitation wavelength that can excite fluorophores in the sample 106. The illumination beam 124 may contain a broad spectrum of light including the excitation wavelength, or it may be a narrowband spectrum centered on the excitation wavelength. In some embodiments, the light source 118 may produce a narrow spectrum of light centered on (or near) the excitation wavelength. In some embodiments, a filter (not shown) in the illumination optical system 120 can be used to restrict the illumination beam 124 to a wavelength close to the excitation wavelength. When excited by the illumination beam 124, the fluorophores in the sample 106 may emit light (which may be centered on a given emission wavelength). A focusing path (e.g., a focusing optical system 130) may include one or more filters that can be used to restrict the light reaching the detector 132 to a wavelength close to the emission wavelength.

[0036] The illumination optics system 120 can couple light from the light source 118 to the illumination objective lens 122. For example, the illumination optics system 120 may include an optical fiber that propagates light from the light source 118 to the rear end of the illumination objective lens 122. In some embodiments, the illumination optics system 120 can couple light between the light source 118 and the objective lens 122 without substantially altering the light provided by the light source 118. In some embodiments, the illumination optics system 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 system 120 may receive broadband light from the light source 118 and filter the light (e.g., using filters, diffraction gratings, acousto-optic modulators, etc.) to provide narrowband light to the objective lens 122.

[0037] In some embodiments, the illumination optical system 120 may include a scanning optical system (e.g., a scanning mirror), and the illumination light can be scanned using the scanning optical system. In some embodiments, the illumination beam 124 can be generated in the form of a light sheet using the scanning optical system (e.g., by scanning the light back and forth along one axis and not scanning along the other axis). In some embodiments, the position of the field of view relative to the sample 106 can be changed using the scanning optical system.

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

[0039] The illumination path can provide an illumination beam 124, which is an illumination beam that is a light sheet as part of a light sheet microscope or light sheet fluorescence microscope (LSFM). The light sheet may have a substantially elliptical cross-section and have 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 perpendicular to the first axis. The illumination optical system 120 may include an optical system that reshapes the light received from the light source 118 into an illumination sheet. For example, the illumination optical system 120 may include one or more cylindrical optical systems that focus light along one axis but not along perpendicular axes.

[0040] In some embodiments, the illumination optical system 120 may include a scanning optical system that can be used to scan the illumination beam 124 over the sample 106. For example, the area illuminated by the illumination beam may be smaller than the desired focal area 126. In this case, the illumination optical system 120 can rapidly vibrate the illumination beam 124 over the entire desired focal area 126 to ensure illumination of the focal area 126.

[0041] The illumination objective lens 122 may include one or more lenses that provide the illumination beam 124. For example, the illumination objective lens 122 may focus the illumination beam 124 toward a focal region 126. The sample holder 108 can position the sample 106 such 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 lens may be a commercially available objective lens including one or more internal optical elements. In some embodiments, the illumination objective lens 122 may be surrounded by the ambient environment (e.g., air), and the illumination objective lens 122 may be an air objective lens. The illumination objective lens 122 may be characterized by one or more numerical apertures, which may be based on the angle at which the light converges in the focal region 126. In some embodiments, the illumination objective lens 122 may be an immersion objective lens, and at least a portion of the illumination objective lens 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 oriented 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 represent an area imaged by the illumination beam 124 from which the focusing objective lens 128 can collect light. In some embodiments, the focal region 126 can represent a single field of view of the focusing objective lens 128. In some embodiments, the focal region 126 can represent an area from which the field of view of the focusing objective lens 128 can be scanned.

[0043] The sample 106 can be supported by the upper surface of the sample holder 108. In some embodiments, the sample 106 may be placed directly on the upper 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 with the sample holder 108. In some embodiments, the sample 106 may be processed before imaging on the optical system 100. For example, the sample 106 may be washed, sliced, and / or labeled before imaging.

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

[0045] In some embodiments, sample 106 may be a non-biological sample. For example, sample 106 may be a fluid and may contain one or more components for investigation. For example, 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, sample 106 may contain one or more types of fluorophores. The fluorophores may be specific to sample 106 (e.g., DNA and proteins in a biological sample) or may be fluorescent labels applied to sample 106 (e.g., acridine orange, eosin). Sample 106 may contain a mixture of specific 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 the excitation wavelength (e.g., redshifted therefrom).

[0047] The sample holder 108 can support the sample 106 on a material that is generally transparent to light collected from the illumination beam 124 and the focal region 126 of the sample 106. In some embodiments, the sample holder 108 may have a window of transparent material on which the sample 106 can be positioned, and the rest 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 to support 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, and different sample holders 108 may be interchangeable depending on the type of sample, the type of imaging, the wavelength of the illuminated / collected light, and combinations thereof.

[0049] The sample holder 108 may have a second surface (e.g., a bottom surface) opposite to the surface of the sample holder 108 that supports the sample 106. In some embodiments, an immersion chamber 110 that holds the immersion fluid 112 may be located below the second surface of the sample holder 108. In some embodiments, the immersion chamber 110 may have an open top and the immersion fluid 112 may be in contact with the second surface of the sample holder 108. In some embodiments, the second surface of the sample holder 108 may be in contact with the immersion fluid 112, while the first surface of the sample holder 108 (supporting the sample 106) may 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 may be capable of moving the sample holder 108 in one or more directions. In some embodiments, the sample holder 108 may 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 may be movable along the x, y, and / or z axes, and / or rotatable (e.g., slope, tilt, etc.). Moving the sample holder 108 can change the position of the focal region 126 in the sample 106, and / or move the sample holder 108 between a loading position and an imaging position. In some embodiments, the actuator may be a manual actuator such as a screw or a coarse / fine adjustment knob. In some embodiments, the actuator may be automated and may be an electric motor, etc., capable of responding to manual input and / or commands from a controller 104. In some embodiments, the actuator 109 can respond to both manual adjustment and automatic control (for example, it may be a knob that responds to both manual rotation and commands from the controller 104).

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

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

[0053] In the NODO geometric configuration shown in Figure 1, the focusing path may have a principal optical axis positioned at an angle γ with respect to the plane of the sample holder 108 (e.g., the XY plane in Figure 1). In some embodiments, such as those shown in Figure 1, the angle γ may be approximately 90°. That is, the focusing path may have a principal optical axis that is nearly perpendicular to the plane of the sample holder 108. The angle γ may be sufficiently close to 90°, i.e., nearly perpendicular, as long as the quality of the image collected by the focusing objective lens remains diffraction-limited. In other words, when using the Strehr ratio as the figure of merit, the Strehr ratio is greater than approximately 0.8. As will be apparent to those skilled in the art, the Strehr ratio may depend on many parameters potentially applicable to a given OTLS microscope system, in addition to the angle α, such as refractive index mismatch (i.e., optical path difference, or the product of the difference in refractive index between the holder and the immersion medium / clear tissue sample and the thickness of the holder), the NA of the illumination and focusing objective lenses, the field of view of the objective lens, the wavelength of the illumination light and / or collected light, and the specific objective lens used.

[0054] The illumination path may have a principal optical axis positioned at an angle θ with respect to the principal optical axis of the focusing path. The angle θ may be non-orthogonal, i.e., acute. Several considerations may limit the range of acceptable values ​​for angle θ. For example, if the angle is 90°, or close to 90°, i.e., almost parallel to the plane of the specimen holder, it may be impractical because it intersects the specimen holder and restricts the lateral dimensions of the specimen. Also, even if the NA of the illumination beam is relatively low, the constraints on refractive index matching may become very strict. Other factors, including physical constraints imposed by the mechanical housing of the focusing objective lens, may limit the lower limit of the range of values ​​for angle θ. Exemplary constraints imposed by the geometric arrangement of the objective lens are described in more detail in Figure 2.

[0055] The geometric shape of the focal region 126 may be partially defined by the field of view of the focusing path, while the field of view may partially depend on the numerical aperture of the focusing objective lens 128. Similar to the illumination objective lens 122, the focusing objective lens 128 may be a commercially available objective lens comprising one or more lenses. In some embodiments, the focusing objective lens 128 may be an air objective lens. In some embodiments, the focusing objective lens 128 may be an immersion objective lens (e.g., an oil immersion objective lens). In some embodiments, the focusing objective lens 128 may use a different immersion medium than the immersion fluid 112 used in the illumination path. In some embodiments, the focal region where the focusing path is focused and the focal region where the illumination path is focused may generally overlap in the focal region 126. In some embodiments, the illumination path and the focusing path may have different shapes, sizes, and / or locations of their respective focal regions.

[0056] The focusing path includes a focusing optical system 130, which can redirect light from the focusing objective lens onto the detector 132. For example, the focusing optical system 130 may be a tube lens designed to focus light from the rear end of the focusing objective lens onto an image projected onto the detector 132. In some embodiments, the focusing optical system 130 may include one or more elements that modify the light received from the focusing objective lens 128. For example, the focusing optical system 130 may include a filter, a mirror, a death scanning optical system, or a combination thereof.

[0057] The focusing optical system 130 may include an optical system capable of reorienting the view of the focal region 126. Since the axis of the focusing objective lens 128 is at an angle θ with respect to the focal region 126, image distortion may occur. The focusing optical system 130 may include one or more features that allow the image to be reoriented, taking angle θ into account, before the image is projected onto the detector 132. For example, the focusing optical system 130 may include a remote focus, where a first lens projects an image of the light collected by the focusing objective lens 128, and a second lens images a remote image at an angle that cancels out angle θ. This allows distortion due to angle θ to be corrected before the light reaches the detector 132. Other exemplary embodiments may use other methods for reorienting the image.

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

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

[0060] The controller 104 includes one or more input / output devices 142, which may enable the user to receive feedback from the controller 104, view data from the microscope 102, provide commands to the controller 104, provide commands to the microscope 102, or a combination thereof. For example, the input / output devices 142 may include a digital display, a touchscreen, a mouse, a keyboard, or a combination thereof.

[0061] The controller 104 includes a processor 140 capable of executing one or more instructions stored in memory 144. These instructions may include control software 152, which may contain 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 actuators 109. The instructions may also include image processing software 150, which can be used to process a "live" image 146 from the detector 132 or an image 146 pre-stored in memory 144. The image processing software 150 can, for example, remove background noise from the image 146. The instructions may also 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) by 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 where the focal region 126 is located. The controller 104 can then instruct the detector 132 to collect another image, and the process can be repeated until a set number of images in the stack and / or a set total displacement in the z-direction are achieved. The analysis software 148 can then combine the depth stacks of images to enable 3D (or pseudo-3D) imaging of the sample 106. Similarly, multiple fields of view can be collected using various other translations. For example, the sample may be scanned along the x, y, and / or z axes. The position of the objective lens (and other optical systems) below the sample holder 108 may allow for less restrictive scanning in these directions, so the geometric arrangement of the OTLS may be particularly useful for scanning in the X or Y direction.

[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 instance, in a first imaging mode, light may be collected from the focusing objective lens 128, while in a second imaging mode, light may be collected through a different focusing objective lens (not shown in Figure 1). Exemplary embodiments having multiple imaging modes are described in more detail in Figures 3 to 6B.

[0064] Figures 2A to 2D are schematic diagrams of some OTLS microscopes according to several embodiments of the present disclosure. Figure 2A shows the layout of the OTLS microscope, and Figures 2B to 2D show detailed views of some parts of the microscope in Figure 2A. In some embodiments, the OTLS microscope 200 may be included in the microscope 102 of Figure 1. For simplicity, details and operations already described with respect to Figure 1 will not be repeated with respect to Figure 2.

[0065] Figure 2 shows a schematic diagram of a microscope focusing on the illumination optical system and the interaction between the focusing optical system and the immersion chamber and sample, along with a portion of the focusing optical system. Microscope 200 may include additional components omitted from Figure 2 for clarity of the drawing.

[0066] The microscope 200 includes an illumination objective lens 202, which receives illumination light from an illumination source and other illumination optics (not shown) as an optional means, and directs the illumination beam 218 into the immersion fluid 222 through a lens 224. The immersion fluid 222 is contained by an immersion chamber 220. The immersion light 218 enters the sample 228 by passing through the bottom surface of the sample holder 226. The collected light 216 exits the sample 228 and enters the focusing objective lens 204 through the sample holder 226 and the immersion fluid 222. The principal axis of the illumination light 218 may be non-orthogonal to the plane of the sample holder 226, while the principal 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 the microscope 200, an exemplary redirection optical system 230 is shown, which can be used to correspond to a non-orthogonal angle θ between the illumination light 218 and the collected light 216. Various types of redirection optical systems 230 can be used. The example in Figure 2 shows a particular implementation of the redirection optical system 230 with a remote image 212. However, other embodiments may use other methods to achieve the redirection optical system 230. Various exemplary redirection optical systems that can be used instead of the redirection optical system 230 in Figure 2 are described in Figures 8A to 8D.

[0068] In the redirection optical system 230 of Figure 2, the condensing objective lens 204 can direct light into the second condensing objective lens 206 through the transmission optical system 210, which is an optional means. The second condensing objective lens 206 can generate a remote image 212 that can be imaged by the third condensing objective lens 208. The second condensing objective lens 206 and the third condensing objective lens 208 can form a predetermined angle. This angle may be based on the angle between the collected light 216 and the illumination light 218. The third condensing objective lens 208 can direct light into a detection system (not shown).

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

[0070] In some embodiments, the lens 224 can be shaped to reduce the refraction of light as it passes from the air through the material of the lens 224 into the immersion fluid 222. In some embodiments, the lens 224 can be made of a material having a refractive index matched to the refractive index of the immersion fluid 222. In some embodiments, the lens 224 may have one or more surfaces shaped to match the wavefront of the light passing through it, thereby eliminating / reducing the refraction of light passing through the lens 224. For example, the lens 224 may be a solid immersion (SIL) lens or a solid immersion meniscus lens (SIMlens), as disclosed in U.S. Patent No. 1,0409,052 and International Publication No. 2020 / 150239, which are incorporated herein by reference.

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

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

[0073] The collected light 216 can pass through the sample holder 226 and the immersion fluid 222 before leaving the sample 228 and entering the proximal lens of the focusing objective lens 204. The focusing objective lens may be an immersion objective lens in which the proximal lens is in contact with the immersion fluid 222. The distal end of the focusing objective lens 204 may be positioned outside the immersion fluid 222 in the ambient environment (e.g., air). The collected light 216 may represent a portion of the light that leaves the sample 228, passes through the focusing objective lens 204 and other focusing optics, and reaches the detector. The size and geometric shape of the collected light 216 may be based, at least in part, on the focusing objective lens 204 and other focusing optics.

[0074] The proximal lens of the focusing objective lens 204 can be located much closer to the sample 228 than the proximal lens of the illumination objective lens 202. This can allow the focusing objective lens 204 to be an objective lens with a higher numerical aperture (NA) than the illumination objective lens 202. Since the focusing objective lens 204 is nearly perpendicular to the plane of the sample holder 226, refraction can be reduced (compared to the illumination beam 218). In some embodiments, the focusing objective lens 204 may be an air objective lens 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 condensing objective lens 204 can direct light to a second condensing objective lens 206 through a transmission optical system 210, which is an optional means. In some embodiments, the transmission optical system 210 may include one or more lenses. For example, the transmission optical system 210 may be a 4f relay system.

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

[0077] For example, as shown in Figure 2B, the exemplary mechanical housing of the focusing objective lens allows for 45° illumination light paths on both sides and has a maximum cone angle β (e.g., the angle between the optical axis of the illumination light 218 and the edge of the focusing objective lens 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 of the objective lens 204, and other angles may be used in other exemplary embodiments.

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

[0079] A condensing objective lens with a shallower angle and a larger housing may further restrict the range of illumination path angles. From another perspective, the illumination light sheet may be tilted to such an extent that it does not remain within the confocal parameter (depth of focus) of the condensing objective lens for the desired field of view (i.e., not orthogonal). Considering this, a reasonable range for the angle θ may be 40° to 70°. For example, the illumination path may be along a first optical axis at an angle of 45° with respect to the bottom surface of the sample holder, while the condensing path may be along a second optical axis at an angle of 90° with respect 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] Figures 3A to 3C are schematic diagrams of OTLS microscopes according to several embodiments of the present disclosure. Figure 3A shows microscope 300, and Figures 3B and 3C show enlarged views of different arrangements of illumination objective lenses and condensing objective lenses, respectively, which may be used with the microscope of Figure 3A. Microscope 300 may, in some embodiments, be included in microscope 102 of Figure 1 and / or microscope 200 of Figure 2. Microscope 300 may be substantially similar to the microscopes described above, except that microscope 300 includes an additional imaging path using a condensing objective lens orthogonal to the illumination sheet, in addition to the non-orthogonal imaging paths described in Figures 1 and 2.

[0081] Since the non-orthogonal double objective (NODO) path of microscope 300 can generally be similar to the operation and components of microscope 102 in Figure 1 and microscope 200 in Figure 2, for simplicity, features and components already described with respect to Figures 1 and 2 will not be repeated with respect to Figure 3.

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

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

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

[0085] The path from the light source (not shown) through the illumination objective lens 302 to the sample 308, and the path from the sample 308 through the fourth focusing objective lens 306 to the second detector 328, can form an ODO optical path. The illumination optical path and the light collected by the fourth focusing objective lens 306 (for example, the optical axis of the illumination objective lens 302 and the optical axis of the fourth focusing objective lens 306) can form an angle that is orthogonal to each other.

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

[0087] In some embodiments, the illumination path may be adjustable and may be adjusted between ODO imaging mode and NODO imaging mode. For example, an illumination optical system (not shown), such as the illumination optical system 120 in Figure 1, may include adjustable components that can adjust the size and shape of the illumination light sheet based on whether a first focusing objective lens 304 or a fourth focusing objective lens 306 is used. For example, the illumination optical system may include a variable beam expander that can be used to adjust the characteristics of the illumination light sheet, such as the NA and / or width of the light sheet. The adjustment of 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 Figure 5.

[0088] In exemplary operation, a sample can be placed on a microscope and screened using an ODO optical path. While the ODO path may have lower resolution and magnification than the NODO path, it may offer a larger field of view. Therefore, screening a sample using the ODO path may be more efficient. In some embodiments, the sample 308 can be scanned (e.g., by the movement of the focal region relative to the sample, by the 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 Figure 1). In some embodiments, the sample 308 can be scanned in three dimensions to construct a volumetric image of the sample. In some embodiments, scanning can be performed manually.

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

[0090] In NODO mode, the microscope 300 may have higher resolution and magnification, but a smaller field of view. NODO mode can be useful for determining one or more characteristics of a region of interest. For example, a clinician can 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 allows the microscope 300 to have a relatively wide operating range with different performance characteristics.

[0091] In some embodiments, information collected in a first operating (imaging) mode (e.g., NODO mode) can be combined with information collected in a second operating (imaging) mode (e.g., ODO mode). This process can be automated. For example, a controller (e.g., 104 in Figure 1) can image a region 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 region is a region of interest. The controller can then use NODO mode to image the region of interest in more detail. In some embodiments, the process may be manual, and a user can identify the region of interest. In some embodiments, a mixture of manual and automated processes (e.g., automated image processing, but manual identification of the region of interest) can also be used.

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

[0093] Figure 4 shows a sample holder of a microscope according to several embodiments of the present disclosure. The sample holder 400 in Figure 4 may, in some embodiments, be included in a microscope such as the microscope 300 in Figure 3, which uses NODO and ODO optical paths. Since Figure 4 shows a diagram focusing on the interaction between the objective lens and the sample holder 400, various components of the microscope have been omitted. For simplicity, the operations, features, and components described above with respect to Figures 1 to 3 will not be repeated again with respect to Figure 4.

[0094] The sample holder 400 supports the immersion fluid 420. The microscope, including the sample holder 400, includes a NODO optical path and an ODO optical path. The illumination objective lens 402 provides an illumination light sheet to the focal region 410 along the illumination optical axis 403. The illumination objective lens 402 may be an air objective lens 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 to the immersion fluid 420.

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

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

[0097] As can be seen from Figure 4 and as described above, the objective lenses 402, 404, and 406 can be positioned so that the NODO focusing objective lens 404 does not block light from the illumination objective lens 402 or the ODO focusing objective lens 406. The angles of the light from the illumination objective lens 402 and the light collected by the ODO focusing objective lens 406 may be based in part on the respective NAs of these objective lenses. Therefore, the NAs of these objective lenses, as well as the size and shape of the NODO objective lens 404, can be designed so as not to interfere with each other.

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

[0099] Figures 5A and 5B each show the respective illumination light sheets 502a / 502b and the respective collected light cones 504a / 504b. The illumination light sheets and collected light interact to generate a field of view (FOV). The illumination light sheets can be adjusted between operating modes to accommodate different geometric shapes of collected light.

[0100] Figure 5A shows the illumination light sheet 502a and the cone 504a of collected light. The illumination light sheet 502a may have 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 Figures 1 to 4. Figure 5B shows the illumination light sheet 502b and the cone 504b of collected light. The illumination light sheet 502b may have 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 lens may have a higher NA than the focusing objective lens used in ODO mode, so the collected light 504a may form a wider cone than the collected light 504b. The illumination light sheet 502a may have a higher NA and a smaller width W compared to the illumination light sheet 502b. Therefore, the FOV in optical mode 500a is smaller than the FOV in optical mode 500b. This can be due to the adjustment of the illumination optics. For example, the W and NA of the illumination light sheet can also be changed between modes using an adjustable beam expander.

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

[0103] Figures 6A and 6B show additional imaging modes that can be used to utilize three objective lenses (e.g., an illumination objective lens, a NODO focusing objective lens, and an ODO focusing objective lens) that can orient and receive light from the sample. Instead of providing illumination to the NODO path through a single illumination objective lens, the system can also be configured in a mode in which illumination can be provided through the ODO focusing objective lens. Figure 6A shows a microscope operating in a first mode in which illumination light is provided by the first objective lens 602 and received by the focusing objective lens 604 at a non-orthogonal angle. Figure 6B shows the same microscope operating in a second mode in which illumination light is provided by the second objective lens 606 and received by the focusing objective lens 604 at a non-orthogonal angle. In some embodiments, images taken using both modes can be combined, for example, to improve imaging performance compared to a single image.

[0104] The microscopes in Figures 6A and 6B include 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 transmission optics (not shown) to reach an optical system that can redirect the collected image. Since there are two illumination paths, each having a different angle (e.g., +45° and -45°) relative to the focusing objective lens 604, the redirecting optics may need to be corrected for two different angles. The exemplary microscopes in Figures 6A and 6B include 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. Depending on the imaging mode, the fourth objective lens 608 can produce a remote image that can be imaged by either the fifth objective lens 610 or the sixth objective lens 612.

[0105] In ODO imaging mode (not shown in Figures 6A-6B), illumination can pass between objective lenses 602 and 606. For example, objective lens 602 can function as an illumination objective lens, providing an illumination light sheet to the sample, and objective lens 606 can image the sample at an angle nearly perpendicular to the illumination light sheet. The opposite configuration can also be used, where objective lens 606 provides the illumination light sheet and objective lens 602 collects at an angle nearly perpendicular to it.

[0106] In some embodiments, various optical components along the illumination path can be shared between the modes shown in Figures 6A and 6B. For example, there may 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 may be shared components in the focusing 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, the light sheet may have different angles with respect to the collected light, depending on whether objective lens 602 or 606 is used to provide the illumination light sheet. For example, if the angle between the axis of objective lens 602 and the axis of objective lens 604 is θ, then the angle between the axis of objective lens 606 and the axis of objective lens 604 may be -θ(90°-θ). Here, the negative sign indicates that the angle is in the opposite direction to angle θ (with respect to the axis of objective lens 604). In some embodiments, angle θ may be about 45°, and the two objective lenses 602 and 606 may have axes that are about 45° (but in opposite directions) from the axis of the condensing objective lens 604, respectively.

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

[0109] Using two different observation modes with different illumination angles can be useful in correcting distortion caused by the angle between illumination and collected light (in NODO mode). For example, the point-spread function (PSF) of an image can be deconvoluted 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 combine information from images acquired with different illumination modes by executing instructions stored in memory (e.g., 144 in Figure 1). For example, a fusion deconvolution algorithm can be used.

[0110] The microscopes in Figures 6A–6B are shown using a particular set of re-orienting optical systems, in particular the re-orienting optical system 230 in Figure 2 and / or the re-orienting optical system 340 in Figure 3, which are described in more detail. However, other exemplary embodiments using different field modes at different viewing angles may also use one or more re-orienting optical systems in addition to (or instead of) the re-orienting optical systems shown in Figures 6A–6B. For example, one of the re-orienting optical systems described in Figures 8A–8D may be used instead.

[0111] Figure 7 is a block diagram of a method for illuminating a sample using a microscope, according to several embodiments of the present disclosure. Method 700 can generally be carried out by one or more of the optical systems described in Figures 1 to 6.

[0112] Method 700 can generally begin with block 710, which describes oriented an illumination light sheet to the focal region of a sample through an illumination objective lens. For example, an illumination optical system (e.g., 120 in Figure 1) can generate an illumination light sheet and orient it to the rear end of the illumination objective lens. In some embodiments, the properties of the illumination light sheet (e.g., width, NA) can be adjusted based on the operating mode of the microscope. An illumination objective lens (e.g., 122 in Figure 1) can orient the illumination light sheet to the sample. In some embodiments, the illumination light sheet can pass through the material of the sample holder on its way to the sample. In some embodiments, the illumination light sheet can pass through the immersion fluid between the illumination objective lens and the sample. In some embodiments, the illumination objective lens may be an air objective lens, and the illumination light sheet can pass through a lens or window (e.g., SIMlens, SIL) between the illumination objective lens and the immersion fluid.

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

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

[0115] In some embodiments, the focusing optical system can direct the collected light towards a remote image, and an additional objective lens can image the remote image at an angle based on the angle between the illumination axis and the focusing axis.

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

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

[0118] Figures 8A to 8D are schematic diagrams of different redirecting optical systems according to several embodiments of the present disclosure. Figures 8A to 8C are schematic diagrams of redirecting optical systems that may be used to redirect non-orthogonal angles between the illumination axis and the focusing axis, and Figure 8D is a schematic diagram showing in more detail the exemplary operation of the redirecting optical system 800c in Figure 8C. Any of the redirecting optical systems in Figures 8A to 8D can be used in any of the microscopes described herein. For example, any of the redirecting optical systems 800a to 800c may be included in the redirecting optical system 230 in Figure 2 and / or the redirecting optical system 340 in Figure 3 in some embodiments. Since Figures 8A to 8D illustrate many features already described with respect to the previous figures, some components and features will not be described again for the sake of simplicity.

[0119] Figure 8A shows a redirection optical system 800a that uses a detector 810 tilted with respect to the optical path of the collected light. A condensing objective lens 802 (e.g., 204 in Figure 2) collects light at an angle non-orthogonal to the illuminated focal plane 801. Tube lenses 804 and 806 direct the light to an additional condensing objective lens 808, which generates an image of the light on the detector 810. Unlike the redirection optical systems shown in Figure 230 and Figure 340, the redirection optical system 800a projects the distant focus directly onto the detector 810 at a predetermined angle, rather than using additional optics to image the distant focus at a predetermined angle.

[0120] The re-orientation optical system 800a can offer certain advantages. For example, the remote focus should ideally be approximately equal to the refractive index of the sample, which is in the range of 1.33 to 1.56. Thus, the image plane at the remote focus is approximately the same size as the image plane in the sample. 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 to perform Nyquist sampling, the detector also needs to have very small pixels of the same size. One potential advantage of placing the tilted detector at this position, rather than the intermediate image plane, is that the image plane remains at 45° and does not tilt any further.

[0121] Figure 8B shows the re-orientation optical system 800b, where the second tube lens 806 and objective lens 808 are omitted. Instead, tube lens 804 directly images onto the detector 810, which is at a predetermined angle with respect 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 relative to this image plane can be in the range of approximately 10x. This change in magnification can be undesirable, as it further tilts the tilted image plane at the detector. However, this can be mitigated, particularly by using a detector with a small pixel size.

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

[0123] Naturally, it should be understood that any of the examples, embodiments, or processes described herein can be combined with one or more other examples, embodiments, and / or processes, or can be performed between separable and / or separate devices or device parts according to this system, device, and method.

[0124] Finally, the above description is intended to be illustrative of the system and should not be construed as limiting the attached claims to any particular embodiment or group of embodiments. Therefore, while the system has been described in particular detail with reference to exemplary embodiments, it should be understood that numerous modifications and alternative embodiments may be devised by those skilled in the art without departing from the broader intended purpose and scope of the system as described in the following claims. Accordingly, the specification and drawings should be considered illustrative and not intended to limit the scope of the attached claims.

Claims

1. An illumination objective lens configured to orient an illumination light sheet within the sample along the illumination axis, A focusing objective lens configured to receive light from the imaging plane of the sample along the focusing axis, Equipped with, The illumination axis and the focusing axis are non-orthogonal to each other. The image quality collected by the aforementioned focusing objective lens is diffraction-limited, with a Strehr ratio greater than approximately 0.

8. Device.

2. The apparatus according to claim 1, further comprising a second focusing objective lens configured to receive light from the imaging plane of the sample along a second focusing axis, wherein the second focusing axis is substantially perpendicular to the illumination axis.

3. The apparatus according to claim 2, further comprising an illumination optical system configured to generate the illumination light sheet, wherein the illumination optical system is adjustable between a setting based on the condensing objective lens and a setting based on the second condensing objective lens.

4. The apparatus according to claim 2, wherein the focusing objective lens has a first numerical aperture (NA), and the second focusing objective lens has a second NA that is lower than the first NA.

5. The apparatus is A third objective lens configured to receive light from the aforementioned condensing objective lens and generate a remote image, A fourth objective lens configured to form the remote image at an angle based on a non-orthogonal angle between the illumination axis and the focusing axis, The apparatus according to claim 1, further comprising:

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

7. The apparatus according to claim 6, further comprising a sample holder configured to support the sample, wherein at least a portion of the sample holder is in contact with the immersion fluid.

8. The apparatus according to claim 6, further comprising a lens positioned between the illumination objective lens and the immersion fluid.

9. The apparatus according to claim 8, wherein the lens is a solid immersion lens (SIL) or a solid immersion meniscus lens (SIMlens).

10. The apparatus according to claim 1, further comprising a sample holder having a first side surface configured to support the sample and a second side surface opposite to the first side surface, wherein the illumination objective lens and the focusing objective lens are positioned below the second side surface.

11. The apparatus according to claim 1, wherein the condensing objective lens has a depth of focus with respect to a given field of view, and the illumination axis is oriented such that the illumination axis does not remain within the depth of focus of the condensing objective lens.

12. A sample holder comprising a first surface and a second surface opposite to the first surface, wherein the first surface is configured to support a sample, An immersion chamber configured to hold the immersion fluid, An illumination objective lens configured to orient an illumination light sheet toward the sample at a non-orthogonal angle to the first surface of the sample holder, wherein the immersion chamber is positioned between the illumination objective lens and the second surface of the sample holder such that the illumination objective lens orients the illumination light sheet toward the focal region of the sample through the ambient medium, through the immersion fluid, and through the material of the sample holder, A focusing objective lens configured to collect light along a focusing axis substantially perpendicular to the first surface, wherein the light has passed through the material of the sample holder and the immersion fluid, A device equipped with the following features.

13. The apparatus according to claim 12, wherein the illumination objective lens and the focusing objective lens are positioned below the second surface.

14. The apparatus according to claim 12, further comprising a second focusing objective lens configured to collect light along a second focusing axis substantially perpendicular to the illumination light sheet.

15. The apparatus according to claim 12, wherein the focusing axis forms an acute angle with respect to the illumination light sheet.

16. The apparatus according to claim 15, wherein the acute angle is approximately 40° to 70°.

17. The apparatus according to claim 12, further comprising a solid immersion lens (SIL), wherein the illumination objective lens is configured to orient the illumination light sheet into the immersion fluid through the SIL.

18. The apparatus according to claim 12, further comprising immersion meniscus lenses (SIMlens), wherein the illumination objective lens is configured to orient the illumination light sheet into the immersion fluid through the SIMlens.

19. The apparatus according to claim 12, wherein at least a portion of the focusing objective lens is positioned in the immersion fluid, and the illumination objective lens does not come into contact with the immersion fluid.

20. The steps of aligning an illumination light sheet to the focal region of a sample through an illumination objective lens, A step of collecting light from the focal region through a condensing objective lens, wherein the optical axis of the condensing objective lens is non-orthogonal to the optical axis of the illumination objective lens, The steps include: and, Includes, The image quality collected by the aforementioned focusing objective lens is diffraction-limited, with a Strehr ratio greater than approximately 0.

8. method.

21. The method described above is: A step of collecting light from the focal region through a second focusing objective lens, wherein the optical axis of the second focusing objective lens is substantially perpendicular to the optical axis of the illumination objective lens, The steps include: forming an image of the light collected from the second condensing objective lens, The method according to claim 20, further comprising:

22. The method according to claim 21, wherein the step of collecting light through the focusing objective lens is part of a first operating mode, and the step of collecting light through the second focusing objective lens is part of a second operating mode, and the method further includes the step of adjusting one or more characteristics of the illumination light sheet between the first operating mode and the second operating mode.

23. The method described above is A step of generating a remote image based on collected light, The steps include forming the remote image at an angle based on the non-orthogonal angle between the optical axis of the focusing objective lens and the optical axis of the illumination objective lens, The method according to claim 20, further comprising:

24. The method described above is: The steps include: passing the illumination light sheet from the illumination objective lens through the surrounding medium, through the immersion fluid, and through the material of the sample holder to the focal region of the sample; The steps include collecting light into the focusing objective lens through the material of the sample holder and through the immersion fluid, The method according to claim 20, further comprising:

25. A step of aligning an illumination light sheet to the focal region of a sample through an illumination objective lens, the step of aligning the illumination light sheet comprising passing the illumination light sheet from the illumination objective lens through a surrounding medium, through an immersion fluid, and through the material of the sample holder to the focal region of the sample, A step of collecting light from the focal region through a focusing objective lens, the step of collecting light from the focal region comprising collecting light to the focusing objective lens through the material of the sample holder and through the immersion fluid, wherein the optical axis of the focusing objective lens is non-orthogonal to the optical axis of the illumination objective lens, The steps include: and, Methods that include...

26. The method described above is: A step of collecting light from the focal region through a second focusing objective lens, wherein the optical axis of the second focusing objective lens is substantially perpendicular to the optical axis of the illumination objective lens, The steps include: forming an image of the light collected from the second condensing objective lens, The method according to claim 25, further comprising:

27. ​​The method according to claim 26, wherein the step of collecting light through the focusing objective lens is part of a first operating mode, and the step of collecting light through the second focusing objective lens is part of a second operating mode, and the method further includes the step of adjusting one or more characteristics of the illumination light sheet between the first operating mode and the second operating mode.

28. The method described above is: A step of generating a remote image based on collected light, The steps include forming the remote image at an angle based on the non-orthogonal angle between the optical axis of the focusing objective lens and the optical axis of the illumination objective lens, The method according to claim 25, further comprising:

29. An illumination objective lens configured to orient an illumination light sheet within a sample along the illumination axis, A focusing objective lens configured to receive light from the imaging plane of the sample along the focusing axis, wherein the illumination axis and the focusing axis are non-orthogonal to each other, A third objective lens configured to receive light from the aforementioned condensing objective lens and generate a remote image, A fourth objective lens configured to form the remote image at an angle based on a non-orthogonal angle between the illumination axis and the focusing axis, A device equipped with the following features.

30. The apparatus according to claim 29, further comprising an immersion fluid, wherein the illumination objective lens is not in contact with the immersion fluid, and at least a portion of the focusing objective lens is in contact with the immersion fluid.

31. The apparatus according to claim 30, further comprising a sample holder configured to support the sample, wherein at least a portion of the sample holder is in contact with the immersion fluid.

32. The apparatus according to claim 30, further comprising a lens positioned between the illumination objective lens and the immersion fluid.

33. An illumination objective lens configured to orient an illumination light sheet within a sample along the illumination axis, A condensing objective lens configured to receive light from the imaging plane of the sample along the condensing axis, wherein the illumination axis and the condensing axis are non-orthogonal to each other, the condensing objective lens has a depth of focus for a given field of view, and the illumination axis is oriented such that the illumination axis does not remain within the depth of focus of the condensing objective lens. A device equipped with the following features.

34. The apparatus according to claim 33, further comprising a second focusing objective lens configured to receive light from the imaging plane of the sample along a second focusing axis, wherein the second focusing axis is substantially perpendicular to the illumination axis.

35. The apparatus according to claim 34, further comprising an illumination optical system configured to generate the illumination light sheet, wherein the illumination optical system is adjustable between a setting based on the condensing objective lens and a setting based on the second condensing objective lens.

36. The apparatus according to claim 34, wherein the focusing objective lens has a first numerical aperture (NA), and the second focusing objective lens has a second NA that is lower than the first NA.

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