Curved light sheet microscope 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 microscopic imaging technology, and more specifically, to a microscopic imaging apparatus and method based on curved light sheet illumination.

Background Art

[0002] Light sheet fluorescence microscopy has advantages such as high spatio-temporal resolution, low phototoxicity, and low photobleaching. It is an important tool for three-dimensional imaging of transparent biological samples, and is widely used in fields such as three-dimensional cell biology, developmental biology, and neuroscience. In recent years, with the development of biological tissue clearing technology, it has become possible to optically clear samples ranging from a mouse brain, to a whole mouse, and even to an entire human brain. However, existing light sheet microscopy techniques mainly rely on commercially available microscope objective lenses used in the life science field, and it is very difficult to obtain high-definition three-dimensional structures of centimeter-sized transparent samples. These objective lenses are usually designed for observing small samples, and have a small space-bandwidth product (the number of resolution-sized spots that can fit within the field of view of the objective lens), so they cannot achieve large field of view and high resolution at the same time. Due to this constraint, existing light sheet microscopes require field of view stitching when imaging centimeter-sized samples, which reduces imaging throughput and increases the complexity of post-processing.

[0003] To improve the imaging throughput of light-sheet microscopes, it is necessary to increase the spatial bandwidth product of the objective lens. In recent years, many custom-made mesoscope objective lenses have emerged, offering large fields of view and high-resolution imaging capabilities. However, because a centimeter-sized field of view must be contained within a micrometer-scale depth of field, the field curve makes it difficult for these objective lenses to maintain uniform imaging contrast and resolution across the entire field of view. Correcting the field curve requires increasing the number of lenses, making the objective lens design complex, difficult to manufacture, and costly. Furthermore, the field curve only bends the ideal image plane and does not blur the image. Due to these factors, the field curve is rarely completely corrected in the design of objective lenses, and even commercially available flat-field objective lenses only contain 80% of the field of view within the depth of field. The field curve frequently appears in various custom-made mesoscope objective lenses. In point scanning imaging techniques such as confocal and two-photon microscopes, the field curve has little effect on the final imaging effect if other aberrations (spherical aberration, coma aberration, astigmatism, etc.) are sufficiently corrected. However, in wide-field imaging techniques using cameras, such as light-sheet microscopy, the field curve causes a portion of the field of view to be out of focus, resulting in reduced image contrast and resolution. Therefore, a technical solution is needed to overcome the aforementioned drawbacks in light-sheet microscopy imaging when a field curve is present in the objective lens.

[0004] In typical imaging systems, a plane is usually imaged. However, when a plane is imaged using a simple optical system, the center of the object is closer to the lens, and the edges are further away from the lens, resulting in a curved image plane rather than an ideal plane. When imaging with a plane imaging device (e.g., a camera), the edges are blurred when the central field of view is in focus, and the center is blurred when the edges are in focus. This is called the field curve. To correct the geometric aberrations of the system, the structural design of the objective lens needs to be more complex, and to achieve a certain resolution while keeping the field of view aberration-free, a portion 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 employ a structural design that uses a planar light sheet for illumination and a planar detector for imaging, and are limited by the spatial bandwidth product of the system, making it difficult to achieve high imaging throughput. Therefore, we broke away from the conventional "planar illumination-planar imaging" method and proposed a new "curved illumination-planar imaging" method in which a curved light sheet is formed as the illumination light and an objective lens with a field curve is designed so that a planar image can be obtained 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, imaging a large field of view of 10 mm with a resolution of 1 μm and achieving a spatial bandwidth product of 420 million pixels. This is an improvement of two orders of magnitude compared to the spatial bandwidth product of most existing objective lenses. Furthermore, this objective lens consists of only three lenses, making its structure extremely simple compared to other objective lenses, and its single-lens information throughput is also far superior to other objective lenses. This objective lens enables diffraction-limited imaging in a wide range of refractive indices (1.33 to 1.6). Therefore, this imaging system can be applied to all current tissue clearing techniques. [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0006] In a first aspect, the present invention provides a curved light sheet microscope imaging apparatus 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, and the entire field of view is in focus. Furthermore, the narrow strip of field of view defined by the curved light sheet illumination module and the fluorescence imaging module is detected by a time-delay integration camera and scanned to image the sample.

[0007] In a second aspect, the present invention provides a finite-focus corrected objective lens having 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 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 solution in the imaging chamber. By controlling the distance between the imaging objective lens and the imaging chamber, and the thickness of the imaging buffer solution that passes through during imaging, aberrations when imaging transparent samples with different refractive indices are corrected. Specifically, diffraction-limited resolution can be achieved if the refractive index of the sample is in 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 is characterized by focusing an annular beam to form an annular focal point. Specifically, the knife-edge prism mirror divides the annular focal point equally, forming symmetrical double-sided curved light sheet illumination. Preferably, the focusing directions of the x-direction and y-direction focusing cylindrical lenses are orthogonal to each other, and their focal planes overlap. More preferably, by adjusting the distance between the conical lens and the x-direction 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 that of an objective lens whose focal plane is curved. This makes it possible to achieve imaging of objective lenses with different focal plane curvatures.

[0009] In a particular embodiment, the present invention provides a curved light sheet microscope imaging apparatus 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 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 form an image of the sample. The curved light sheet generated by the microscope imaging module overlaps with the curved focal plane of the microscope imaging module, and imaging is performed by a time-delayed integrating camera by scanning the sample.

[0010] Specifically, in the curved light sheet microscope imaging device, the microscope imaging module includes an imaging objective lens, a filter, and a time-delay integrating camera. Preferably, the curved light sheet illumination module is either bilateral or unilateral 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 subject 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 consists of an x-direction focusing cylindrical lens, a conical lens, a knife-edge prism mirror, and a y-direction focusing cylindrical lens, and forms symmetrical bilateral curved light sheet illumination by spectrally spectroscopy and focusing an annular beam.

[0011] Furthermore, the present invention 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, a microscope imaging module has a curved focal plane and forms an image of 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 is detected by a time-delayed integrating camera.

[0012] Furthermore, the present invention provides a method for fixing a sample in curved light sheet imaging, characterized in that a relatively hard sample is directly fixed to a support and immersed in an imaging medium for imaging, a relatively soft sample is embedded and placed in a cuvette, the cuvette is sealed around with a coverslip, the front end of the imaging surface of the sample is not covered, the sample is stably fixed to the support, and a time-delay integrating camera synchronizes with the scanning of the sample at a constant velocity to perform imaging.

[0013] Furthermore, the present invention 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 sets of doublet lenses, with the first surface being a single lens. The field diameter of the objective lens is 1 cm or more, and the numerical aperture is 0.25. When imaging a transparent sample, the image difference between 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 solution that passes through during imaging. [Brief explanation of the drawing]

[0014] [Figure 1] The schematic optical configuration of the curved light sheet microscope imaging apparatus according to the embodiment of this disclosure is shown. [Figure 2] A schematic diagram of the microscope imaging module of the curved light sheet microscope imaging apparatus according to an embodiment of this disclosure is shown. [Figure 3] A schematic diagram of the curved light sheet illumination module of the curved light sheet microscope imaging apparatus according to an embodiment of this disclosure is shown. [Figure 4] This shows a magnified view of the area near the focal plane of a curved light sheet microscope imaging device. [Figure 5] This document shows the optical simulation design of the imaging objective lens of a curved light sheet microscope imaging apparatus according to an embodiment of the present disclosure. [Figure 6] The field-of-view dependence of the equal root wavefront error for the imaging objective lens according to the embodiments of this disclosure at refractive indices of 1.33 to 1.60 is shown. [Figure 7] The field curve test results of the imaging objective lens processed and manufactured based on the simulation design in the embodiments of this disclosure are shown. [Figure 8] The resolution test results of the imaging objective lens processed and manufactured based on the simulation design in the embodiments of this disclosure are shown. [Figure 9-1] This shows the optical simulation design (side view) of a curved light sheet lighting module according to an embodiment of the present disclosure. [Figure 9-2]Shows an optical simulation design (top view) of a curved light sheet illumination module according to an embodiment of the present disclosure. [Figure 10] 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] Shows the axial resolution of a curved light sheet microscopic imaging apparatus according to an embodiment of the present disclosure. [Figure 12] Shows the contrast of the entire field of view of a curved light sheet microscopic imaging apparatus according to an embodiment of the present disclosure. [Figure 13] Shows a flow diagram of a curved light sheet microscopic imaging method according to an embodiment of the present disclosure. [Figure 14] Shows a sample fixing apparatus for a curved light sheet microscopic imaging method according to an embodiment of the present disclosure. [Figure 15] Shows a three-dimensional imaging result of a mouse brain (labeled with green fluorescent protein) cleared by an oily clearing method using the curved light sheet microscopic imaging apparatus according to an embodiment of the present disclosure. [Figure 16] Shows a three-dimensional imaging result of an entire mouse brain (stained with propidium iodide) cleared by an aqueous clearing method using the curved light sheet microscopic imaging apparatus according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF EMBODIMENTS

[0015] To allow those skilled in the art to better understand the technical solutions of the present disclosure, embodiments of the present invention are described in detail below with reference to the drawings. The embodiments described below with reference to the drawings are illustrative, and are used for the purpose of describing the present invention, and shall 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 the following 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-orthogonal-focused by a laser beam spreader 009, then passes through a cylindrical lens 003 that focuses in the y direction, a reflector M1, and a conical lens 002, and is split into two paths by a knife-edge right-angle prism reflector 006. One path passes through reflectors M2 and M3 and a cylindrical lens 004 that focuses in the x direction, and the other path passes through reflectors M4 and M5 and a cylindrical lens 005 that focuses in the x direction, forming symmetrical double-sided curved light sheet illumination in the detection area within the imaging chamber 011. The fluorescence excited by this passes through the objective lens 001 and filter 010 and is then imaged by a time-delay integrating camera 007. The microscope imaging system of this embodiment is capable of achieving a field of view of 10 mm and a resolution of 1 μm, and the spatial bandwidth product of the entire system exceeds 4 × 10⁸.

[0017] As shown in Figure 4, in a magnified view of the vicinity of the focal plane of a curved light sheet microscope, A is the curved focal plane, B is the imaging field, C and C' are the curved light sheet illumination, D is the sample scan, E is the sample, and 001 is the imaging objective lens. The focal plane of the microscope imaging module is the curved surface A, and the curved light sheets (C and C') generated by the curved light sheet illumination module overlap with the focal plane of the microscope imaging module, causing fluorescence excitation. The narrow strip of field B passes through the imaging objective lens 001 and filter 010 and is projected directly 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, where the x direction defines the propagation length of the light sheet, the y direction defines the width of the light sheet, and the z direction defines the imaging optical axis.

[0018] As shown in Figure 2, the microscope imaging module consists of an imaging objective lens 001, a filter 010, and a time-delay integrating camera 007. The imaging objective lens 001 images a transparent sample immersed in the imaging chamber 011 and does not come into contact with the imaging buffer solution, thus preventing contamination and damage to the objective lens, reducing maintenance costs, and promoting widespread adoption. As shown in Figure 5, the main parameters of the optical simulation design of the imaging objective lens 001 of the curved light sheet microscope apparatus in this embodiment are a working distance of 20 mm, a numerical aperture of 0.25, a magnification of 8, a distance from the object plane to the image plane of 974 mm, an imaging field of view of 13 mm, a focal length of 118 mm, a primary working wavelength of 500-530 nm, and a total working wavelength of 470-700 nm. The objective lens directly projects the magnified image from the focal plane to the image plane, eliminating the need to use a large tube lens. The objective lens is optimized to image through a sample chamber wall (for magnified display) simulated with approximately 20 mm thick imaging buffer solution and 1 mm thick fused silica glass. This simplified design reduces the risk of contamination of the objective lens by the clearing reagent. Table 1 shows the detailed lens parameters. The difficulty of designing and manufacturing the objective lens is greatly reduced because there is no need to consider the field curve in the design of the imaging objective lens. The imaging objective lens 001 consists of five lenses (one single lens and two doublet lenses), has finite distance correction, and does not use a large-diameter tube lens, further reducing costs. By adjusting the curvature of the curved focal plane, the thickness of the imaging buffer solution that passes through during imaging, and the distance from the imaging objective lens 001 to the imaging chamber 011, the imaging field of view of the imaging objective lens 001 exceeds 1 cm and the resolution reaches the diffraction limit when using an imaging buffer solution with a refractive index of 1.33 to 1.60 (meeting the sample imaging requirements of all clearing methods) and an operating wavelength range of 470 to 700 nm (see Figure 6). The working distance of the imaging objective lens 001 (thickness of the imaging solution that passes through during imaging) is approximately 2 cm, allowing imaging of the cleared mouse whole brain without cutting it.

[0019] [Table 1]

[0020] As shown in Figure 7, field curve test results of the imaging objective lens 001 manufactured based on the simulation of this embodiment showed that when the refractive index of the imaging buffer solution was 1.33 and 1.50, the curvature of the corresponding curved focal plane was 40.6 mm and 46.2 mm, respectively. By imaging a 500 nm diameter fluorescent bead, it was confirmed that the lateral resolution of the imaging objective lens 001 reached the diffraction limit across a 1 cm field of view. The resolution was 1 μm at wavelengths of 500-530 nm and 1.2 μm at wavelengths of 590-610 nm (Figure 8).

[0021] While the embodiments of the present invention show a configuration of a high spatial bandwidth integration objective lens 001 having a curved focal plane, the invention is not limited thereto. Those skilled in the art can select any objective lens with a curved focal plane depending on the actual application. In this embodiment, a curved light sheet is generated using a double-sided illumination method to achieve more uniform sample illumination, and microscopic imaging is performed using this method. However, the method of microscopic imaging of a curved light sheet is not limited to a double-sided illumination method, and microscopic imaging of a sample can also be achieved with a single-sided illumination method.

[0022] As shown in Figure 3, the curved light sheet illumination module receives a laser beam output from a continuous wave laser 008 (488 nm or 561 nm), which is then quasi-rectangularly focused by a beam spreader 009. This beam then passes through a y-direction focusing cylindrical lens 003 and a conical lens 002 to form a y-direction focused annular beam. A knife-edge prism reflector 006 divides the annular beam equally in the x-direction, passing through x-direction focusing cylindrical lenses 004 and 005, respectively, to form symmetrical double-sided curved light sheet illumination in the detection region within the imaging chamber 011. When the focal planes of the x-direction and y-direction focusing cylindrical lenses overlap in the detection region, uniform light sheet illumination is obtained. Figures 9-1 and 9-2 show the optical simulation design (side view and top view) of the curved light sheet illumination module of the curved light sheet microscope imaging apparatus of this embodiment, and Table 2 shows the 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, allowing the curved light sheet and the curved focal plane of the microscope imaging module to overlap 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 is not limited to this. Those skilled in the art can select any other method, such as a spatial light modulator, to generate the annular beam depending on the actual application, as long as the function of the conical lens 002 in this embodiment of the present invention is realized.

[0023] [Table 2]

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

[0025] The aforementioned narrow-band field of view (80 μm × 1 cm) is mainly determined by the field of view range that the time-delay integration camera 007 can image. In the x-direction, the confocal length of the light fragments generated by the curved light sheet illumination module matches the field of view size defined by the time-delay integration camera 007, thereby obtaining the optimal optical section imaging effect.

[0026] The sample scanning module uses an electric stage to reciprocate the sample 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 system to obtain uniform imaging contrast in the x-direction, effectively extending the exposure time and improving imaging sensitivity by imaging a moving sample using the time-delay integration camera 007. The scanning distance of the sample determines the size of the x-direction imaging field of view of the curved light-sheet microscope imaging system, for example, 5 × 5 × 5 cm. 3 When using the imaging chamber, a scanning distance of approximately 2 cm can be obtained. Since the field of view in the y-direction is 1 cm, the imaging field of the device is 2 × 1 cm. 2This eliminates the need for image stitching when imaging centimeter-scale samples such as the whole brain of a clearened mouse, improving imaging throughput and significantly reducing the complexity of image post-processing. The sample scanning speed determines the imaging speed; for example, at a scanning speed of 1 cm / s, 1 × 1 cm 2 It takes 1 second to image the field of view. Furthermore, the sample scanning module achieves three-dimensional imaging of the sample by scanning in the z direction using another motorized parallel transport table.

[0027] A curved light sheet microscope imaging apparatus according to an embodiment disclosed by the present invention has been described in detail with reference to Figures 1 to 12. Below, a curved light sheet microscope imaging method according to an embodiment disclosed by the present invention will be described with reference to Figures 13 to 16. Figure 13 is a flowchart of the curved light sheet microscope imaging method according to an embodiment disclosed by the present invention. As shown in Figure 13, this curved light sheet microscope imaging method includes the following steps.

[0028] Step S01: Fixation of the cleared sample and refractive index matching. The curved light sheet microscopy imaging method according to the embodiment of the present invention can be used to image samples cleared by all clearing methods. As shown in Figure 14, hard samples (samples cleared by the oil-based clearing method) are fixed directly with UV-curing resin, and soft samples (samples cleared by the aqueous clearing method) are embedded in a fixation device. After sample fixation, the sample is placed in the imaging chamber 011, which is filled with imaging buffer solution. The refractive index of the imaging buffer solution must match that of the cleared sample to avoid aberrations due to refractive index mismatch.

[0029] Step S02: Determining the optimal conditions for the imaging objective lens 001. A fluorescent bead with a diameter of 500 nm is imaged, and the imaging effect in the central field of view is observed. The distance between the imaging objective lens 001 and the imaging chamber 011 and the thickness of the imaging solution passing through during imaging are repeatedly adjusted until the central field of view is clearly imaged and the resolution reaches the diffraction limit.

[0030] Step S03: Determination of the curvature of the curved light sheet. Based on Step S02, the distance between the conical lens 002 of the curved light sheet illumination module and the focusing cylindrical lenses in the x and y directions is adjusted 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 perfectly coincide.

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

[0032] Figure 15 shows the three-dimensional imaging result of a mouse brain (labeled with green fluorescent protein) cleared by an oil-based clearing method using a curved light-sheet microscope imaging apparatus according to an embodiment of the present invention. During the imaging process, the sample movement speed was 10 mm / s, and the camera's synchronization line scanning speed was 16 kHz. The total imaging time for the sample was approximately 3.5 hours, and the voxel size was 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 connections, has micrometer-level resolution, uniform resolution and contrast across the entire field of view, and allows for clear observation of the morphological structure of individual neurons. This connection-free method not only saves time on post-processing but also avoids traces and incorrect connections caused by connections.

[0033] Figure 16 shows the three-dimensional imaging results of a mouse whole brain (stained with propidium iodide) cleared by aqueous clearing 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 to approximately 1.25 times its original size and was rotated 90 degrees so that the longest dimension aligned with the direction of light sheet illumination. When upright, it exceeds the imaging field of view of 1 cm. The curved light sheet microscope imaging device according to an embodiment of the present invention has a resolution of 1 μm and a horizontal field of view of the sample (10.24 × 15.31 mm). 2It images the entire brain in one go without requiring sync. This is impossible with other microscopes of the same resolution. During the imaging process, the sample movement speed is 5 mm / s and the camera's synchronization line scanning speed is 8 kHz. Three different brain regions are shown magnified in the figure, and single cells can be accurately identified in each region. Applying this curved light sheet microscope to whole-brain cell imaging will make a significant contribution to achieving whole-cell counting in mouse whole-brain tissue, as well as whole-brain distribution mapping of neuronal and glial cell types. Furthermore, it can be used for more precise definition of brain region compartments and identification of components, enabling a deeper understanding of brain structure.

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

[0035] (Note) (Note 1) A curved light sheet microscope imaging apparatus 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 tissue sample, the microscope imaging module has a curved focal plane and images 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 imaging is performed by a time-delayed integrating camera by scanning the sample.

[0036] (Note 2) The curved light sheet microscope imaging apparatus according to Appendix 1, characterized in that the microscope imaging module includes an imaging objective lens, a filter, and a time-delay integrating camera.

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

[0038] (Note 4) The curved light sheet microscope imaging apparatus according to 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 sample to be examined.

[0039] (Note 5) The curved light sheet microscope imaging apparatus described in Appendix 2, characterized in that a curved light sheet is generated using one conical lens and two cylindrical lenses, the conical lens forms an annular beam, and symmetrical illumination of both sides of the curved light sheet is obtained by spectrally spectroscopy at the center of the annular beam.

[0040] (Note 6) 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 is characterized in that it forms symmetrical double-sided curved light sheet illumination by spectrally spectroscopy and focusing an annular beam, as described in Appendix 2 of the curved light sheet microscope imaging apparatus.

[0041] (Note 7) A curved light sheet microscopy 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, a 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 is detected by a time-delayed integrating camera.

[0042] (Note 8) A method for fixing a sample in curved light sheet imaging, characterized in that relatively hard samples are directly fixed to a support and immersed in an imaging medium for imaging, relatively soft samples are embedded and placed in a cuvette, the cuvette is sealed around the edges with a coverslip, the front end of the imaging surface of the sample is not covered, the sample is stably fixed to the support, and a time-delay integrating camera synchronizes with the scanning of the sample at a constant velocity to perform imaging.

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

[0044] (Note 10) The objective lens for curved light sheet illumination as 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] (Note 11) The objective lens for curved light sheet illumination described in Appendix 10, characterized in that the field diameter of the objective lens is 1 cm or more and the numerical aperture is 0.25.

[0046] (Note 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 solution that passes through during imaging.

Claims

1. A curved light sheet microscope imaging apparatus 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 and images the subject tissue 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 imaging is performed by a time-delayed integrating camera by scanning the sample.

2. The curved light sheet microscope imaging apparatus according to claim 1, characterized in that the microscope imaging module includes an imaging objective lens, a filter, and a time-delay integrating camera.

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

4. The curved light sheet microscope imaging apparatus according to 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 subject tissue sample.

5. The curved light sheet microscope imaging apparatus according to claim 2, characterized in that a curved light sheet is generated using one conical lens and two cylindrical lenses, the conical lens forms an annular beam, and symmetrical illumination of both sides of the curved light sheet is obtained by spectrally spectroscopy at the center of the annular beam.

6. 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 is characterized in that it forms symmetrical double-sided curved light sheet illumination by spectrally spectroscopy and focusing an annular beam, as described in claim 2.

7. A curved light sheet microscopy 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, a microscope imaging module has a curved focal plane that images the subject tissue 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 is detected by a time-delayed integrating camera.

8. Curved light sheet imaging is performed by imaging a subject tissue sample using the curved light sheet microscopy imaging method described in Claim 7. A method for fixing a tissue sample in curved light sheet imaging, characterized in that a relatively hard tissue sample is directly fixed to a support and immersed in an imaging medium for imaging, a relatively soft tissue sample is embedded and placed in a cuvette, the cuvette is sealed around with a coverslip, the front end of the imaging surface of the tissue sample is not covered, the tissue sample is fixed to a support, and a time-delay integrating camera synchronizes with the constant-velocity scanning of the tissue sample to perform imaging.

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

10. The objective lens for curved light sheet illumination according to claim 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.

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 according to 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 solution that passes through during imaging.

Images