Reducing spherical aberration in a microscope using a lens having a convex rear surface and flattening light sheets in the context of light sheet microscopes

Abstract

Spherical aberration in microscopes can be eliminated or reduced by using an air objective to image samples that are sitting in immersion oil within a container. The front surface of a lens sits in the immersion oil, and a convex rear surface of the lens sits outside the container. The air objective is positioned and aligned so that light exiting the convex rear surface of the lens enters the front element of the air objective. The lowest spherical aberration occurs when the indices of refraction of the lens and the immersion oil match.

Description

Atty. Docket No. 1448-0265wo01REDUCING SPHERICAL ABERRATION IN A MICROSCOPE USING A LENS HAVING A CONVEX REAR SURFACE AND FLATTENING LIGHT SHEETS IN THE CONTEXT OF LIGHT SHEET MICROSCOPESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of US Provisional Applications 63 / 750,076 (filed January 27, 2025), 63 / 822,672 (filed June 12, 2025), and 63 / 906,947 (filed October 28, 2025), each of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under MH 119423 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Microscopes universally rely on a detection objective lens to capture sample images. These detection objectives can be broadly classified into two categories: immersion objectives and air objectives.

[0004] Immersion objectives are designed for use with specific immersion media, such as oil (of a defined refractive index) or water, often equipped with a correction collar to accommodate a range of media. These objectives typically offer higher numerical aperture (NA), resulting in superior imaging resolution and minimized optical aberrations. However, they are generally very expensive (ranging from $10,000 to $40,000) and have a very limited working distance, which restricts the achievable imaging depth.

[0005] Air objectives are optimized for operation in air, and are significantly more affordable and feature ultra-long working distances, enabling deeper imaging of large samples. However, when imaging large specimens that are typically immersed in a medium, air objectives suffer from severe spherical aberrations. This results in substantial degradation of imaging resolution, both laterally (by a factor of two or more) and axially (by orders of magnitude). Consequently, using air objectives in advanced microscopy techniques (such as lightsheet microscopy) has heretofore compromised image quality.

[0006] Furthermore, certain microscopes operate by projecting a light sheet through a sample (e.g., in a cleared sample or in vivo), and detecting fluorescent light that emanates from the illuminated portion of the sample. By sweeping the light sheet through the sampleand combining the images obtained at different relative positions, it becomes possible to generate a 3D image of the sample. And the resolution of the resulting image can be improved by reducing the thickness of the light sheet.SUMMARY OF THE INVENTION

[0007] One aspect of this application is directed to a first microscope that comprises a container that is filled with a volume of immersion oil, a lens, and an air objective. The lens has a front surface and a convex rear surface with a radius of curvature R, and the lens is mounted so that (i) the front surface of the lens is positioned within the container, sitting in the immersion oil and (ii) the rear surface of the lens is positioned outside the container. The air objective has a front element and a working distance WD. The front element of the air objective is positioned behind the rear surface of the lens, and the air objective is aligned so that light exiting the rear surface of the lens can enter the front element of the air objective. The immersion oil and the lens have indices of refraction that match within ± 20%.

[0008] In some embodiments of the first microscope, the immersion oil and the lens have indices of refraction that match within ± 10%. In some embodiments of the first microscope, the immersion oil and the lens have indices of refraction that match within ± 5%. In some embodiments of the first microscope, the immersion oil and the lens have indices of refraction that match within ± 2%.

[0009] In some embodiments of the first microscope, the lens is a plano-convex lens that has a flat front surface and a spherical rear surface.

[0010] In some embodiments of the first microscope, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 20%. Optionally, these embodiments can further comprise a light sheet source configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.

[0011] In some embodiments of the first microscope, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%. Optionally, these embodiments can further comprise a light sheet source configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.24898-7566-6314, v. 9

[0012] Some embodiments of the first microscope further comprise a light sheet source configured and positioned to project a light sheet into the container so that the light sheet is positioned Rx(l+l / n) in front of a rearmost point on the convex rear surface of the lens, where n is a refractive index of the immersion oil.

[0013] Some embodiments of the first microscope further comprise a tube lens and a camera. In these embodiments, the tube lens is configured and positioned to route light that emanates from the back aperture of the air objective into the camera.

[0014] Some embodiments of the first microscope further comprise a light sheet source that is configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.

[0015] Another aspect of this application is directed to a second microscope that comprises a container, a lens, and an air objective. The container is configured to hold a volume of immersion oil within the container. The lens has front surface and a convex rear surface with a radius of curvature R, and the lens is mounted so that (i) the front surface of the lens is positioned within the container so that when the container is filled with the immersion oil, the front surface of the lens will sit in the immersion oil and (ii) the rear surface of the lens is positioned outside the container. The air objective has a front element and a working distance WD. The front element of the air objective is positioned behind the rear surface of the lens, with the air objective aligned so that light exiting the rear surface of the lens can enter the front element of the air objective.

[0016] In some embodiments of the second microscope, the lens is a plano-convex lens that has a flat front surface and a spherical rear surface.

[0017] In some embodiments of the second microscope, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 20%. In some embodiments of the second microscope, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

[0018] Some embodiments of the second microscope further comprise the volume of immersion oil. Preferably, in these embodiments, the immersion oil and the lens have indices of refraction that match within ± 20%.34898-7566-6314, v. 9

[0019] Some embodiments of the second microscope further comprise a tube lens and a camera. In these embodiments, the tube lens is configured and positioned to route light that emanates from the back aperture of the air objective into the camera.

[0020] Some embodiments of the second microscope further comprise a light sheet source that is configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.

[0021] Another aspect of this application is directed to a first apparatus for flattening a projected light sheet. The first apparatus comprises a container, a lens, and an air objective. The container is filled with a volume of immersion oil. The lens has a front surface and a convex rear surface with a radius of curvature R, and the lens is mounted so that (i) the front surface of the lens is positioned within the container, sitting in the immersion oil and (ii) the rear surface of the lens is positioned outside the container. The air objective has a front element and a working distance WD. The rear surface of the lens is positioned in front of the front element of the air objective, with the lens aligned so that when the light sheet is being projected out of the air objective via the front element, the light sheet enters the rear surface of the lens. The immersion oil and the lens have indices of refraction that match within ± 20%.

[0022] In some embodiments of the first apparatus, the immersion oil and the lens have indices of refraction that match within ± 10%. In some embodiments of the first apparatus, the immersion oil and the lens have indices of refraction that match within ± 5%. In some embodiments of the first apparatus, the immersion oil and the lens have indices of refraction that match within ± 2%.

[0023] In some embodiments of the first apparatus, the lens is a plano-convex lens that has a flat front surface and a spherical rear surface. In some embodiments of the first apparatus, the rear surface of the lens is spaced apart from the front element of the air objective by a distance D, wherein D equals WD minus R, ± 20%. In some embodiments of the first apparatus, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

[0024] Some embodiments of the first apparatus further comprise a detection objective, a tube lens and a camera that are respectively positioned and focused to capture images of portions of a sample that are being illuminated by the light sheet while the sample is positioned within the container.44898-7566-6314, v. 9

[0025] Another aspect of this application is directed to a second apparatus for flattening a projected light sheet. The second apparatus comprises a container, a lens, and an air objective. The container is configured to hold a volume of immersion oil within the container. The lens has a front surface and a convex rear surface with a radius of curvature R, and the lens is mounted so that (i) the front surface of the lens is positioned within the container so that when the container is filled with the immersion oil, the front surface of the lens will sit in the immersion oil and (ii) the rear surface of the lens is positioned outside the container. The air objective has a front element and a working distance WD. The rear surface of the lens is positioned in front of the front element of the air objective, with the lens aligned so that when the light sheet is being projected out of the air objective via the front element, the light sheet enters the rear surface of the lens.

[0026] In some embodiments of the second apparatus, the lens is a plano-convex lens that has a flat front surface and a spherical rear surface. In some embodiments of the second apparatus, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 20%. In some embodiments of the second apparatus, the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

[0027] Some embodiments of the second apparatus further comprise the volume of immersion oil. Optionally, in these embodiments, the immersion oil and the lens have indices of refraction that match within ± 20%.

[0028] Some embodiments of the second apparatus further comprise a detection objective, a tube lens and a camera that are respectively positioned and focused to capture images of portions of a sample that are being illuminated by the light sheet while the sample is positioned within the container.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 depicts an embodiment in which light rays from the focal plane are incident orthogonally to the rear curved surface of a lens, thus minimizing refraction which is the main cause of spherical aberration.

[0030] FIG. 2 is a ray tracing that shows how the convex rear surface of the lens in FIG. 1 is used to minimize spherical aberrations.

[0031] FIG. 3 is a ray tracing that shows how systems without the lens that is present in FIG. 1 has a surface that induces refractions that result in spherical aberration.54898-7566-6314, v. 9

[0032] FIG. 4 depicts empirical measurements of a point spread function after spherical aberration correction for an air objective with NA 0.28.

[0033] FIG. 5 is a schematic of a projected light sheet microscopy (pLSM) setup.

[0034] FIG. 6 is a comparative summary of key performance characteristics of immersion, air, and SCOPE-enabled air objectives.

[0035] FIG. 7 depicts classical hemispherical and Weierstrass solid immersion lenses positioned in front of respective objective lenses.

[0036] FIG. 8 is a schematic of the HySIL concept and its implementation as the modular SCOPE and Super-SCOPE imaging devices.

[0037] FIG. 9 depicts Zemax optical simulations comparing detection performance for air-objective imaging in air, immersion media, SCOPE, and Super-SCOPE configurations.

[0038] FIG. 10 depicts quantifications of normalized irradiance and diffraction-encircled energy for the Huygens PSFs shown in FIG. 9.

[0039] FIG. 11 depicts a tolerance analysis of air-objective detection in immersion, SCOPE, and Super-SCOPE configurations.

[0040] FIG. 12 depicts a modular integration of the SCOPE imaging device with the compact, low-cost pLSM platform.

[0041] FIG. 13 depicts quantifications of lateral and axial full width at half maximum (FWHM) for two objectives across the field of view.

[0042] FIG. 14 depicts Zemax optical simulations comparing detection using an air objective in air, in immersion media (standard configuration), and with the SCOPE imaging device.

[0043] FIG. 15 depicts a wavefront error analysis for the SCOPE and air immersion cases.

[0044] FIG. 16 depicts a system that relies on a reduction in spherical aberration to reduce the thickness of the light sheet in a light sheet microscope.64898-7566-6314, v. 9

[0045] FIG. 17 depicts a system that combines a reduced-thickness light sheet in the illumination arm with a reduction in spherical aberration in the detection arm.

[0046] Various embodiments are described in detail below with reference to the accompanying drawings, wherein like reference numerals represent like elements.DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Part 1 - Reducing Spherical Aberration in a Microscope Using a Lens Having a Convex Rear Surface

[0048] The embodiments described herein employ off-the-shelf components to facilitate simple and cost-effective solutions to correct spherical aberrations in air objectives. These embodiments enhance imaging resolution, surpassing the native resolution of their air objectives by increasing their effective NA. This enables high-quality imaging of both large, cleared samples and smaller, living specimens.

[0049] FIG. 1 depicts a microscope that eliminates or at least minimizes spherical aberration. The microscope uses an air objective 40 to image a sample that is immersed in a volume of immersion oil 25. A container 20 is filled with a volume of immersion oil 25. A lens 30 has a front surface and a convex rear surface with a radius of curvature R, and the lens 30 is mounted so that (i) the front surface of the lens is positioned within the container 20, sitting in the immersion oil 25 and (ii) the rear surface of the lens is positioned outside the container 20. The immersion oil 25 and the lens 30 have indices of refraction that match within ± 20% (or, in some embodiments, within ± 15%, ± 10%, ± 5%, ± 2%, ± 1%, or ± 0.5%).

[0050] In the embodiment depicted in FIG. 1, the lens 30 is a plano-convex lens that has a flat front surface and a spherical rear surface. Note, however, that only the shape of the rear surface of the lens 30 is important, for the reasons described below. On the other hand, the shape of the front surface is not critical, and other types of lenses that do not have a flat front surface (e.g., a double convex lens) can be substituted for the plano-convex lens depicted in FIG. 1.

[0051] An air objective 40 has a front element 40F, and this front element 40F is positioned behind the rear surface of the lens 30, with the air objective 40 aligned so that light exiting the rear surface of the lens 30 can enter the front element 40F of the air objective 40.74898-7566-6314, v. 9

[0052] The air objective 40 has a working distance WD, and the front element of the air objective is spaced apart from the rear surface of the lens by a distance D that nominally equals WD minus R. In some embodiment, this distance D equals WD minus R, ± 20%. But in other embodiments, this tolerance can vary from ± 20%, and could be, for example, ± 40%, ± 30%, ± 25%, ± 15%, ± 10%, ± 5%, ± 2%, or ± 1%. In practice, the position of the air objective 40 can be moved forward or backwards (e.g., by turning a knob that operates a mechanical linkage) to change the distance D until an operator sees that the resulting image is in focus.

[0053] The air objective 40 has a back aperture 40B, and a tube lens 50 is positioned to the rear of the back aperture 40B. The tube lens is configured and positioned to route light that emanates from the back aperture 40B of the air objective into a camera 60 (which can be, e.g., a CMOS or CCD-based camera).

[0054] A light sheet source (not shown) is configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens. The light sheet source could be, for example, a laser whose beam is flattened into a sheet by a cylindrical lens. Alternatively, the light sheet source could be a laser whose output beam is scanned into a virtual sheet by an oscillating galvanometer mirror.

[0055] Light from the light sheet source causes a region within the sample to emit fluorescent light. This fluorescent light travels rearwards through the immersion oil 25 until it reaches the lens 30. As explained above, the immersion oil 25 within the container 20 and the lens 30 have indices of refraction that match within the tolerances noted above. This ensures minimal refraction at the boundary between the immersion oil 25 and the front surface of the lens 30 when the light enters the lens 30.

[0056] The light then continues in a rearward direction through the lens 30, and exits the convex rear surface of the lens. The rear surface of the lens 30 has a radius of curvature R. The light then continues across an air gap and enters the front element 40F of the air objective 40.

[0057] The lens 30 and the air objective 40 are positioned such that the focal plane of the air objective 40 will be a distance R in front of the curved rear surface of the lens 30. This is accomplished by setting the size of the air gap to a distance D that nominally equals WD minus R (with any of the tolerances noted above). Due to this arrangement, the light rays84898-7566-6314, v. 9emerging from light emitting points within the sample will arrive perpendicularly to the curved rear surface of the lens 30.

[0058] FIG. 2 is a ray tracing that shows how the convex rear surface of the lens in FIG. 1 is used to minimize spherical aberrations. The perpendicular orientation of the light that arrives at the curved rear surface of the lens 30 will advantageously eliminate spherical aberrations (or at least minimize spherical aberrations when the indices of refraction of the immersion oil 25 and the lens 30 do not match exactly and / or when the focal plane is not precisely positioned at a distance R from the rear surface of the lens 30). Thus, the rear surface of the lens 30 acts as an imaging window for correcting spherical aberrations.

[0059] FIG. 3 is a ray tracing that shows how systems without the lens 30 that is present in FIG. 1 will result in spherical aberration. More specifically, because the rearwardtraveling light in the FIG. 1-2 is perpendicular to the rear surface of the lens 30 when it exits the lens 30 and enters the air gap, it will NOT be refracted when it enters the air gap. In contrast, in the FIG. 3 counterexample, the light WILL be refracted when it exits the immersion medium 25 and enters the air gap that is positioned in front of the objective lens. And this refraction manifests itself as spherical aberration. Heatmaps 200 and 300 show the results of a Zeemax simulation of spherical aberration in the two contrasting systems of FIGS. 2 and 3, respectively.

[0060] Notably, the effective NA of the detection objective in the embodiment depicted in FIG. 1 is increased by a factor corresponding to the RI of the immersion oil, which results in a very significant enhancement in resolution. This advantageously enables an inexpensive air objective to achieve performance similar to a much more expensive oil immersion objective.

[0061] Three examples of suitable combinations of materials for the lens 30 and corresponding immersion oils include: (1) using a plano-convex lens made of N-BK7 glass (RI of 1.515), and filling the container 20 with immersion oil 25 having matching RI of 1.515; (2) using a plano-convex lens made of fused silica glass (RI of 1.46) and filling the container 20 with immersion oil 25 having matching RI of 1.46; and (3) using a plano-convex lens made of Magnesium Fluoride (RI 1.38) and filling the container 20 with immersion oil 25 (or a water-based liquid) having matching RI. Note that in each of these examples, the RI of the immersion oil can deviate to some extent from the RI of the lens 30. For example, a94898-7566-6314, v. 9sample immersed in iDISCO tissue clearing media having an RI of 1.52 can still be nicely imaged using the lenses described in examples (1) and (2) above.

[0062] FIG. 4 depicts empirical measurements of point spread function (PSF) after spherical aberration correction for an air objective with NA 0.28. The axial full width at half maximum (FWHM) is measured to be about 9.5 microns and lateral FWHM about 1 micron, showing complete correction with further enhanced resolution. The scale bar on the lower right corner of the figure is 50 microns.

[0063] It is important to note that the vertical orientation depicted in FIG. 1 is not the only orientation that can be used. To the contrary, the entire system depicted in FIG. 1 could be rotated clockwise with respect to the page e.g., by 90° or 270° (in which case the light sheet would be vertical and the optical axis of the objective would be horizontal), and the system will operate as described above. The entire system could also be rotated by other angles.

[0064] Note also that the explanation of the system described above in connection with FIG. 1 assumes that the container 20 has been filled with the immersion oil 25 at some prior point in time. But of course the system can be provided to an end-user with an empty container 20, as long as the end-user realizes that the container 20 must be filled with the correct type of immersion oil prior to use. In this situation, the end-user need only be provided with the following three items to make the microscope: (1) a container 20 configured to hold a volume of immersion oil within the container; (2) a lens 30 having front surface and a convex rear surface with a radius of curvature R, wherein the lens is mounted so that (i) the front surface of the lens is positioned within the container so that when the container is filled with the immersion oil, the front surface of the lens will sit in the immersion oil and (ii) the rear surface of the lens is positioned outside the container; and (3) an air objective 40 having a front element 40F and a working distance WD. The front element 40F of the air objective 40 is positioned behind the rear surface of the lens 30, with the air objective 40 aligned so that light exiting the rear surface of the lens 30 can enter the front element 40F of the air objective 40. After the end user fills the container 20 with the correct type of immersion oil 25, the microscope can be used as described above in connection with FIG. 1.104898-7566-6314, v. 9

[0065] A variation of the FIG. 1 embodiment is depicted on the right side of FIG. 8. In that embodiment, the air objective 40 has a working distance WD, R is the radius of curvature of the convex rear surface of the lens 30, and n is the refractive index (RI) of the immersion oil 25. But the distances are adjusted with respect to the FIG. 1 embodiment so that the light sheet source (not shown) is configured and positioned to project a light sheet into the container so that the light sheet is positioned Rx(l+l / n) in front of the rearmost point on the convex rear surface of the lens. In practice, the position of the air objective 40 can be moved forward or backwards (e.g., by turning a knob that operates a mechanical linkage) to change the distance between the front element of the air objective and the rear convex surface of the lens until an operator sees that the resulting image is in focus.

[0066] The inventor has successfully used the microscopes depicted in FIGS. 1 and 8 to correct spherical aberrations in the context of light-sheet microscopy. And the remainder of this Part 1 describes some additional examples, referred to herein as SCOPE (Solid-Liquid hybrid immersion-Corrected Optics for Phase Errors) that implement the concepts described above.

[0067] The Hybrid Solid-Liquid Immersion Lens (HySIL) framework described in this Part 1 pairs an off-the-shelf solid optical component with a refractive index-matched liquid to precompensate aberrations and enhance resolution. HySIL combined with SCOPE achieved submicron lateral resolution (<0.75 pm) across centimeter-scale samples using inexpensive air objectives with >30 mm working distances. Integration with a low-cost LSM platform yielded a compact, scalable system demonstrated through multi-immersion, multicolor, subcellular-resolution mapping of cleared or expanded mouse, salamander, and cavefish brains, human iPSC-derived organoids, and 3D histopathology of breast tissue. HySIL and SCOPE establish an accessible foundation for scalable, high-resolution volumetric imaging, advancing data-driven biological discovery.

[0068] Breakthroughs such as AlphaEold and ESM3 for protein structure prediction, advances in computational histopathology, and generative modeling of virtual cells exemplify the transformative power of data-driven approaches in life sciences. Extending this paradigm from molecular to multicellular systems require imaging technologies capable of routinely generating subcellular-resolution volumetric datasets from intact tissues and organs. Recent developments in tissue clearing and expansion microscopy (ExM) have provided unprecedented optical access to intact specimens. In parallel, light-sheet microscopy (LSM)114898-7566-6314, v. 9has emerged as the leading modality for high-speed volumetric imaging, offering minimal phototoxicity and exceptional optical efficiency. Open-source initiatives such as openSPIM and mesoSPIM have broadened access to LSM, while the inventor’s open-source projected light-sheet microscopy (pLSM) platform has further reduced costs by orders of magnitude through repurposing consumer-grade components.

[0069] However, achieving scalability in imaging volume, sample throughput, and widespread adoption while maintaining subcellular resolution remains constrained by fundamental limitations in detection optics. Immersion objectives enable aberration-free, high numerical aperture (NA) detection necessary for achieving submicron resolution but also impose severe constraints: short working distances limiting imaging depth, high costs, narrow refractive index (RI) correction ranges limiting multi-immersion compatibility, and operational complexity requiring sealed chambers. On the other hand, air objectives offer long working distances, cost reductions of several orders of magnitude, and mechanical simplicity for integration into low-cost, portable platforms. However, they suffer from severe spherical aberrations, reduced photon collection, and degraded resolution when imaging immersed samples, limiting performance to cellular resolution. Existing correction approaches including adaptive optics, custom meniscus lenses, and specialized corrected air objectives, mitigate some of these effects but remain system-specific, require custom engineering, or are expensive. Consequently, existing LSM systems often sacrifice imaging performance for portability, performing adequately for cellular-resolution applications but leaving efficient subcellular-resolution imaging of large volumes inaccessible. This limitation has become particularly pressing with the advent of ExM, which can increase specimen dimensions by an order of magnitude, creating urgent demand for aberration-free imaging over extended working distances.

[0070] To address this challenge, we developed the Hybrid Solid-Liquid Immersion Lens (HySIL) framework. HySIL goes beyond the solid immersion lens (SIL) concept, which enhances resolution by positioning a high-RI hemispherical solid element in contact with specimens, increasing effective detection NA by n (the refractive index of the lens). While SILs have proven valuable in data storage, semiconductor photoluminescence, and superresolution microscopy applications, their use has been limited to surface imaging. The HySIL approach, instead, combines a simple, off-the-shelf solid lens element with a precisely RL matched liquid to form a continuous solid-liquid hybrid optical system. This architecture124898-7566-6314, v. 9establishes two complementary optical environments: a solid interface enabling use of air detection optics, and a liquid interface allowing flexible sample positioning and imaging.

[0071] SCOPE and Super-SCOPE (i.e., a variant of SCOPE that utilizes a Weierstrass super-hemispherical geometry) are objective-agnostic modular imaging devices that (a) increase effective detection NA (scaled by n and n2for SCOPE and Super-SCOPE, respectively), (b) correct spherical aberrations, (c) provide tolerance to diverse immersion media, and (d) preserve the long working distance, chromatic correction, low cost, and simplicity of air objectives.

[0072] We validated SCOPE through optical simulations and point spread function (PSF) measurements, demonstrating aberration-free, submicron resolution (<0.75 pm) imaging using long working distance (~34 mm), inexpensive air objectives with high performance across diverse tissue clearing media. We then integrated SCOPE with our low-cost pLSM platform to create pLSM-SCOPE, a compact, cost-effective, and scalable light sheet microscopy system for submicron resolution imaging of centimeter- scale samples. The pLSM platform is described in WO2025 / 199301, which is incorporated herein by reference in its entirety. pLSM-SCOPE enabled imaging of diverse specimens, including expanded mouse and salamander brains, CUBIC-R cleared intact mouse brains, iDISCO cleared cavefish brains, brain organoids with microglia, and 3D histopathology of human breast tissue. In all cases, pLSM-SCOPE produced high-contrast, color-corrected, aberration-free imaging throughout entire sample volumes. Together, SCOPE and pLSM provide a simple, low-cost, off-the-shelf, and scalable solution that transforms air objectives into high-resolution, aberration-free detectors, bridging affordability and performance for nextgeneration, data-driven biological discovery.

[0073] RESULTS

[0074] Design and Theoretical Validation of SCOPE and Super-SCOPE

[0075] FIGS. 5-12 depict the design and validation of the HySIL and its imaging device implementations, SCOPE and Super-SCOPE. FIG. 5 is a schematic of the projected light sheet microscopy (pLSM) setup illustrating the use of air versus immersion detection objectives. The plot on the right half of FIG. 5 compares numerical aperture (NA) and working distance (WD) for air and immersion objectives, highlighting the trade-off between resolution and scalability. FIG. 6 is a comparative summary of key performance134898-7566-6314, v. 9characteristics of immersion, air, and SCOPE-enabled air objectives. FIG. 7 is a conceptual illustration of classical hemispherical and Weierstrass solid immersion lenses (SILs), showing NA scaling, chromatic properties, and limitations to surface-proximal imaging.

[0076] FIG. 8 is a schematic of the HySIF concept and its implementation as the modular SCOPE (left half of FIG. 8) and Super-SCOPE (right half of FIG. 8) imaging devices. A truncated hemispherical quartz lens is paired with refractive-index (Rl)-matched immersion oil (RI ~ 1.46 in this study) to form a hybrid solid-liquid optical system that corrects aberrations while maintaining the long WD, ease of integration, and low cost of air objectives. FIG. 9 depicts Zemax optical simulations comparing detection performance for air-objective imaging in air, immersion media, SCOPE, and Super-SCOPE configurations. Corresponding estimated Huygens point-spread functions (PSFs) are shown on the right. FIG.10 depicts quantifications of normalized irradiance and diffraction-encircled energy for the Huygens PSFs shown in FIG. 9.

[0077] FIG. 11 depicts a tolerance analysis of air-objective detection in immersion, SCOPE, and Super-SCOPE configurations. Root-mean- square (RMS) wavefront error is plotted as a function of sample RI and thickness. FIG. 12 depicts a modular integration of the SCOPE imaging device with the compact, low-cost pESM platform.

[0078] Achieving high-resolution, large-volume imaging with inexpensive, long-working-distance air objectives requires overcoming the spherical aberrations, resolution loss, and signal-collection inefficiency that arise when imaging through high-RI media typical of cleared tissues (FIGS. 5-6). To address these challenges, we developed the HySIF framework (FIGS. 7-8), which consists of a solid truncated spherical lens coupled with a precisely Rl-matched immersion oil to form a continuous hemispherical optical interface. Similar to a classical hemispherical SIE (FIG. 7), HySIF precompensates phase errors introduced by high-RI specimens and increases effective detection NA. However, unlike conventional SIEs, which are inherently limited to surface-proximal imaging (FIG. 7), HySIF (FIG. 8) decouples the solid lens from both the sample and the objective, enabling aberration-free imaging throughout large, cleared specimens.

[0079] The SCOPE and Super-SCOPE variants depicted in FIG. 8 can be seamlessly integrated into most existing ESM implementations. SCOPE incorporates an off-the-shelf plano-convex quartz lens (RI ~ 1.46) paired with a precisely Rl-matched immersion oil (RI ~144898-7566-6314, v. 91.46) to form a hybrid solid-liquid immersion system with hemispherical geometry that increases effective NA by n. Super-SCOPE utilizes Weierstrass super-hemispherical geometry to achieve NA scaling by n2, providing enhanced resolution, though with limited chromatic correction. The RI of ~ 1.46 was specifically selected to ensure broad compatibility with commonly used tissue clearing methods, from aqueous (RI ~ 1.33) to solvent-based (RI ~ 1.56) protocols, without requiring modifications.

[0080] In the original SCOPE variant (on the left side of FIG. 8), the chamber geometry is calibrated such that the distance between (i) the light- sheet illumination plane, entering through the center of the illumination window (either flat or HySIL-enabled for thinner light sheets) and (ii) the curved surface of the solid lens equals the lens radius of curvature. This is one way to ensure optimal wavefront correction and alignment of the illumination and detection planes.

[0081] In the Super-SCOPE variant (on the right side of FIG. 8), the chamber geometry is calibrated such that the distance between (i) the light- sheet illumination plane, entering through the center of the illumination window (either flat or HySIL-enabled for thinner light sheets) and (ii) the rearmost point on the curved surface of the solid lens 30 equals Rx(l+l / n) (where R is the radius of curvature of the convex rear surface of the lens 30 and n is the RI of the immersion oil 25). This is another way to ensure optimal wavefront correction and alignment of the illumination and detection planes.

[0082] We performed Zemax optical simulations to evaluate SCOPE performance for high-resolution imaging of large samples cleared with diverse tissue-clearing methods (FIGS.9-11). The detection system was modeled with an idealized optical path comprising a 40 mm focal length detection lens (NA_air = 0.28, matching the Mitutoyo objective used in this work) and a 200 mm tube lens (TTL200, Thorlabs). For direct comparison of performance across four configurations, including air, immersion, SCOPE, and Super SCOPE, a hemispherical curvature of 16 mm was selected. To emulate the configuration used experimentally and to utilize most of the detection working distance, a radius of curvature of 27.5 mm was also simulated (FIGS. 14-15), yielding comparable results.

[0083] As shown in FIGS. 9-11, the HySIL-based SCOPE and Super-SCOPE optical designs markedly reduced spherical aberrations, achieving diffraction-limited performance (FIGS. 9-10) and resolution enhancement approximately by factors of n and n2, respectively154898-7566-6314, v. 9(compare PSFs in FIGS. 9-10), as expected. FIG. 15 depicts a wavefront error analysis that further confirmed effective compensation of optical path differences across both sagittal and tangential planes, demonstrating efficient precompensation of phase errors introduced when imaging samples immersed in high-RI media.

[0084] We next assessed the tolerance of SCOPE (system RI - 1.46) across a range of sample RIs representative of common tissue clearing protocols (1.38 to 1.54) and sample thicknesses of 2 to 6 mm. As shown in FIG. 11, the root mean square (RMS) wavefront error remained consistently below or near the Marechai criterion (X / 14) under all tested conditions for SCOPE, confirming high correction efficiency and broad adaptability without hardware adjustments. As expected, Super-SCOPE exhibited more limited tolerance to RI variation but still outperformed conventional air objective imaging in immersion media.

[0085] Collectively, these optical simulations establish HySIL as a generalizable framework for aberration-free, high-resolution imaging of large, cleared samples using inexpensive, long-working-distance air objectives. The passive, easy-to-integrate design of SCOPE and Super-SCOPE chambers eliminates the need for sealed immersion assemblies, allowing seamless integration into a wide range of imaging systems, including the low-cost pLSM platform depicted in FIG. 12.

[0086] Experimental Validation of SCOPE through PSF Characterization

[0087] Wide-field fluorescence imaging of 150 nm gold nanoparticles embedded in 0.5% agarose (FEO) was performed using a 10x / 0.28 NA / 34 mm WD air objective.Comparison between conventional flat-window imaging and SCOPE configurations showed correction of optical aberrations across a 1.2 mm field of view (FOV) and substantially higher detected signal intensity under identical illumination power. It also showed the improved PSF achieved with SCOPE.

[0088] An equivalent comparison using a 5x / 0.14 NA / 34 mm WD air objective across a 2.7 mm FOV demonstrated aberration correction and enhanced signal detection with SCOPE. FIG. 13 depicts the Quantification of lateral and axial full width at half maximum (FWHM) for both objectives across the FOV. SCOPE achieves lateral resolutions of -0.75 pm (lOx) and -1.2 pm (5x), and axial FWHM values of -11 pm and -18 pm, respectively. The horizontal dashed lines indicate theoretical diffraction-limited (Abbe) performance for these air objectives. Multicolor light-sheet imaging of polychromatic fluorescent beads164898-7566-6314, v. 9demonstrated preserved chromatic alignment across three channels (455 nm, 520 nm, and 640 nm).

[0089] To experimentally validate SCOPE performance, we measured and characterized the detection point spread function (PSF) using two inexpensive long-working-distance air objectives: Mitutoyo 10x / 0.28 NA / 34 mm WD and 5x / 0.14 NA / 34 mm WD. The SCOPE imaging device incorporated a plano-convex fused quartz lens (RI - 1.46; radius of curvature of 27.5mm) paired with an Rl-matched immersion oil to form the hybrid solidliquid immersion interface depicted in FIG. 8.

[0090] We first imaged sub-resolution (150 nm) gold nanoparticles embedded in 0.5% agarose (H2O) under wide-field illumination to assess the detection PSF of SCOPE. Identical illumination power was applied for both SCOPE and conventional air-immersion setups to enable direct comparison of resolution and signal collection efficiency. SCOPE effectively corrected spherical aberrations across multi-millimeter scale fields of view (1.2 mm for lOx and 2.7 mm for 5x objectives) and produced higher signal intensity relative to the Air- Immersion configuration.

[0091] Returning to FIG. 13, we quantified the detection PSF by measuring the lateral and axial full width at half maximum (FWHM), yielding lateral resolutions of -0.75 pm (lOx) and -1.2 pm (5x), and axial FWHM values of -11 pm (lOx) and -18 pm (5x). Similar to a classical SIE, the resolution achieved with SCOPE exceeded the theoretical diffraction limits predicted by Abbe's criterion for the design NA (in air) of these objectives across the entire field of view. Notably, these FWHM values approach those obtained with high-NA immersion objectives, typically orders of magnitude more expensive and limited by much shorter working distances, demonstrating that the hybrid solid-liquid immersion strategy effectively transforms inexpensive air objectives into high-resolution, aberration-free imaging systems.

[0092] Finally, we integrated the SCOPE imaging device into the pESM system shown in FIG. 12 and imaged fluorescent beads to evaluate performance under light- sheet illumination. The resulting data confirmed aberration-free, high-resolution imaging with preserved chromatic alignment across multiple excitation channels. Moreover, SCOPE maintained high performance for deep imaging, up to 1 cm depth and 2.6 mm field of view, demonstrating that its integration into light-sheet microscopy enables standard air objectives174898-7566-6314, v. 9to achieve enhanced resolution and achromatic imaging across large volumes while retaining long working distance.

[0093] Collectively, these experiments demonstrate that SCOPE enables aberration-free, submicron-resolution imaging across multi-millimeter fields of view and centimeterscale imaging depths using standard, inexpensive air objectives. Together with the low-cost pLSM platform, pLSM-SCOPE establishes a scalable and cost-effective solution for high-throughput, high-resolution volumetric imaging - a key advance toward democratizing advanced optical microscopy and accelerating Al-driven biological discovery.

[0094] Integration of Expansion Microscopy with SCOPE for Large-Volume, Nanoscale Imaging

[0095] Recent advances in ExM enable nanoscale interrogation of biological structures by physically enlarging specimens, achieving effectively sub-diffraction imaging with conventional optics. However, ExM imposes additional demands: multi-fold expansion dilutes fluorophores, markedly reducing signal per voxel; expanded tissues are mechanically fragile; and intact-sample imaging requires long-working-distance objectives with well-corrected detection and mounting strategies that preserve resolution and signal over large volumes.

[0096] Leveraging SCOPE'S ability to pair long-working-distance air objectives with NA enhancement and improved signal collection efficiency, we integrated SCOPE with ExM for nanoscale imaging of large, expanded tissues. Using ~4x expanded Thyl-GFP mouse brain sections (see the Methods section below), we systematically optimized final immersion formulation and mounting strategy to balance optical performance, fluorophore stability, and structural integrity while maintaining isotropic expansion. Optimal conditions employed 65-85% glycerol supplemented with >2.5 mg / mL DABCO (l,4-diazabicyclo[2.2.2]octane), which preserved fluorophore brightness, minimized photobleaching, and stabilized the expanded tissue. Samples were mounted in quartz cuvettes (10 x 10 mm or 10 x 20 mm base, scaled as needed) to maintain RI continuity with the SCOPE chamber.

[0097] Under these optimized conditions, expanded Thyl-GFP mouse brain sections were imaged using a 10x / 0.28 NA / 34 mm WD air objective for detection and a 5x / 0.14 NA / 34 mm WD air objective for light-sheet illumination within the pLSM-SCOPE setup. SCOPE enabled aberration-free, high-resolution imaging of neuronal architecture, clearly184898-7566-6314, v. 9resolving dendritic spines and fine neural processes across large tissue volumes. The resulting data were readily compatible with automated reconstruction tools, enabling comprehensive neuronal and dendritic spine tracing. Given the measured lateral PSF (-0.75 pm) and ~4x expansion, the effective pre-expansion resolution was -190 nm, sufficient for automated neurite and spine quantification

[0098] We next adapted the ExM-SCOPE workflow for whole intact salamander brains, an emerging model in regenerative and comparative neurobiology. By optimizing gelation, digestion, and immersion, we achieved robust 4x isotropic expansion with preserved morphology. Brains immunostained for tyrosine hydroxylase (TH) were imaged on the pLSM-SCOPE platform, yielding aberration-free, high-resolution volumetric datasets of the entire expanded brain and revealing both mesoscale organization and nanoscale anatomical detail across the dopaminergic system.

[0099] Together, these results demonstrate that integrating SCOPE'S aberration correction, resolution enhancement, and long-working-distance optics with cost-effective light-sheet microscopy platforms such as pLSM enables scalable, high-resolution imaging of large expanded tissues. The combined pLSM-SCOPE-ExM framework provides a practical route to organ-level nanoscale mapping with low-cost, high-throughput, portable instrumentation.

[0100] SCOPE Enables Multi-Immersion Imaging Across Diverse Clearing Methods

[0101] Advanced tissue-clearing methods exhibit wide variability in their optical properties: aqueous-based protocols such as CLARITY yield RIs of -1.44- 1.48, whereas solvent-based methods such as iDISCO and ECi reach RI values approaching 1.56. High-quality, aberration-free imaging across this RI range typically requires different specialized objectives or hardware modifications.

[0102] Optical simulations of SCOPE (system RI - 1.46; FIG. 11) indicated broad tolerance to variations in sample RI while maintaining effective aberration correction across an RI range of 1.38-1.54. This performance eliminates the need for hardware adjustments and supports a single, generalizable workflow for diverse biological and clinical specimens. To experimentally assess this versatility, we evaluated SCOPE (implemented at RI - 1.46) across samples immersed in media spanning from aqueous to organic-solvent conditions.194898-7566-6314, v. 9

[0103] SCOPE enabled color-corrected deep imaging (up to 1 cm) of polychromatic fluorescent beads embedded in 0.5% agarose / ffcO (RI - 1.33), despite the large RI mismatch between the aqueous sample and the hybrid solid-liquid immersion lens implemented with RI -1.46. We also applied SCOPE to image CUBIC-R-cleared mouse brains (RI - 1.51) from the fosTRAP2-tdTomato transgenic line. The pLSM-SCOPE approach provided high-contrast, aberration-free imaging throughout the entire brain volume. The resulting datasets were directly compatible with standard neuroinformatics pipelines, including registration to the Allen Brain Atlas and automated segmentation and quantification of tdTomato-positive neurons, revealing the expected brain-wide activation patterns. Next, we imaged iDISCO-cleared (RI - 1.56) blind cavefish (A. mexicanus) brains stained with nuclear dye TO-PRO-3. SCOPE-enabled imaging yielded high-contrast, single-nucleus-resolved images throughout the entire brain, including densely labeled regions, confirming its effective performance in high-RI, solvent-cleared tissues.

[0104] Further, we demonstrated the high-throughput imaging potential for collections of human iPSC-derived brain organoids with relevance for phenotype screening. pLSM-SCOPE imaging of Day 94 iPSC-derived cortical organoids incorporated with microglia (labelled with anti-IBAl) showed complete colonization of the organoid after 2 weeks of co-culture. The images obtained showed wide distribution and even tiling throughout the organoid, as well as varying morphology, including amoeboid, elongated, and ramified, indicating possible heterogeneity in inflammatory states.

[0105] Together, these results demonstrate that the SCOPE imaging device provides robust, multi-immersion compatibility across tissue-clearing methods ranging from aqueous to solvent-based immersion media without requiring hardware changes, optical realignment, or system reconfiguration. Combined with cost-efficient light-sheet microscopy platform pLSM, SCOPE establishes a scalable, general-purpose imaging framework that seamlessly bridges research and clinical applications, enabling high-resolution volumetric imaging for neuroscience, regenerative biology, and translational pathology.

[0106] Scalable High-Resolution 3D Histopathology with pLSM-SCOPE

[0107] Histopathological analysis remains the cornerstone of preclinical disease research, clinical diagnosis, therapeutic monitoring, and drug discovery. Traditional workflows rely on microscopic examination of thin tissue sections stained with hematoxylin204898-7566-6314, v. 9and eosin (H&E) or immunohistochemical markers. While these methods have been indispensable for over a century, they inherently disrupt the native three-dimensional (3D) tissue architecture, limiting insight into spatial cell-cell and cell-matrix relationships that underpin disease progression - a critical limitation in heterogeneous pathologies such as cancer.

[0108] Recent studies have demonstrated that deep learning models trained on volumetric histopathological datasets markedly outperform 2D-based models for key clinical tasks, including cancer detection, tumor grading, surgical margin assessment, and recurrence prediction. These findings underscore the transformative potential of 3D histopathology for precision medicine. Nevertheless, widespread adoption of 3D imaging techniques faces persistent barriers, including prohibitive costs, operational complexity, and limited accessibility.

[0109] To address these challenges, we evaluated the pLSM-SCOPE platform for rapid, high-resolution 3D imaging of intact pathological tissues. As an initial application, we focused on normal and cancerous human breast tissue. We implemented fluorescent H&E analog labeling of fresh-frozen breast specimens, using TO-PRO-3 for nuclear staining and eosin for cytoplasmic labeling. Samples were cleared and imaged in ECi (RI ~ 1.56), and data were acquired in two channels using pLSM-SCOPE. Through a false-coloring procedure, the two-channel fluorescence images were converted into conventional H&E-like representations for compatibility with standard pathology workflows.

[0110] The pLSM-SCOPE system was successfully used to obtain rapid, high-quality 3D imaging of large, intact human breast tissue samples at nuclear resolution. The volumetric datasets revealed the mesoscopic organization of terminal duct-lobular units (TDLUs) into tree-like networks while resolving subcellular structures within individual acini. Imaging of cancerous tissue further demonstrated the platform’s capability to capture diagnostic histopathological features of malignancy, including abnormal nuclear morphology, fibrotic foci, disrupted and tissue architecture, highlighting the potential of pLSM-SCOPE for scalable, high-resolution 3D histopathology and disease characterization.

[0111] The ability to capture mesoscale 3D tissue architecture while resolving nuclear and cytoplasmic morphology using a low-cost, highly portable system represents a significant advance toward democratizing 3D histopathology. This approach enables large-scale214898-7566-6314, v. 9generation of volumetric tissue datasets with spatial context preserved, ultimately advancing digital pathology, Al-driven diagnostics, and precision oncology for improved clinical outcomes.

[0112] DISCUSSION

[0113] We present the HySIL optical framework, which enables sophisticated wavefront control through seamless integration of a solid optical element with a precisely RI-matched liquid medium. This hybrid configuration demonstrates exceptional performance when combined with long-working-distance, low-cost air objectives positioned at the solid interface for signal collection, while a specimen is positioned in the liquid compartment (housed in Rl-matched glass cuvettes) to enable aberration-corrected, high-resolution 3D imaging of large samples. This principle was realized as modular SCOPE and Super-SCOPE imaging devices (FIGS. 5-12) that integrate readily into existing open- source and commercial LSM platforms. Integration of SCOPE with the economical pLSM platform yields a highly compact, portable system (pLSM-SCOPE) for subcellular-resolution imaging across large volumes.

[0114] A key advantage of SCOPE is its remarkable robustness to sample RI variations, maintaining excellent performance across a broad RI range (1.33-1.56) without hardware modifications while preserving chromatic correction and providing wide detection fields (up to 2.6 mm). We validated this multi-immersion versatility through aberration-free imaging of samples spanning aqueous to organic solvent media, including 1-cm-thick volumes of polychromatic fluorescent beads in 0.5% agarose / IUO, ExM-expanded mouse and salamander brains, CUBIC-cleared intact mouse brains, iDISCO-cleared cavefish brains, and high-throughput screening of brain organoids revealing subcellular details of microglial morphology across different activation states.

[0115] High-resolution volumetric imaging of expanded mouse brain was performed. More specifically, light- sheet imaging of a ~4x expanded Thyl-GFP mouse brain section was done using the pLSM-SCOPE system. 3D depth color-coded rendering of a selected region of interest within the hippocampal area, corresponding to the whole-section dataset were obtained. These renderings revealed fine axonal and dendritic processes, including spines. Automated tracing of individual neurons, dendritic arbors, and dendritic spines was done. Dendritic shafts highlighting individual spines were clearly visible.224898-7566-6314, v. 9

[0116] Brain-wide, high-resolution imaging of the dopaminergic system in the salamander brain was also performed. More specifically, we obtained volumetric rendering of a whole-brain dataset from an adult salamander (~4x expanded), immunolabeled for tyrosine hydroxylase (TH) and imaged using the pLSM-SCOPE system. We obtained two orthogonal views; depth color-coded, high-resolution 3D rendering of a selected region of interest; and maximum-intensity projections of raw data from the hindbrain, midbrain, and forebrain regions that revealed fine neuronal features across the dopaminergic network.

[0117] We also obtained high-resolution, multi-immersion imaging across diverse tissue clearing methods. More specifically, we performed whole-brain imaging of a CUBIC-R-cleared fosTRAP2-tdTomato mouse brain using the pLSM-SCOPE system. We obtained a volumetric rendering of the entire mouse brain. We implemented registration to the Allen Brain Atlas (ABA) and automated segmentation enable anatomical annotation and quantification of labeled neuronal populations. In one example, we observed the top 5% of tdTomato+cells within ABA-defined regions of interest (ROIs).

[0118] Imaging of an iDISCO-cleared Astyanax mexicanus (blind cavefish) brain stained with the nuclear label TO-PRO3 was performed. Volumetric renderings revealed dense, high-resolution nuclear labeling throughout the brain. We obtained sagittal optical section highlighting image quality across distinct, densely labeled brain regions. High-throughput imaging of human iPSC-derived day-94 cortical organoids co-cultured with microglia and immunolabeled for IB Al was performed. We obtained a clear image of a cuvette illustrating parallel imaging of multiple organoids in a single experiment, depth color-coded sub-volume projection showing extensive microglial coverage and spatial tiling, and magnified views revealing diverse microglial morphologies - amoeboid, elongated, and ramified - consistent with heterogeneous activation states.

[0119] Beyond basic science research applications, we demonstrated pLSM-SCOPE as a scalable solution for volumetric histopathological imaging of clinically relevant specimens. This capability directly overcomes a fundamental constraint of traditional microtome sectioning - the destruction of three-dimensional spatial relationships critical for disease characterization. Our proof-of-concept studies with human breast tissue achieved nuclear-resolution imaging of intact volumes, with datasets amenable to false-coloring algorithms that produce H&E-equivalent visualizations compatible with existing diagnostic workflows. The capacity to simultaneously resolve mesoscale tissue organization and234898-7566-6314, v. 9cellular / nuclear features using an affordable, transportable platform represents meaningful progress toward accessible and scalable 3D histopathology, enabling generation of large-scale 3D tissue repositories that can accelerate advances in computational pathology, machine learning-based diagnostics, and precision medicine.

[0120] Scalable, high-resolution 3D histopathology of human breast tissue was obtained using pLSM-SCOPE. More specifically, we successfully obtained volumetric rendering of an intact human breast tissue sample imaged using the pLSM-SCOPE system following fluorescent H&E analog labeling (TO-PRO-3 for nuclei and eosin for cytoplasm), ECi clearing, and false H&E-like coloring. Renderings with maximum-intensity projection and minimum-intensity projection, revealed nuclear-resolution architecture of terminal duct-lobular units (TDLUs) and their mesoscopic organization into tree-like structures. We also obtained 3D rendering of a cancerous human breast tissue sample showing invasive carcinoma with fibrotic focus features, visualized at three different depth levels.

[0121] The performance of HySIL and SCOPE is robust, and these systems can optionally be enhanced in a number of ways. First, while HySIL amplifies effective NA in proportion to the hybrid lens RI, fundamental limits on resolution and light collection are imposed by the air objective used. The Super-SCOPE configuration, which employs Weierstrass super-hemispherical geometry, achieves NA enhancement scaling with n2, offering improved resolution at the cost of stricter optical tolerances and narrower spectral bandwidth, making it most suitable for monochromatic or narrowband imaging. Moreover, incorporating higher-refractive-index solid components with matched immersion media, along with use of higher NA air objectives (such as Mitutoyo Plan Apochromat 20x / 0.42 NA / 20 mm WD), could provide imaging resolution and photon efficiency on par with the highest NA immersion objectives which only provide a few hundred microns working distance. Second, although we demonstrated compatibility across widely used clearing methods, samples with extreme RI values (<1.33 or >1.56) may require alternative HySIL configurations using different optical materials or liquid formulations to maintain aberration correction. Advantageously, SCOPE'S modular and easy-to-swap architecture facilitates straightforward use of multiple SCOPE chambers to accommodate extreme RI requirements. Third, although the implementations discussed herein are described in the context of horizontal benchtop LSM systems, the underlying HySIL principles are geometry-244898-7566-6314, v. 9independent and can be adapted to upright or inverted configurations through appropriate chamber redesign.

[0122] In summary, the HySIL framework and SCOPE implementation establish a practical and cost-effective foundation for next- generation volumetric microscopy, bridging the gap between cutting-edge optical performance, scalability, and widespread accessibility. This advance marks a significant step toward the democratization of high-resolution biological imaging and the acceleration of data-driven scientific discovery in preclinical research as well as in potential clinical applications in 3D histopathology.

[0123] METHODS

[0124] Design, Simulations and PSF Characterization of SCOPE

[0125] Optical Simulations

[0126] All optical simulations were performed in Ansys Zemax OpticStudio (2024). Three representative fluorescence emission wavelengths commonly used in biological imaging - 520 nm, 590 nm, and 640 nm - were simulated. As illustrated schematically in FIGS. 9 and 14, the optical model comprised a paraxial lens (serving as the detection objective), a Thorlabs TTL200 tube lens (black-box model available from Thorlabs), and a plano-convex fused-silica lens (radius of curvature of 16 mm (FIG. 9) or 27.5 mm (FIG. 14); Coming 7980 substrate) acting as the solid element of the hybrid immersion lens. The RI of the immersion liquid was set to match that of the fused-silica lens (n ~ 1.46), forming a continuous solid-liquid optical system. The sample was modeled as a planar slab of defined thickness and RI.

[0127] The optical path difference (OPD) wavefront error was used to quantify residual aberrations. Simulations were performed at 520 nm with a NA of 0.28, corresponding to the Mitutoyo lOx objective, and a 700 pm field of view (FOV) at the sample plane, which is typical for light sheet detection at this magnification. The STOP parameter, corresponding to an NA of 0.28 in the air objective used in air, was fixed across all configurations to match the experimental condition of using the same objective lens. Tangential and sagittal planes were analyzed along orthogonal field axes to capture off axis aberrations. Results were compared between the center and edge of the FOV for both the SCOPE and the conventional flat window based air immersion configuration.254898-7566-6314, v. 9

[0128] Deep, aberration-free multicolor imaging was successfully achieved with SCOPE across a large field of view and refractive index mismatch. FIG. 14 depicts Zemax optical simulations comparing detection using an air objective in air, in immersion media (standard configuration), and with the SCOPE imaging device. The radius of curvature of the truncated hemispherical (plano-convex) lens used in the simulation matches the practical configuration employed in all imaging experiments. In FIG. 14, moving from left to right, we see, for each of the three systems: optical ray traces; estimated Huygens point- spread functions (PSFs); simulated image formation; and quantification of normalized irradiance profiles of the Huygens PSFs.

[0129] Three-dimensional volumetric imaging of polychromatic fluorescent beads embedded in 0.5% agarose / H2O (RI ~ 1.33) was performed using the SCOPE imaging chamber (system RI ~ 1.46). Despite the large refractive-index mismatch between the aqueous sample and the hybrid solid-liquid immersion system, SCOPE achieved aberration-free, chromatically aligned imaging across the full sample depth (~1 cm demonstrated) and width (~2.6 mm demonstrated). Merged multichannel images and individual fluorescence channels (red, green, and blue) were successfully obtained.

[0130] RMS wavefront error (FIG. 11) was used to evaluate aberration correction robustness. Calculations were performed at 520 nm while varying both sample RI and sample thickness. The optical system was re-optimized after each modification to ensure best performance. RI values were tested in increments of ± 0.02 around the baseline (n = 1.46) of the system. Across the variation in the RI, the RMS error remained below the Marechai criterion (1 / 14), indicating that spherical-aberration correction was preserved, confirming the robustness of the HySIL configuration.

[0131] SCOPE Device Implementation

[0132] The SCOPE imaging device consisted of an off-the-shelf plano-convex lens (Edmund Optics cat #84-283; R = 27.5 mm, 0 = 30 mm) as the solid component and a Cargille Laboratories Rl-matching immersion oil (n ~ 1.46, Code: 50350) as the liquid component, forming a hybrid solid-liquid seamless system. The chamber body was 3D-printed using black resin and sealed with optical-grade epoxy to ensure mechanical stability and prevent leakage. The lens was fixed to the chamber window using UV-curable adhesive. The assembled chamber was integrated into custom projected light-sheet microscope (pLSM)264898-7566-6314, v. 9and its commercial implementation, SLICE (MBF Bioscience). For detection, Mitutoyo 10x / 0.28 NA and 5x / 0.14 NA long WD air objectives were used; illumination was provided by Mitutoyo 5x / 0.14 NA for most experiments.

[0133] PSF Imaging and Characterization

[0134] To measure the system PSF, gold nanoparticles (Sigma-Aldrich, #742058; 150 nm diameter) were suspended in 0.5% agarose to a final concentration of 5% (v / v) in 1.5 mF total volume. For wide-field PSF imaging, the pESM illumination light sheet was expanded to full extent to result in illumination of entire volume. The scattered light was collected in the detection arm without emission filter. Z-stacks were acquired at 2 pm step size using identical laser power for all conditions to enable quantitative signal comparison. For fluorescence-based PSF measurements, polychromatic and 1 pm fluorescent beads (Polysciences, Fluoresbrite Microparticles, cat. number: 18660) were embedded in 0.5% agarose under identical conditions. Appropriate excitation and emission filters were selected for each spectral channel.

[0135] PSF FWHM Analysis. Image stacks were analyzed to determine the lateral (X-Y) and axial (Z) FWHM of individual PSFs. All beads or nanoparticles in an image volume were manually marked by using a custom Python-based GUI from maximum-intensity projection image. Center coordinates were further refined by locating the pixel of maximum intensity in the immediate neighborhood. Intensity profiles along the X, Y, and Z axes were extracted and fit to a Gaussian model, from which FWHM values were calculated. The resulting lateral and axial resolutions were compared across objectives and configurations (SCOPE vs. Air-Immersion).

[0136] Sample Preparations

[0137] Expansion Microscopy of Salamander Brain

[0138] Building upon multiple published expansion microscopy (ExM) protocols, we systematically optimized experimental parameters to establish a reliable method for wholebrain expansion in the adult salamander, as described below.

[0139] An adult salamander was anesthetized by immersion in 0.1% MS-222, followed by transcardial perfusion with 10 mF of cold lx phosphate-buffered saline (PBS) to274898-7566-6314, v. 9flush out the blood, and then with 10 mL of cold 4% paraformaldehyde (PFA) in PBS for fixation. The brain was carefully extracted and post-fixed overnight in 4% PFA at 4°C.Immunolabeling was performed according to the salamander immunohistochemistry protocol. To label dopaminergic neurons and their processes, we used a rabbit anti-tyrosine hydroxylase primary antibody (1:1000, Sigma- Aldrich AB 152), followed by a goat antirabbit Alexa Fluor 546-conjugated secondary antibody (1:1000, Fisher A-l 1035).

[0140] Following immuno staining, the sample was transferred from PBS to MES-buffered saline [2-(N-morpholino)ethanesulfonic acid] to enhance penetration of the anchoring reagent. This step was adapted from a whole-brain ExM protocol for mouse brain. The brain was incubated in Acryloyl-X (AcX) at 4°C for 48 hours - adjusted for the smaller size of the salamander brain. The sample was then washed twice in PBS at 4°C for 1 hour each, followed by an overnight wash. For hydrogel embedding, the Stock X monomer solution was adapted from the TissUExM protocol, with increased acrylamide concentration based on our optimization experiments. The final monomer composition was: 23% sodium acrylate, 10% acrylamide, 0.1% N,N'-methylenebisacrylamide, and lx PBS. This solution was activated with 0.12% (w / v) VA-044, and the brain was incubated in the activated monomer mix at 4°C for 2 days.

[0141] For gelation, the sample was degassed in a vacuum chamber for 20 minutes, purged with nitrogen gas, and then incubated in an airtight container at 37 °C for approximately 3 hours, or until complete polymerization was observed. The gelled brain was then removed from the container, trimmed, and briefly washed twice in PBS (5 minutes each).

[0142] The embedded sample was incubated in digestion buffer containing 8 U / mL of Proteinase K at room temperature for 24 hours, or until the tissue appeared fully cleared. Following digestion, the gel was washed twice in PBS (1 hour each), followed by an overnight wash at room temperature.

[0143] Expansion was initiated by immersing the sample in deionized water, with water changes every 30-60 minutes. Expansion progress was monitored visually and halted once the desired expansion factor was achieved. The sample was then re-immersed in PBS to stabilize the gel and reduce over-expansion. To better match the RI of the expanded sample for optimal imaging, the tissue was equilibrated in a graded series of glycerol / PBS solutions,284898-7566-6314, v. 9culminating in 65% glycerol in PBS (RI ~ 1.42). To prevent photobleaching during imaging and long-term storage, 0.5% DABCO (l,4-diazabicyclo[2.2.2]octane) was added to all glycerol solutions. The fully expanded, Rl-matched sample was imaged using the pLSM / SLICE microscope equipped with the SCOPE imaging chamber.

[0144] Expansion Microscopy of Mouse Brain Sections

[0145] An adult (8-10 weeks old) transgenic Thyl-GFP (C57BL / 6) mouse was transcardially perfused with 4% PFA, and the brain was extracted and post-fixed overnight in 4% PFA at 4°C. The brain was then washed in PBS three times for 30 minutes each. A 400 pm-thick vibratome section was prepared for expansion microscopy. For processing, the same overall protocol described for the salamander brain was used, with adjustments to incubation times to account for the reduced tissue thickness. Specifically, AcX treatment was carried out overnight in PBS instead of MES-buffered saline, as the lower pH was not required for thin section penetration. The brain section was then incubated in activated Stock X monomer solution for 1 hour at 4°C, followed by gelation and digestion as described above. Digestion was carried out for 24 hours at room temperature in the dark. The embedded sample was expanded by immersion in deionized water for a total of 3 hours, with water changes every 30-60 minutes and was subsequently returned to PBS. For imaging, the sample was Rl-matched using the same 65% glycerol in PBS solution with 0.5% DABCO and imaged using the SEICE microscope with SCOPE imaging chamber.

[0146] CUBIC-R Clearing of FosTRAP Whole Mouse Brain

[0147] FosTRAP transgenic mice, aged 3-5 months, were exposed to a novel complex environment for 1 hour to induce neuronal activity. Immediately following this exposure, mice received an intraperitoneal injection of 4-hydroxytamoxifen (4-OHT; 50 mg / kg). One week later, animals were transcardially perfused with saline followed by 4% formaldehyde. Brains were extracted, post-fixed in 4% formaldehyde for 24 hours at 4°C, and then transferred to PBS. Tissue clearing was performed using a modified version of the CUBIC-R protocol. Neurons that expressed Fos during the 1-hour stimulation period prior to tamoxifen administration were permanently labeled with tdTomato, enabling visualization of activity-tagged neuronal populations across the entire brain. Cleared brains were imaged using the pESM / SEICE microscope in conjunction with the SCOPE imaging chamber.294898-7566-6314, v. 9

[0148] iDISCO cleared blind cavefish brains. Adult Astyanax mexicanus (blind cavefish) were anesthetized in an ice bath for 1 minute. Fish were euthanized via decapitation at the gill plate using a scalpel, and the heads were briefly immersed in ice-cold lx phosphate-buffered saline (PBS) for 30 seconds to remove residual blood. Samples were then fixed in 4% paraformaldehyde (PFA) in lx PBS at 4°C overnight (18-20 hours) with gentle shaking. The following day, heads were washed three times in lx PBS at room temperature (RT) for 30 minutes each, with shaking. Brains were carefully dissected and dehydrated through a graded methanol / Milli-Q water series (20%, 40%, 60%, 80%, 100%, 100%) and stored in 100% methanol at 4°C until further processing for iDISCO+ staining.

[0149] For staining, brains were bleached overnight at 4°C in 5% hydrogen peroxide in methanol without shaking (16-18 hours). The next day, samples were rehydrated through a reverse methanol / Milli-Q water gradient (80%, 60%, 40%, 20%) and finally into lx PBS. Samples were then washed twice in 0.2% Triton X-100 in PBS (PBSTx) for 30 minutes each at RT with rocking. Brains were permeabilized in a solution of 2.3% glycine and 20% dimethyl sulfoxide (DMSO) in PBSTx at 37°C for 24 hours without shaking. Blocking was performed in 6% normal donkey serum and 10% DMSO in PBSTx at 37°C for an additional 24 hours without shaking. Samples were then incubated in TO-PRO-3 iodide nuclear stain (Invitrogen, Cat#: T3605) at a 1:10,000 dilution in PTwH buffer (0.1% heparin, 0.2% Tween-20 in lx PBS) containing 3% normal donkey serum and 5% DMSO, for 24 hours at 37°C without shaking. After staining, brains were washed 4-5 times in PTwH over the course of a day at RT on a rocker. This wash step was repeated the following day under the same conditions, with samples protected from light.

[0150] For clearing, the brains were dehydrated again through a methanol / Milli-Q water gradient (20%, 40%, 60%, 80%, 100%, 100%) for 30 minutes per step at RT on a rocker. Samples were then incubated in 66% dichloromethane (DCM) in methanol for 3 hours at RT with rocking, followed by two 15-minute washes in 100% DCM. Final clearing was performed in dibenzyl ether (DBE) for at least 24 hours at RT in the dark. All tubes were filled completely with DBE to prevent air exposure during storage and imaging. The cleared sample was mounted in a cuvette and imaged with pLSM / SLICE equipped with SCOPE imaging chamber.304898-7566-6314, v. 9

[0151] Preparation of iPSC-Derived Cortical Organoids with Microglia

[0152] Cortical organoids with microglia were generated from the 1535-2 iPSC line. On Day 0, 9,000 iPS cells were seeded per well in a 96-well U-bottom plate in DMEM / F12, 15% KSR, 5% FBS, 1% NEAA, 1% Glutamax, lOOuM beta- mercaptomethanol, lOOnM LDN-193189, lOuM SB-431542, 2uM XAV-939, and 50uM Y27632. On day 2, the media was replenished without Y27632. On days 4-8, the media was replenished every other day without FBS. On day 10, the media was changed to 50% DMEM / F12, 50% Neurobasal, 0.5% N2, 1% B27 without RA, 0.5% NEAA, 1% Glutamax, 0.25% insulin, 1% pen-strep, and 50uM beta-mercaptoethanol, replenished every other day through day 16. On day 18, the media was changed to 50% DMEM / F12, 50% Neurobasal, 0.5% N2, 1% B27 with vitamin A, 0.5% NEAA, 1% glutamax, 0.25% insulin, 50uM beta-mercaptoethanol, 1% pen-strep, 20ng / mL BDNF, and 200uM ascorbic acid. The media was replenished every other day until day 26, after which the media was changed every 4-5 days. Microglial progenitors were generated as previously described. Progenitors were harvested and co-cultured with day 80 cortical organoids at 50,000 cells / organoid in IL-34 (lOOng / mL) and M-CSF (10 ng / mL) for 2 weeks before analysis.

[0153] Organoids were fixed in 4% PFA overnight and washed with PBS 3x 10 minutes, followed by simultaneous blocking and permeabilization in 5% serum / 0.5% TritonX in PBS for 2 hr at room temperature. The organoids were incubated with chicken anti-IBAl (Synaptic Systems) at 1:500 in 5% serum / 0.1% Triton-X overnight, were washed in 0.05% Tween-20 / PBS 3x 10 minutes and incubated with goat anti-chicken 647 secondary antibody in 5% serum / 0.1% Triton-X overnight. Finaly, the organoids were washed 0.05% Tween-20 / PBS 3x 10 minutes and stored in PBS with azide.

[0154] Organoids were aligned in 1% agarose and cleared using F-disco as previously described. They were placed sequentially for 1 hour at 4°C in 50% THF / dH20, 70% THF / dH20, 80% THF / dH20, 100% THF / dH20, 100% THF / dH20, and 100% dibenzyl ether. Cleared organoids were stored in 100% dibenzyl ether at 4°C.314898-7566-6314, v. 9

[0155] 3D Histopathology

[0156] Fluorescent H&E-Like Staining and Clearing of Human Breast Tissue

[0157] Frozen, de-identified human breast tissue specimens were obtained and stored at -80 °C. Samples were thawed at room temperature (RT) for 30 minutes and pre-washed in 70% ethanol (v / v in deionized water) to remove residual cryoprotectant. Rehydration was performed by immersing the samples in 70% ethanol within 50 mL Falcon tubes and incubating them on an orbital shaker at 60-80 rpm for a minimum of 3 hours at RT.

[0158] For fluorescent H&E-like staining, a staining buffer was prepared consisting of 70% ethanol, 10 mM NaCl, and adjusted to pH 4 using 0.1 N HC1. TO-PRO-3 (1:500 v / v) and eosin (1:100 v / v) were diluted into this buffer to prepare the staining solution. Samples were incubated in the staining solution for 48 hours at RT with gentle agitation. Following staining, tissues were dehydrated by incubation in 100% ethanol for 1 hour at RT, followed by a second incubation in fresh ethanol overnight to ensure complete dehydration. For optical clearing, samples were immersed in 10 mL of ethyl cinnamate (ECi) for 2 hours at RT with gentle agitation, followed by replacement with fresh ECi and an additional 2-hour incubation. For imaging, samples were transferred into 10x 10 mm cuvettes containing 1.5 mL of ECi and imaged using the SLICE microscope equipped with the SCOPE imaging chamber.

[0159] Imaging Experimentations

[0160] All imaging experiments were performed using the pLSM system and SLICE -a commercial implementation of pLSM developed by MBF Bioscience. Both systems were equipped with the SCOPE imaging device, which incorporates a hybrid solid-liquid immersion lens system composed of a fused quartz plano-convex lens with a 27.5 mm radius of curvature and immersion oil matched to a RI of 1.46. Samples were mounted in quartz cuvettes (10 x 20 mm or 10 x 10 mm base dimensions) to ensure RI continuity across optical interfaces. Image detection was carried out using Mitutoyo long WD air objectives (10x / 0.28 NA / 34 mm WD; 5x / 0.14 NA / 34 mm WD). For most experiments, a 5x / 0.14 NA / 34 mm WD illumination objective was used. Multicolor imaging was performed using laser excitation lines at 455 nm, 520 nm, and 640 nm. Appropriate emission bandpass filters were selected based on the spectral characteristics of the fluorophores used.324898-7566-6314, v. 9

[0161] Data Analysis and Visualization

[0162] All stitching and visualization of imaging data were performed using BrightSLICE software (MBF Bioscience). Detailed volumetric renderings were generated using ImageJ / FIJI, Neurolucida 360, and Amira. Automated neuronal tracing and 3D reconstructions were carried out with Neurolucida 360. Whole-brain registration and cellular quantification were performed using NeuroInfo software (MBF Bioscience).

[0163] Conclusion of Part 1

[0164] Note that the concepts described herein are not limited to light sheet microscopy. To the contrary, the approaches described herein are versatile and are compatible with a wide variety of other types of microscopy systems (including but not limited to confocal microscopes) without requiring major modifications to the microscope itself. For instance, in confocal microscopy, the plano-convex lens depicted in the FIG. 1 embodiment can serve as the imaging window for a specially designed sample mounting device.

[0165] Part 2 - Flattening Light Sheets in the Context of Light Sheet Microscopes

[0166] FIG. 16 depicts a light sheet microscope that reduces the thickness of the illumination light sheet by eliminating or minimizing spherical aberration in the illumination arm of the light sheet microscope. The light sheet microscope includes an illumination arm 100, a detection objective 40, and additional components (not shown) beyond the detection objective. The illumination arm 100 includes the illumination objective 140 and additional components 110 positioned prior to the illumination objective. The illumination arm 100 can use a variety of approaches for forming the light sheet, including but not limited to using a cylindrical lens to flatten a laser beam into a sheet, using an oscillating galvanometer mirror to scan a pencil beam, thereby forming a virtual sheet, etc.

[0167] The final component of the illumination arm 100 of the light sheet microscope is an air objective 140. The sample is immersed in a volume of immersion oil 25 within a container 20. A lens 130 has front surface and a convex rear surface with a radius of curvature R, and the lens 130 is mounted so that (i) the front surface of the lens is positioned within the container 20, sitting in the immersion oil 25 and (ii) the rear surface of the lens is positioned outside the container 20. The immersion oil 25 and the lens 130 have indices of334898-7566-6314, v. 9refraction that match within ± 20% (or, in some embodiments, within ± 15%, ± 10%, ± 5%, ± 2%, ± 1%, or ± 0.5%).

[0168] In the embodiment depicted in FIG. 16, the lens 130 is a plano-convex lens that has a flat front surface and a spherical rear surface. Note, however, that only the shape of the rear surface of the lens 130 is important, for the reasons described herein. On the other hand, the shape of the front surface is not critical, and other types of lenses that do not have a flat front surface (e.g., a double convex lens) can be substituted for the plano-convex lens depicted in FIG. 16.

[0169] The air objective 140 has a front element 140f, and this front element 140f is positioned behind the rear surface of the lens 130, with the air objective 140 aligned so that the light sheet that exits the front element 140f of the air objective 140 will pass through the lens 130 before it reaches the sample. The air objective 140 has a working distance WD2, and the front element of the air objective is spaced apart from the rear surface of the lens by a distance D2 that nominally equals WD2 minus R. In some embodiment, this distance D2 equals WD2 minus R2, ± 20%. But in other embodiments, this tolerance can vary from ± 20%, and could be, for example, ± 40%, ± 30%, ± 25%, ± 15%, ± 10%, ± 5%, ± 2%, or ± 1%.

[0170] The lens 130 and the air objective 140 are positioned such that the focal plane of the air objective 140 will be a distance R2 in front of the curved rear surface of the lens 130. This is accomplished by setting the size of the air gap to a distance D2 that nominally equals WD2 minus R2 (with any of the tolerances noted above). Due to this arrangement, the light rays that eventually reach the waist of the light sheet within the sample will be perpendicular to the curved rear surface of the lens 130. This dramatically reduces spherical aberration, which yields a thinner light sheet with improved axial resolution (from the perspective of the detection arm).

[0171] FIG. 16 includes ray tracings that show how the convex rear surface of the lens 130 is used to minimize spherical aberrations. Tracing backwards from the waist of the light sheet located within the sample, the perpendicular orientation of the light at the curved rear surface of the lens 130 will advantageously eliminate spherical aberrations (or at least minimize spherical aberrations when the indices of refraction of the immersion oil 25 and the lens 130 do not match exactly and / or when the focal plane is not precisely positioned at a344898-7566-6314, v. 9distance R2 from the rear surface of the lens 130). This is analogous to the situation described above in connection with FIG. 1, except that the light is travelling into the immersion oil 25 (as opposed to exiting the immersion oil).

[0172] In either case, light from the light sheet source causes a region within the sample to emit fluorescent light. This fluorescent light is captured by the detection objective 40 and eventually routed towards a camera (e.g., as described above in connection in Part 1).

[0173] The same examples described above in Part 1 regarding suitable combinations of materials for the lens 130 and corresponding immersion oils apply in this Part 2 as well. And here again, the explanation of the system described above in connection with FIG. 16 assumes that the container 20 has been filled with the immersion oil 25 at some prior point in time. But of course the system can be provided to an end-user with an empty container 20, as long as the end-user realizes that the container 20 must be filled with the correct type of immersion oil prior to use.

[0174] Note that the horizontal orientation depicted in FIG. 16 is not the only orientation that can be used. To the contrary, the entire system depicted in FIG. 16 could be rotated clockwise with respect to the page e.g., by 90° or 270°, and the system will operate as described above. The entire system could also be rotated by other angles.

[0175] Part 3 - A Light Sheet Microscope That Has a Flattened Light Sheet in the Illumination Arm and Reduced Spherical Aberration in the Detection Arm

[0176] FIG. 17 depicts a system that combines the concepts described above in Part 2 for generating a flattened light sheet with the concepts described above in Part 1 for reducing spherical aberration in the detection arm in a single system. The reference numbers in FIG.17 correspond to similarly-numbered reference numbers in FIGS. 1, 8, and 16.

[0177] While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.354898-7566-6314, v. 9

Claims

WHAT IS CLAIMED IS:

1. A microscope comprising:a container filled with a volume of immersion oil;a lens having front surface and a convex rear surface with a radius of curvature R, wherein the lens is mounted so that (i) the front surface of the lens is positioned within the container, sitting in the immersion oil and (ii) the rear surface of the lens is positioned outside the container; andan air objective having a front element and a working distance WD, wherein the front element of the air objective is positioned behind the rear surface of the lens, with the air objective aligned so that light exiting the rear surface of the lens can enter the front element of the air objective,wherein the immersion oil and the lens have indices of refraction that match within ± 20%.

2. The microscope of claim 1, wherein the immersion oil and the lens have indices of refraction that match within ± 10%.

3. The microscope of claim 1, wherein the immersion oil and the lens have indices of refraction that match within ± 5%.

4. The microscope of claim 1, wherein the immersion oil and the lens have indices of refraction that match within ± 2%.

5. The microscope of claim 1, wherein the lens is a plano-convex lens that has a flat front surface and a spherical rear surface.

6. The microscope of claim 1, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 20%.

7. The microscope of claim 6, further comprising a light sheet source configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.364898-7566-6314, v.

98. The microscope of claim 1, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

9. The microscope of claim 8, further comprising a light sheet source configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.

10. The microscope of claim 1, further comprising a light sheet source configured and positioned to project a light sheet into the container so that the light sheet is positioned Rx(l+l / n) in front of a rearmost point on the convex rear surface of the lens, where n is a refractive index of the immersion oil.

11. The microscope of claim 1, further comprising a tube lens and a camera, wherein the air objective has a back aperture, andwherein the tube lens is configured and positioned to route light that emanates from the back aperture of the air objective into the camera.

12. The microscope of claim 1, further comprising a light sheet source configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.

13. A microscope comprising:a container configured to hold a volume of immersion oil within the container;a lens having front surface and a convex rear surface with a radius of curvature R, wherein the lens is mounted so that (i) the front surface of the lens is positioned within the container so that when the container is filled with the immersion oil, the front surface of the lens will sit in the immersion oil and (ii) the rear surface of the lens is positioned outside the container; andan air objective having a front element and a working distance WD, wherein the front element of the air objective is positioned behind the rear surface of the lens, with the air objective aligned so that light exiting the rear surface of the lens can enter the front element of the air objective.374898-7566-6314, v.

914. The microscope of claim 13, wherein the lens is a plano-convex lens that has a flat front surface and a spherical rear surface.

15. The microscope of claim 13, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 20%.

16. The microscope of claim 13, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

17. The microscope of claim 13, further comprising the volume of immersion oil.

18. The microscope of claim 17, wherein the immersion oil and the lens have indices of refraction that match within ± 20%.

19. The microscope of claim 13, further comprising a tube lens and a camera, wherein the air objective has a back aperture, andwherein the tube lens is configured and positioned to route light that emanates from the back aperture of the air objective into the camera.

20. The microscope of claim 13, further comprising a light sheet source configured and positioned to project a light sheet into the container so that the light sheet intersects a center of curvature of the convex rear surface of the lens.

21. An apparatus for flattening a projected light sheet, the apparatus comprising:a container filled with a volume of immersion oil;a lens having a front surface and a convex rear surface with a radius of curvature R, wherein the lens is mounted so that (i) the front surface of the lens is positioned within the container, sitting in the immersion oil and (ii) the rear surface of the lens is positioned outside the container; andan air objective having a front element and a working distance WD, wherein the rear surface of the lens is positioned in front of the front element of the air objective, with the lens384898-7566-6314, v. 9aligned so that when the light sheet is being projected out of the air objective via the front element, the light sheet enters the rear surface of the lens, andwherein the immersion oil and the lens have indices of refraction that match within ± 20%.

22. The apparatus of claim 21, wherein the immersion oil and the lens have indices of refraction that match within ± 10%.

23. The apparatus of claim 21, wherein the immersion oil and the lens have indices of refraction that match within ± 5%.

24. The apparatus of claim 21, wherein the immersion oil and the lens have indices of refraction that match within ± 2%.

25. The apparatus of claim 21, wherein the lens is a plano-convex lens that has a flat front surface and a spherical rear surface.

26. The apparatus of claim 21, wherein the rear surface of the lens is spaced apart from the front element of the air objective by a distance D, wherein D equals WD minus R, ± 20%.

27. The apparatus of claim 21, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

28. The apparatus of claim 21, further comprising a detection objective, a tube lens and a camera that are respectively positioned and focused to capture images of portions of a sample that are being illuminated by the light sheet while the sample is positioned within the container.

29. An apparatus for flattening a projected light sheet, the apparatus comprising:a container configured to hold a volume of immersion oil within the container;a lens having a front surface and a convex rear surface with a radius of curvature R, wherein the lens is mounted so that (i) the front surface of the lens is positioned within the container so that when the container is filled with the immersion oil, the front surface394898-7566-6314, v. 9of the lens will sit in the immersion oil and (ii) the rear surface of the lens is positioned outside the container; andan air objective having a front element and a working distance WD, wherein the rear surface of the lens is positioned in front of the front element of the air objective, with the lens aligned so that when the light sheet is being projected out of the air objective via the front element, the light sheet enters the rear surface of the lens.

30. The apparatus of claim 29, wherein the lens is a plano-convex lens that has a flat front surface and a spherical rear surface.

31. The apparatus of claim 29, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 20%.

32. The apparatus of claim 29, wherein the front element of the air objective is spaced apart from the rear surface of the lens by a distance D, wherein D equals WD minus R, ± 5%.

33. The apparatus of claim 29, further comprising the volume of immersion oil.

34. The apparatus of claim 33, wherein the immersion oil and the lens have indices of refraction that match within ± 20%.

35. The apparatus of claim 29, further comprising a detection objective, a tube lens and a camera that are respectively positioned and focused to capture images of portions of a sample that are being illuminated by the light sheet while the sample is positioned within the container.404898-7566-6314, v. 9

Images