Curved light sheet microscopy imaging apparatus and method

Abstract

SOLUTION: An apparatus includes a microscopy imaging module, a curved light sheet lighting module, and a sample scanning module. The curved light sheet lighting module generates a curvature-adjustable curved light sheet and operates in conjunction with a wide-field microscopy imaging apparatus having a field curvature, thereby enabling the entire imaging field to be maintained in focus. By integrating with the sample scanning module, high-throughput microscopy imaging of a centimeter-scale transparent sample is achieved without the need for field-of-view stitching.EFFECT: An apparatus and method are useful for solving the technical problems associated with existing light sheet microscopy technology, which suffers from low throughput and requires field-of-view stitching when imaging a centimeter-scale transparent sample. They also contribute to addressing a field curvature issue in microscope objective lens design, thereby enabling reduced design complexity and production cost for a high-throughput objective lens.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of microscopy imaging, and more particularly to a microscopy imaging device and method based on curved light sheet illumination. [Background technology]

[0002] Light sheet microscopy, with its advantages of high spatiotemporal resolution, low phototoxicity, and minimal photobleaching, is an important tool for 3D imaging of transparent biological samples and is widely used in fields such as 3D cell biology, developmental biology, and neuroscience. Recent advances in tissue clearing technology have made it possible to optically transparent whole mouse brains, even whole human brains. However, existing light sheet microscopy techniques rely primarily on commercially available microscope objectives used in the life sciences, making it extremely difficult to capture high-resolution 3D structures of centimeter-sized transparent samples. These objectives are typically designed for observing tiny samples and have a small spatial-bandwidth product (the number of resolution-sized spots that can fit within the objective's field of view), making it impossible to simultaneously achieve a large field of view and high resolution. Due to this limitation, existing light sheet microscopes require field-of-view consolidation when imaging centimeter-sized samples, reducing imaging throughput and increasing the complexity of post-processing.

[0003] To improve the imaging throughput of light sheet microscopes, the spatial-bandwidth product of the objective lens must be increased. In recent years, many custom-made mesoscopic objectives have emerged, offering large fields of view and high-resolution imaging capabilities. However, because centimeter-sized fields of view must be contained within micrometer-scale depth of focus, these objectives struggle to maintain uniform imaging contrast and resolution across the entire field of view due to the effects of field curve. Correcting the field curve requires an increased number of lenses, which complicates the objective lens design, making it difficult to fabricate and costly. Furthermore, the field curve only bends the ideal image plane and does not blur the image. Due to these factors, field curves are rarely perfectly corrected in objective lens designs, and even commercially available flat-field objectives only fit 80% of the field of view within the depth of focus. Field curves also frequently appear in various custom-made mesoscopic objectives. In point-scanning imaging techniques such as confocal and two-photon microscopy, the field curve has little effect on the final imaging effect if other aberrations (e.g., spherical aberration, coma, and astigmatism) are adequately corrected. However, in camera-based wide-field imaging techniques, such as light sheet microscopy, the field curvature causes parts of the field of view to be out of focus, resulting in reduced imaging contrast and resolution. Therefore, a technical solution to overcome the above drawbacks in light sheet microscopy imaging when the objective lens has a field curvature is desired.

[0004] Conventional imaging systems typically image a plane. However, when a plane is imaged using a simple optical system, the center of the object is close to the lens and the edges are far from the lens, resulting in a curved image plane rather than an ideal plane. When imaging a plane using a planar imaging device (e.g., a camera), the edges become blurred when the central field of view is in focus, and the center becomes blurred when the edges of the field of view are in focus. This phenomenon is called field curvature. To correct the system's geometric aberrations, the structural design of the objective lens must become more complex. To achieve a certain resolution while eliminating aberrations within the field of view, part of the imaging field must be sacrificed. This is why the spatial-bandwidth product of current objective lenses cannot be further increased. Current light-sheet microscopes use a structural design that forms a planar light sheet for illumination and then uses a planar detector for imaging. This structural design limits the system's spatial-bandwidth product, making it difficult to achieve high imaging throughput. Therefore, we overturned the conventional "planar illumination-planar imaging" imaging method and proposed a new "curved illumination-planar imaging" method. By forming a curved light sheet as illumination and designing an objective lens with a field curve, we can obtain a planar image at the image plane when the curvature of the curved light sheet matches the field curve of the objective lens. Based on this idea, we designed an imaging objective lens with a fixed field curve, which can image a large 10 mm field of view with 1 μm resolution and achieve a spatial-bandwidth product of 420 million pixels. This is a two-order improvement over most existing objective lenses. Furthermore, this objective lens consists of only three lenses, making its structure extremely simple compared to other objective lenses, and the information throughput of a single lens also far exceeds that of other objective lenses. This objective lens is capable of diffraction-limited imaging in media with a wide range of refractive indices (1.33–1.6). Therefore, this imaging system is applicable to all current tissue clearing techniques. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above problems, the present invention provides a novel light sheet microscope imaging device and method that uses curved light sheet illumination and combines it with a microscope objective lens with a field curve to keep the entire imaging field in focus. By combining sample scanning with line-scan camera detection, high-throughput microscope imaging of centimeter-sized samples is achieved without field-of-view merging. [Means for solving the problem]

[0006] In a first aspect, the present invention provides a curved light sheet microscope imaging device including a fluorescence imaging module, a curved light sheet generation module, and a sample scanning module. The curved light sheet generated by the curved light sheet illumination module overlaps with the curved focal plane of the fluorescence imaging unit, resulting in an entire field of view being in focus. Furthermore, the narrow strip-shaped field of view defined by the curved light sheet illumination module and the fluorescence imaging module is detected and scanned by a time-delay integration camera to image the sample.

[0007] In a second aspect, the present invention provides a finite-correction objective lens with a curved focal plane. This objective lens is composed of five lenses (one single lens and two doublet lenses), with the first surface being a single lens. Preferably, this objective lens has a field of view diameter of 1 cm or more and a numerical aperture of 0.25. More preferably, it images a transparent sample whose refractive index matches that of the imaging buffer liquid in the imaging chamber. By controlling the distance between the imaging objective lens and the imaging chamber and the thickness of the imaging buffer liquid passing through during imaging, aberrations are corrected when imaging transparent samples with different refractive indices. Specifically, diffraction-limited resolution can be achieved if the refractive index of the sample is within the range of 1.33 to 1.60.

[0008] In a third aspect, the present invention provides a curved light sheet illumination device comprising an x-direction focusing cylindrical lens, a conical lens, a knife-edge prism mirror, and a y-direction focusing cylindrical lens. This device focuses an annular beam to form an annular focus. Specifically, the knife-edge prism mirror divides the annular focus equally, forming a symmetrical double-sided curved light sheet illumination. Preferably, the focusing directions of the x- and y-direction focusing cylindrical lenses are orthogonal to each other, and the focal planes overlap. More preferably, by adjusting the distance between the conical lens and the x- and y-direction focusing cylindrical lenses, the curvature of the curved light sheet generated by the curved light sheet illumination device can be adjusted to match the focal plane of an objective lens with a curved surface. This allows imaging with objective lenses with different focal plane curvatures.

[0009] In a specific embodiment, the present invention provides a curved light sheet microscope imaging device, comprising: a curved light sheet illumination module, a sample scanning module, and a microscope imaging module, wherein the curved light sheet illumination module generates a curved light sheet illumination with a variable curvature, the sample scanning module scans a tissue sample of a subject, and the focal plane of the microscope imaging module is curved to image the sample. The curved light sheet generated by the microscope imaging module overlaps with the curved focal plane of the microscope imaging module, and the sample is scanned to form an image using a time delay integration camera.

[0010] Specifically, in the curved light sheet microscope imaging device, the microscope imaging module includes an imaging objective, a filter, and a time delay integration camera. Preferably, the curved light sheet illumination module is either a two-sided curved light sheet illumination or a one-sided curved light sheet illumination. More specifically, in the curved light sheet illumination module, the emission direction of the laser beam is parallel to the scanning direction of the specimen sample. The curvature of the curved light sheet generated by the curved light sheet illumination module is adjustable. More specifically, the curved light sheet illumination module is composed of an x-direction focusing cylindrical lens, a conical lens, a knife-edge prism mirror, and a y-direction focusing cylindrical lens, and forms a symmetrical two-sided curved light sheet illumination by dispersing and focusing an annular beam.

[0011] The present invention also provides a curved light sheet microscope imaging method, characterized in that a curved light sheet illumination module generates a curved light sheet illumination with adjustable curvature, a sample scanning module scans a subject tissue sample, the focal plane of a microscope imaging module is curved to image the sample, and the curved light sheet generated by the curved light sheet illumination module overlaps with the curved focal plane of the microscope imaging module and is detected by a time delay integration camera.

[0012] The present invention also provides a method for fixing a sample in curved light sheet imaging, characterized in that a relatively hard sample is fixed directly to a support and imaged by immersing it in an imaging medium, and a relatively soft sample is embedded and placed in a cuvette, the periphery of the cuvette is sealed with a cover glass, the front end of the sample's imaging surface is not covered, the sample is stably fixed to the support, and a time delay integration camera performs imaging synchronously when the sample is scanned at a constant speed.

[0013] The present invention also provides an objective lens for curved light sheet illumination used in a microscope imaging device, characterized in that the focal plane of the objective lens is curved. The objective lens includes five lenses, consisting of one single lens and two pairs of doublet lenses, with the first surface being a single lens. The field of view diameter of the objective lens is 1 cm or more, and the numerical aperture is 0.25. When imaging a transparent sample, image aberrations in transparent samples with different refractive indices are corrected by controlling the distance between the imaging objective lens and the sample and the thickness of the imaging buffer solution passed through during imaging. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows a schematic optical configuration of a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 2] 1 shows a schematic diagram of a microscope imaging module of a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 3] 1 shows a schematic diagram of a curved light sheet illumination module of a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 4] An enlarged view of the vicinity of the focal plane of the curved light sheet microscope imaging device is shown. [Figure 5] 1 shows an optical simulation design of the imaging objective lens of a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 6] 1 shows the field dependence of the root mean square wavefront error of an imaging objective lens according to an embodiment of the present disclosure when the refractive index is 1.33 to 1.60. [Figure 7] 10 shows the results of a field curve test of an imaging objective lens processed and manufactured based on a simulation design in an embodiment of the present disclosure. [Figure 8] 10 shows the results of a resolution test of an imaging objective lens processed and manufactured based on a simulation design in an embodiment of the present disclosure. [Figure 9-1] 1 shows an optical simulation design (side view) of a curved light sheet illumination module according to an embodiment of the present disclosure. [Figure 9-2]1 shows an optical simulation design (top view) of a curved light sheet illumination module according to an embodiment of the present disclosure. [Figure 10] 1 shows an example of a curved light sheet generated by a curved light sheet illumination module according to an embodiment of the present disclosure. [Figure 11] 1 illustrates the axial resolution of a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 12] 1 shows the contrast across the field of view of a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 13] 1 shows a flow diagram of a curved light sheet microscope imaging method according to an embodiment of the present disclosure. [Figure 14] 1 illustrates a sample fixation device for a curved light sheet microscope imaging method according to an embodiment of the present disclosure. [Figure 15] 1 shows the results of three-dimensional imaging of a mouse brain (labeled with green fluorescent protein) cleared using the oil-based clearing method using a curved light sheet microscope imaging device according to an embodiment of the present disclosure. [Figure 16] 1 shows the results of 3D imaging of a mouse whole brain (propidium iodide stained) cleared using aqueous clearing method using a curved light sheet microscope imaging device according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0015] In order to allow those skilled in the art to better understand the technical solution disclosed herein, the embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below with reference to the drawings are illustrative and are used for the purpose of explaining the present invention, but should not be construed as limiting the present invention.

[0016] Figure 1 is a schematic diagram of a curved light sheet microscope imaging device provided by this embodiment. The curved light sheet microscope imaging device according to this embodiment includes three modules: a microscope imaging module (Figure 2), a curved light sheet illumination module (Figure 3), and a sample scanning module. A continuous-wave laser 008 (wavelength 488 nm or 561 nm) is quasi-rectangularly expanded by a laser beam expander 009, passes through a cylindrical lens 003 focusing in the y direction, a reflecting mirror M1, and a conical lens 002, and is then split into two paths by a knife-edge right-angle prism reflecting mirror 006. One path passes through reflecting mirrors M2 and M3 and a cylindrical lens 004 focusing in the x direction, while the other path passes through reflecting mirrors M4 and M5 and a cylindrical lens 005 focusing in the x direction, forming a symmetrical double-sided curved light sheet illumination at the detection region in the imaging chamber 011. The excited fluorescence passes through an objective lens 001 and a filter 010, and is then imaged by a time-delay-integration camera 007. The microscope imaging system of this example is capable of achieving a 10 mm field of view and 1 μm resolution, with the spatial-bandwidth product of the entire system exceeding 4×10 8 .

[0017] As shown in Figure 4, in the enlarged view near the focal plane of the curved light sheet microscope device, A is the curved focal plane, B is the imaging field of view, C and C' are the curved light sheet illumination, D is the sample scanning, E is the sample, and 001 is the imaging objective lens. The focal plane of the microscope imaging module is curved surface A, and the curved light sheet (C and C') generated by the curved light sheet illumination module overlaps with the focal plane of the microscope imaging module to perform fluorescence excitation. The narrow strip-shaped field of view B passes through the imaging objective lens 001 and filter 010 and is directly projected onto the time delay integration camera 007, where sample scanning imaging is performed. In this invention, the x, y, and z directions are defined as shown in Figure 4, with the x direction defining the propagation length of the light sheet, the y direction defining the width of the light sheet, and the z direction defining the imaging optical axis.

[0018] As shown in Figure 2, the microscope imaging module consists of an imaging objective 001, a filter 010, and a time-delay integration camera 007. The imaging objective 001 images a transparent sample immersed in an imaging chamber 011 without contacting the imaging buffer solution, preventing contamination and damage to the objective, reducing maintenance costs, and promoting widespread use. As shown in Figure 5, the main parameters of the optical simulation design of the imaging objective 001 of this curved light sheet microscope device are: working distance 20 mm, numerical aperture 0.25, magnification 8, object-to-image plane distance 974 mm, imaging field of view 13 mm, focal length 118 mm, primary operating wavelength 500-530 nm, and full operating wavelength 470-700 nm. The objective projects a magnified image directly from the focal plane to the image plane, eliminating the need for a large tube lens. The objective lens has been optimized to allow imaging through an approximately 20 mm thick imaging buffer solution and a 1 mm thick simulated sample chamber wall (for magnification). This simplified design reduces the risk of the objective lens being contaminated by the clearing reagent. Table 1 shows detailed lens parameters. Since there is no need to consider the field curve in the design of the imaging objective lens, the difficulty of designing and fabricating the objective lens is significantly reduced. The imaging objective lens 001 consists of five lenses (one singlet lens and two doublet lenses), is finite-focus corrected, and does not use a large-aperture tube lens, further reducing costs. By adjusting the curvature of the curved focal plane, the thickness of the imaging buffer solution passing through during imaging, and the distance from the imaging objective 001 to the imaging chamber 011, the imaging field of view of the imaging objective 001 exceeds 1 cm and the resolution reaches the diffraction limit in the imaging buffer solution with a refractive index of 1.33-1.60 (meeting the sample imaging requirements of all clearing techniques) and the operating wavelength range of 470-700 nm (see Figure 6). The working distance of the imaging objective 001 (the thickness of the imaging solution passing through during imaging) is approximately 2 cm, allowing imaging of a cleared mouse whole brain without sectioning.

[0019] [Table 1]

[0020] As shown in Figure 7, the field curve test results for the imaging objective 001 manufactured based on the simulation in this example showed that when the refractive index of the imaging buffer solution was 1.33 and 1.50, the corresponding curved focal plane curvatures were 40.6 mm and 46.2 mm, respectively. By imaging fluorescent beads with a diameter of 500 nm, the lateral resolution of the imaging objective 001 was confirmed to reach the diffraction limit across a 1 cm field of view. At wavelengths between 500 and 530 nm, the resolution was 1 μm, and at wavelengths between 590 and 610 nm, the resolution was 1.2 μm (Figure 8).

[0021] It should be noted that, although the embodiment of the present invention illustrates the configuration of a high spatial bandwidth product imaging objective lens 001 with a curved focal plane, the present invention is not limited thereto. Those skilled in the art can select any objective lens with a curved focal plane according to the actual application situation. In order to achieve more uniform sample illumination, this embodiment uses a double-sided illumination method to generate a curved light sheet and perform microscopic imaging. However, the method of microscopic imaging of a curved light sheet is not limited to a double-sided illumination method, and a single-sided illumination method can also be used to achieve microscopic imaging of a sample.

[0022] As shown in Figure 3, the curved light sheet illumination module uses a continuous-wave laser 008 (488 nm or 561 nm) for quasi-rectangular expansion by a beam expander 009. The beam then passes through a y-direction focusing cylindrical lens 003 and a conical lens 002 to form a focused annular beam in the y-direction. A knife-edge prism reflector 006 divides the annular beam into equal parts in the x-direction, which then pass through x-direction focusing cylindrical lenses 004 and 005, respectively, to form a symmetrical, double-sided curved light sheet illumination at the detection area within the imaging chamber 011. When the focal planes of the x- and y-direction focusing cylindrical lenses overlap at the detection area, uniform light sheet illumination is achieved. Figures 9-1 and 9-2 show the optical simulation design (side view and top view) of the curved light sheet illumination module of this example's curved light sheet microscope imaging device, and Table 2 lists detailed simulation parameters. By adjusting the distance between the conical lens 002 and the y-direction focusing cylindrical lens 003 and the x-direction focusing cylindrical lenses 004 and 005, the curvature of the curved light sheet can be adjusted to overlap the curved light sheet and the curved focal plane of the microscope imaging module within the imaging field of view. This curvature adjustment mechanism can accommodate changes in the curvature of the curved focal plane due to changes in the refractive index of the imaging buffer solution. In this embodiment, the annular beam is generated by the conical lens 002, but this is not limited thereto. Those skilled in the art can choose any other method, such as a spatial light modulator, to generate the annular beam depending on the actual application situation, as long as it can achieve the function of the conical lens 002 in this embodiment of the present invention.

[0023] [Table 2]

[0024] As shown in Figure 10, in an example of a curved light sheet generated by the curved light sheet microscope imaging device of this embodiment, the curved light sheet generated by the curved light sheet illumination module effectively covers a 1 cm field of view in the y direction, demonstrating uniform intensity distribution across the entire field of view. By imaging 500 nm diameter fluorescent beads, we confirmed that the axial resolution (light strip thickness) across the entire field of view was 2.5-3.0 μm when excited by 488 nm and 561 nm lasers when the refractive index of the imaging buffer solution was 1.33 and 1.50 (Figure 11). The narrow strip field of view (80 μm x 1 cm) formed by the curved light sheet passes through the imaging objective lens 001 and filter 010, and is then directly projected onto the time-delay integration camera 007 for sample scanning imaging. Furthermore, by imaging 500 nm diameter fluorescent beads, we confirmed that the light strip illumination generated by the curved light sheet illumination module can ensure uniform imaging contrast across the entire field of view (Figure 12).

[0025] It should be noted that the narrow strip field of view (80 μm × 1 cm) is mainly determined by the field of view range that can be imaged by the time delay integration camera 007. In the x direction, the confocal length of the light fragment generated by the curved light sheet illumination module is consistent with the field of view size defined by the time delay integration camera 007, thereby achieving an optimal optical section imaging effect.

[0026] The sample scanning module uses a motorized stage to scan the sample back and forth in the x-direction within the imaging chamber 011, synchronizing the sample scanning with the line scanning of the time delay integration camera 007. This operating mechanism allows the curved light sheet microscope imaging device to achieve uniform imaging contrast in the x-direction, and imaging the moving sample using the time delay integration camera 007 can effectively extend the exposure time and improve imaging sensitivity. The sample scanning distance determines the size of the x-direction imaging field of view of the curved light sheet microscope imaging device, e.g., 5 x 5 x 5 cm. 3 When using an imaging chamber with a scanning distance of approximately 2 cm, the field of view in the y direction is 1 cm, so the imaging field of the device is 2 × 1 cm. 2This eliminates the need for image stitching when imaging centimeter-scale samples such as cleared mouse whole brains, improving imaging throughput and significantly reducing the complexity of image post-processing. The sample scanning speed determines the imaging speed; for example, a scanning speed of 1 cm / s results in a 1 × 1 cm 2 It takes 1 second to image a field of view of 1. Furthermore, the sample scanning module uses another motorized translation stage to scan in the z-direction, achieving 3D imaging of the sample.

[0027] A curved light sheet microscope imaging device according to an embodiment of the present invention has been described in detail with reference to Figures 1 to 12. Hereinafter, a curved light sheet microscope imaging method according to an embodiment of the present invention will be described with reference to Figures 13 to 16. Figure 13 is a flow diagram of the curved light sheet microscope imaging method according to an embodiment of the present invention. As shown in Figure 13, the curved light sheet microscope imaging method includes the following steps:

[0028] Step S01: Fixing and refractive index matching of the cleared sample. The curved light sheet microscope imaging method according to an embodiment of the present invention can be used to image samples cleared using any clearing method. As shown in FIG. 14, hard samples (samples cleared using an oil-based clearing method) are directly fixed with UV-curable resin, while soft samples (samples cleared using an aqueous clearing method) are embedded in a fixation device. After the sample is fixed, it is placed in an imaging chamber 011, which is filled with an imaging buffer solution. The refractive index of the imaging buffer solution must match the refractive index of the cleared sample to avoid aberrations caused by refractive index mismatch.

[0029] Step S02: Determine the optimal conditions for the imaging objective lens 001. Image a fluorescent bead with a diameter of 500 nm, observe the imaging effect in the central field of view, and repeatedly adjust the distance between the imaging objective lens 001 and the imaging chamber 011 and the thickness of the imaging fluid passing through during imaging until the central field of view is clearly imaged and the resolution reaches the diffraction limit.

[0030] Step S03: Determine the curvature of the curved light sheet. Based on step S02, adjust the distance between the conical lens 002 of the curved light sheet illumination module and the focusing cylindrical lens in the x and y directions to adjust the curvature of the curved light sheet until the entire field of view is clearly imaged and the resolution reaches the diffraction limit. This ensures that the curved light sheet and the curved focal plane are perfectly aligned.

[0031] Step S04: Three-dimensional imaging of the sample. Depending on the size and fluorescence intensity of the sample, the lateral scanning field of view and speed, the axial scanning range and speed, and the line scanning speed of the time delay integration camera 007 are defined to acquire a three-dimensional image of the sample.

[0032] Figure 15 shows the 3D imaging results of a mouse brain (labeled with green fluorescent protein) cleared by the oil-based clearing method using a curved light sheet microscope imaging device according to an embodiment of the present invention. During the imaging process, the sample moving speed is 10 mm / s, and the camera synchronous line scanning rate is 16 kHz. The entire sample imaging time is approximately 3.5 hours, and the voxel size is 0.625 × 0.625 × 1.25 μm. 3 The data volume is 1TB. This curved light sheet microscope can image the entire brain tissue without merging, with micrometer-level resolution, uniform resolution and contrast across the entire field of view, allowing clear observation of the morphological structure of individual neurons. This merging-free method not only saves time in post-processing, but also avoids merging traces and false merging.

[0033] Figure 16 shows the results of three-dimensional imaging of a mouse whole brain (propidium iodide stained) cleared with an aqueous clearing method using a curved light sheet microscope imaging device according to an embodiment of the present invention. After treatment with the clearing reagent, the brain tissue expanded approximately 1.25 times and was rotated 90 degrees so that its longest dimension was aligned with the light sheet illumination direction. When upright, it exceeded the 1 cm imaging field of view. The curved light sheet microscope imaging device according to an embodiment of the present invention was able to capture a horizontal field of view (10.24 × 15.31 mm) of the sample with a resolution of 1 μm. 2) at once, without the need for fusion. This is not possible with other microscopes with the same resolution. During the imaging process, the sample movement speed is 5 mm / s, and the camera's synchronized line scanning rate is 8 kHz. The figure shows three different brain regions enlarged, and single cells can be precisely identified in each region. The application of this curved light sheet microscope to whole-brain cell imaging will make an important contribution to realizing total cell counting in whole mouse brain tissue and whole-brain distribution mapping of neuronal and glial cell types. Furthermore, it can be used to more precisely define brain regions and identify their components, thereby deepening our understanding of brain structure.

[0034] Finally, the above embodiments are merely illustrative examples of the present invention and do not limit the present invention. Those skilled in the art can modify the technical solutions described in the embodiments of the present invention or replace some technical features with equivalent substitutes. Such modifications or equivalent substitutions to the present invention will not deviate from the essence of the corresponding technical solutions and the spirit and scope of the disclosed technical solutions of the present invention.

[0035] (Addendum) (Appendix 1) A curved light sheet microscope imaging device comprising a curved light sheet illumination module, a sample scanning module, and a microscope imaging module, wherein the curved light sheet illumination module generates a curved light sheet illumination with adjustable curvature, the sample scanning module scans a subject tissue sample, the microscope imaging module has a curved focal plane for imaging the sample, the curved light sheet generated by the curved light sheet illumination module overlaps with the curved focal plane of the microscope imaging module, and the sample is scanned to perform imaging using a time delay integration camera.

[0036] (Appendix 2) The curved light sheet microscope imaging device described in Appendix 1, characterized in that the microscope imaging module includes an imaging objective lens, a filter, and a time delay integration camera.

[0037] (Appendix 3) The curved light sheet microscope imaging device described in Appendix 1, characterized in that the curved light sheet illumination module is either a double-sided curved light sheet illumination or a single-sided curved light sheet illumination.

[0038] (Appendix 4) The curved light sheet microscope imaging device described in Appendix 3, characterized in that the direction of the light beam emitted from the curved light sheet illumination module is parallel to the scanning direction of the specimen sample.

[0039] (Appendix 5) A curved light sheet microscope imaging device as described in Appendix 2, characterized in that a curved light sheet is generated using one conical lens and two cylindrical lenses, the conical lenses form an annular beam, and the annular beam is dispersed at the center to obtain symmetrical double-sided curved light sheet illumination.

[0040] (Appendix 6) The curved light sheet microscope imaging device described in Appendix 2 is characterized in that the curved light sheet illumination module is composed of an x-direction focusing cylindrical lens, a conical lens, a knife-edge prism mirror, and a y-direction focusing cylindrical lens, and forms a symmetrical double-sided curved light sheet illumination by dispersing and focusing the annular beam.

[0041] (Appendix 7) A curved light sheet microscope imaging method, characterized in that a curved light sheet illumination module generates a curved light sheet illumination with adjustable curvature, a sample scanning module scans a subject tissue sample, the focal plane of a microscope imaging module images the sample on a curved surface, and the curved light sheet generated by the curved light sheet illumination module overlaps with the curved focal plane of the microscope imaging module and is detected by a time delay integration camera.

[0042] (Appendix 8) A method for fixing a sample in curved light sheet imaging, characterized in that a relatively hard sample is fixed directly to a support and imaged by immersing it in an imaging medium, and a relatively soft sample is embedded and placed in a cuvette, the periphery of the cuvette is sealed with a cover glass, the front end of the sample's imaging surface is not covered, the sample is stably fixed to the support, and a time delay integration camera performs imaging synchronously when the sample is scanned at a constant speed.

[0043] (Appendix 9) An objective lens for curved light sheet illumination used in a microscope imaging device according to any one of appendices 1 to 6, characterized in that the focal plane of the objective lens is a curved surface.

[0044] (Appendix 10) The objective lens for curved light sheet illumination described in Appendix 9, characterized in that the objective lens includes five lenses, consisting of one single lens and two sets of doublet lenses, and the first surface is a single lens.

[0045] (Appendix 11) An objective lens for curved light sheet illumination as described in Appendix 10, characterized in that the field of view diameter of the objective lens is 1 cm or more and the numerical aperture is 0.25.

[0046] (Appendix 12) An objective lens for curved light sheet illumination as described in Appendix 9, characterized in that when imaging a transparent sample, the image difference in transparent samples with different refractive indices is corrected by controlling the distance between the imaging objective lens and the sample and the thickness of the imaging buffer liquid passed through during imaging.

Claims

1. A curved light sheet microscope imaging device comprising a curved light sheet illumination module, a sample scanning module, and a microscope imaging module, wherein the curved light sheet illumination module generates a curved light sheet illumination with adjustable curvature, the sample scanning module scans a subject tissue sample, the microscope imaging module has a curved focal plane for imaging the sample, the curved light sheet generated by the curved light sheet illumination module overlaps with the curved focal plane of the microscope imaging module, and the sample is scanned to perform imaging using a time delay integration camera.

2. The curved light sheet microscope imaging device of claim 1 , characterized in that the microscope imaging module includes an imaging objective lens, a filter, and a time delay integration camera.

3. The curved light sheet microscope imaging device of claim 1 , characterized in that the curved light sheet illumination module is either a double-sided curved light sheet illumination or a single-sided curved light sheet illumination.

4. The curved light sheet microscope imaging device of claim 3, characterized in that the direction of the light beam emitted from the curved light sheet illumination module is parallel to the scanning direction of the specimen sample.

5. The curved light sheet microscope imaging device of claim 2, characterized in that the curved light sheet is generated using one conical lens and two cylindrical lenses, the conical lens forms an annular beam, and the annular beam is split at the center to obtain symmetrical double-sided curved light sheet illumination.

6. The curved light sheet microscope imaging device of claim 2, characterized in that the curved light sheet illumination module is composed of an x-direction focusing cylindrical lens, a conical lens, a knife-edge prism mirror and a y-direction focusing cylindrical lens, and forms a symmetrical double-sided curved light sheet illumination by dispersing and focusing the annular beam.

7. A curved light sheet microscope imaging method, characterized in that a curved light sheet illumination module generates a curved light sheet illumination with adjustable curvature, a sample scanning module scans a subject tissue sample, the focal plane of a microscope imaging module images the sample on a curved surface, and the curved light sheet generated by the curved light sheet illumination module overlaps with the curved focal plane of the microscope imaging module and is detected by a time delay integration camera.

8. A method for fixing a sample in curved light sheet imaging, characterized in that a relatively hard sample is fixed directly to a support and imaged by immersing it in an imaging medium, and a relatively soft sample is embedded and placed in a cuvette, the periphery of the cuvette is sealed with a cover glass, the front end of the sample's imaging surface is not covered, the sample is stably fixed to the support, and a time delay integration camera performs imaging synchronously when the sample is scanned at a constant speed.

9. An objective lens for curved light sheet illumination used in a microscope imaging device described in any one of claims 1 to 6, characterized in that the focal surface of the objective lens is curved.

10. The objective lens for curved light sheet illumination described in claim 9, characterized in that the objective lens includes five lenses, consisting of one single lens and two pairs of doublet lenses, and the first surface is a single lens.

11. The objective lens for curved light sheet illumination according to claim 10, characterized in that the field diameter of the objective lens is 1 cm or more and the numerical aperture is 0.

25.

12. The objective lens for curved light sheet illumination described in claim 9, characterized in that when imaging a transparent sample, the image difference in transparent samples with different refractive indices is corrected by controlling the distance between the imaging objective lens and the sample and the thickness of the imaging buffer liquid passed through during imaging.

Images