A method of super-resolution large-depth three-dimensional scanning light-sheet microscopy

Abstract

The application discloses a kind of methods of super-resolution large depth three-dimensional scanning light sheet microscope, belong to fluorescence imaging technical field, the excitation light is handled by column lens in the present application, the longer excitation objective working distance is obtained using the secondary convergence of the initial light sheet generated, so as to reduce the space conflict between excitation objective and sample;Secondly, the collection objective is placed with a small inclination angle, and is matched with a liquid chamber with the same inclination angle with optical window, and the configuration determines the imaging depth of the collection objective;Then, the collection objective and imaging buffer solution matching the refractive index of the tissue transparent sample are selected, and the configuration can avoid optical aberration to realize high-resolution imaging.By the present application, high-resolution imaging and depth imaging can be realized, and the transverse range of the image can also be improved compared with the prior art, which provides strong support for the research of biological science and clinical medicine.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging technology, and in particular relates to a method for super-resolution large-depth three-dimensional scanning light sheet microscope. Background Technology

[0002] In recent years, three-dimensional high-resolution imaging of biological samples has provided a wealth of information in life science and clinical medical research. Among them, light-sheet microscopy has become the main technical platform for rapid high-resolution three-dimensional fluorescence imaging of transparent large tissues due to its advantages of low photobleaching (photobleaching is the process by which fluorescent groups undergo covalent bond breakage or non-specific chemical reactions caused by light, resulting in permanent fluorescence failure. During the imaging process, the fluorescence signal continuously attenuates under illumination, leading to a decrease in signal-to-noise ratio and resolution) and high-speed imaging.

[0003] A light sheet microscope is a type of microscope based on the principle of fluorescence imaging. The excitation light is modulated into a sheet-like structure through an optical path, which only excites the fluorescent dye near the imaging plane. This avoids the signal attenuation caused by photobleaching and avoids interference from signals in non-imaging areas, thus improving the signal-to-noise ratio. It is suitable for imaging thick samples.

[0004] As research progresses, higher demands are being placed on the spatial resolution and imaging range of light-sheet microscopes. Due to spatial conflicts between the excitation objective, the detection objective, and the sample to be observed, existing light-sheet microscopes struggle to simultaneously achieve high resolution, deep-view imaging, and compatibility with multiple samples, making it difficult to meet the needs of life science and clinical medical research.

[0005] To address this core issue, in most cases, one key performance characteristic must be chosen at the expense of the other. Some light-sheet microscopes opt for low numerical aperture objectives to reduce imaging resolution in exchange for greater imaging depth; others use high numerical aperture objectives but have limited imaging depth, making it difficult to achieve both simultaneously. Summary of the Invention

[0006] The problem to be solved by the present invention is to provide a method for super-resolution large-depth three-dimensional scanning light sheet microscope.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for super-resolution large-depth three-dimensional scanning light sheet microscopy, comprising the following steps:

[0008] S1. Generation of secondary converging excitation light sheet:

[0009] The excitation laser beam is focused before the back focal plane of the excitation objective by a cylindrical lens. The focused beam formed by the excitation objective will generate a primary light plate at the focal plane of the excitation objective. At the same time, a secondary focused excitation light plate with a longer excitation distance will be generated outside the focal plane.

[0010] The secondary converging excitation plate provides more free space for the sample and the collecting objective, allowing the collecting objective to be placed at a smaller tilt angle.

[0011] S2. Optical path setup for collecting objective lens:

[0012] The collecting objective is placed at a small tilt angle and paired with an optically transparent liquid chamber with the same tilt angle, so that the excitation plane can coincide with the imaging plane of the collecting objective, thus avoiding aberrations (the deviation between the actual optical system imaging and the ideal Gaussian optical imaging; the smaller the aberration, the better the imaging effect).

[0013] S3. Sample Transparency Treatment:

[0014] The sample to be observed undergoes tissue transparency treatment to achieve optical transparency by unifying the refractive index.

[0015] S4, 3D scanning imaging execution:

[0016] By selecting an imaging buffer solution and a collecting objective lens that match the refractive index of the transparent sample, the sample is placed in the liquid chamber, the imaging buffer solution is injected, a secondary converging excitation sheet excites the sample surface, and the collecting objective lens simultaneously collects the fluorescence signal emitted from the surface. The camera and the displacement stage are controlled to operate sequentially to achieve high-resolution, deep-range three-dimensional scanning imaging of the sample.

[0017] S5. Image Data Processing:

[0018] The image data acquired in step S4 is processed to obtain a three-dimensional reconstructed image and a high-resolution single image of the sample.

[0019] Further, in step S1, the excitation distance of the secondary converging excitation plate is 20~100mm, with an optimal excitation distance of 40nm. The excitation distance depends on the distance between the back focal plane of the objective lens and the cylindrical lens.

[0020] Furthermore, in step S2, the small tilt angle is 5°~30°, with the optimal angle being 15°.

[0021] Furthermore, in step S5, the resolution of the high-resolution sample single image is 100~300nm, with the optimal resolution being 100nm.

[0022] This invention processes the excitation light through a cylindrical lens, utilizing the secondary convergence of the generated initial light plate to achieve a longer working distance for the excitation objective, thereby reducing spatial conflict between the excitation objective and the sample. Secondly, the collecting objective is placed at a small tilt angle, paired with a liquid chamber with an optical window at the same tilt angle; this configuration determines the imaging depth of the collecting objective. Finally, a collecting objective and imaging buffer solution matching the refractive index of the tissue-cleared sample are selected; this configuration avoids optical aberrations and achieves high-resolution imaging. This invention achieves both high-resolution and depth imaging, while also increasing the lateral imaging range compared to existing technologies. The applicable imaging sample range includes major research types such as tissue sections, adherent cells, and organoids. It provides strong support for research in bioscience and clinical medicine.

[0023] The specific effects of this invention are as follows:

[0024] (1) Balancing high resolution and large depth: By using a secondary converging excitation plate to alleviate the spatial conflict between the excitation objective and the sample, and by using a small tilt angle collection objective and a matching liquid chamber, the high resolution advantage of the high numerical aperture objective is retained, while the large imaging depth is achieved through a long excitation distance, thus overcoming the performance contradiction of the existing technology.

[0025] (2) Improve the effective working distance: By adjusting the tilt setting of the collection objective lens in conjunction with the liquid chamber, the effective working distance of the objective lens (the actual working distance that the objective lens can use in a light sheet microscope) is improved to meet the imaging requirements of thick samples.

[0026] (3) Expanding sample compatibility: Through refractive index matching (sample, imaging buffer solution, collection objective lens) design, it can be adapted to a variety of biological samples such as tissue sections, adherent cells, and organoids, solving the problem of narrow sample applicability of existing technologies.

[0027] (4) Ensure imaging quality: Ensure optical optimization design with a Strehl rate ≥ 0.8, and combine the inherent advantages of low light bleaching of light sheet microscope to ensure imaging signal-to-noise ratio and resolution, meeting the high requirements of scientific research and clinical imaging quality.

[0028] It is evident that this invention can greatly alleviate the spatial conflict between the objective lens and the sample to be observed in a microscope, resolve the contradiction between imaging depth and imaging resolution requirements in the prior art, and bring great convenience to achieving high-resolution imaging of large-size, thick samples that are compatible with various types of samples. Attached Figure Description

[0029] The present invention will be described in detail below with reference to the accompanying drawings and examples. The advantages and implementation methods of the present invention will become more apparent from this description. The accompanying drawings are for illustrative purposes only and do not constitute any limitation on the present invention. In the accompanying drawings:

[0030] Figure 1 This is a flowchart illustrating the present invention.

[0031] Figure 2 This is a schematic diagram of the secondary focusing excitation plate microscope of the present invention.

[0032] Figure 3 This is a top view of the beam propagation after passing through the excitation objective lens in this invention.

[0033] Figure 4 This is a front view of the beam propagation after passing through the excitation objective lens in this invention.

[0034] Figure 5 This is an intensity distribution diagram of the excitation plate obtained from actual measurements using the microscope of this invention.

[0035] Figure 6 This is an optical structure diagram of the microscope of the present invention.

[0036] Figure 7 This is an image showing the imaging effect of the present invention on a sample of fluorescent nanospheres embedded in agarose.

[0037] Figure 8 This is an image of the mouse brain nerve sample processed using dilatation microscopy, based on the present invention.

[0038] In the picture:

[0039] 1. Cylindrical lens; 2. Excitation objective; 3. Collection objective; 4. Liquid chamber; 5. Camera; 6. Optical window. Detailed Implementation

[0040] like Figures 1 to 6 As shown, a method for super-resolution, large-depth three-dimensional scanning light sheet microscopy includes the following steps:

[0041] S1. Generation of secondary converging excitation light sheet:

[0042] like Figures 2 to 5 As shown, the excitation laser beam is focused before the back focal plane of the excitation objective lens 2 by the cylindrical lens 1. The focused beam formed by the excitation objective lens will generate a primary light plate at the focal plane of the excitation objective lens 2. At the same time, a secondary focused excitation light plate with a longer excitation distance will be generated outside the focal plane.

[0043] The secondary converging excitation plate provides more free space for the sample and the collecting objective 3, allowing the collecting objective 3 to be placed at a smaller tilt angle.

[0044] S2, Optical path settings for collecting objective lens 3:

[0045] The collecting objective 3 is placed at a small tilt angle (15° from the vertical direction) and paired with an optically transparent liquid chamber 4 with the same tilt angle, so that the excitation plane can coincide with the imaging plane of the collecting objective 3, thus avoiding aberrations (the deviation between the actual optical system imaging and the ideal Gaussian optical imaging; the smaller the aberration, the better the imaging effect).

[0046] S3. Sample Transparency Treatment:

[0047] The sample to be observed undergoes tissue transparency treatment to achieve optical transparency by unifying the refractive index.

[0048] The oil immersion objective-based imaging scheme is processed using the CUBIC-X method, while the water immersion objective-based sample is processed using standard expansion microscopy techniques to obtain optically transparent imaging samples.

[0049] S4, 3D scanning imaging execution:

[0050] like Figure 6 As shown, an imaging buffer solution and a collecting objective 3 with a refractive index matching the transparent sample are selected. The sample is placed in the liquid chamber 4, and the imaging buffer solution is injected. Excitation light is introduced through the optical window 6 on the tilted side of the liquid chamber 4, and a secondary focusing excitation plate excites the sample surface. The sample is placed in a recessed sample chamber, and agarose is dropped around the sample. After it solidifies, the sample is rigidly connected to the sample chamber. The sample chamber is designed with holes, which can be fixedly connected to a mechanical device that connects to a displacement stage. The displacement of the sample can be controlled by controlling the movement of the displacement stage. The collecting objective 3 simultaneously collects the fluorescence signal emitted from this plane. The camera 5 and the displacement stage are operated sequentially by program control. After the camera 5 completes its acquisition, the displacement stage moves one fixed step. After the displacement stage completes its movement, the camera 5 acquires a new image. This cycle is repeated until the set acquisition task is completed, achieving high-resolution, deep-view 3D scanning imaging of the sample. Both the optical window 6 and the collecting objective 3 are tilted at a small angle, so that the high-resolution objective can retain most of its working distance, thus achieving a balance between high resolution and large imaging depth.

[0051] S5. Image Data Processing:

[0052] The image data acquired in step S4 is processed by scaling, Deskew, and other image data processing techniques to obtain a 3D reconstructed image and a high-resolution single image of the sample.

[0053] like Figure 7As shown, taking a water-based imaging system as an example, the photoluminescence surface area (PSF) of the microscope system was obtained after collecting and processing data from fluorescent nanospheres embedded in agarose. The PSF exhibits a typical Airy spot. The intensity distribution of the PSF along the x, y, and z directions was characterized, showing a typical Gaussian distribution. Through statistical analysis of a large number of fluorescent nanospheres, the half-width at half maximum (FWHM) of the imaging system along the x, y, and z directions were found to be 369.93±2.57 nm, 375.82±2.41 nm, and 1312.77±28.43 nm (n = 134), respectively. The experimental results are close to the theoretical PSF values, indicating that the hydrogel, imaging buffer, and water immersion objective constitute an ideal imaging system.

[0054] like Figure 8 As shown, neurons in mouse brain slices are used as a representation of the actual sample imaging effect, demonstrating consistent high-resolution visualization of neurons across the entire sample depth range. Brightly marked clusters of pyramidal cells delineate the boundaries of the cortical layer (e.g., Figure 8 As shown in a), the nerves are clearly visible at a depth of 15 μm in the slice, with numerous brightly labeled neuronal cell bodies and proximal dendrites, exhibiting high contrast and almost negligible background (as shown in a diagram). Figure 8 b and Figure 8 As shown in c), we observed a small tubular structure within the neural tissue, with a measured diameter of 101.53 nm (as shown in c). Figure 8 (As shown in g). Even at a depth of 87 μm, the fine dendritic projections remain clearly visible, demonstrating that light-section microscopy can effectively perform optical sectioning of brain tissue (as shown in g). Figure 8 (as shown in d). Even at a depth of 131 μm (as shown in d). Figure 8 As shown in e), subcellular details remain clearly visible, appearing as bright filamentous structures with almost no blurring or loss of intensity. At the distal 200 μm of the section (as shown in e...), subcellular details are still clearly visible, exhibiting bright filamentous structures with almost no blurring or loss of intensity. Figure 8 As shown in f), the imaging system still maintains excellent resolution and contrast, indicating that the system performs well in imaging actual samples, clearly distinguishing two adjacent nerve fibers with a distance of 180.81 nm between them (as shown in f). Figure 8 (as shown in g).

[0055] In the 3D reconstructed image, numerous neurons are clearly and completely visible, with their cell bodies and extensive dendritic trees intact (e.g., Figure 8 As shown in h), this indicates that the long and complex protrusions were not interrupted or lost during the capture process, allowing us to easily identify different structures within the volume (such as...). Figure 8 i to Figure 8 (as shown in k).

[0056] Example:

[0057] Equipment and reagent preparation:

[0058] Optical components: 2 is a long working distance excitation objective with a numerical aperture of 0.28 and a high-resolution collecting objective with a numerical aperture of 1.0 and a working distance of 2.0 mm; 1 is a quartz cylindrical lens with a focal length of 50 mm; 4 is made of optical glass and the tilt angle is set to 15°.

[0059] Reagents: The imaging buffer solution used was deionized water; the sample clearing reagent used standard expansion microscopy, and the sample was incubated with 0.1 mg / mL acx-PBS solution before reacting with the hydrogel solution. The monomer solution of the hydrogel was: 1×PBS, 2M NaCl, 8.625% (w / w) sodium acrylate, 2.5% (w / w) acrylamide, 0.15% (w / w) N,N′-methylenebisacrylamide (4-hydroxyTEMPO, TEMED, and APS were added before use to prepare the hydrogel). The digestion buffer was 50 mM Tris-HCl, 1M NaCl, 0.5% Triton X-100, and 0.8M guanidine hydrochloride (proteinase K was added to 8 U / mL before use).

[0060] Control equipment: A PCO SCMOS edge 5.5 USB camera (resolution 2560×2160, 6.5×6.5μm) is used; an electric displacement stage (accuracy 30nm); and a laser light source (wavelengths 488nm, 561nm and 641nm). The synchronous control of the camera 5 and the displacement stage is realized through a Python program.

[0061] A method for super-resolution, large-depth three-dimensional scanning light-sheet microscopy includes the following steps:

[0062] S1. Generation of secondary converging excitation light sheet:

[0063] When the 488nm laser source is turned on, the excitation laser beam is focused before the back focal plane of the excitation objective lens 2 by the cylindrical lens 1. The focused beam formed by the excitation objective lens 2 will generate a primary light plate at the focal plane of the excitation objective lens 2, and at a distance outside the focal plane, a secondary focused excitation light plate with an excitation thickness of 2μm will be generated. The focusing position of the secondary light plate depends on the distance between the back focal plane of the objective lens and the cylindrical lens.

[0064] The secondary converging excitation plate provides more free space for the sample and the collecting objective 3, allowing the collecting objective 3 to be placed at a smaller tilt angle.

[0065] S2, Optical path settings for collecting objective lens 3:

[0066] The collecting objective lens 3 is placed at a 15° tilt angle and paired with an optically transparent liquid chamber 4 with the same tilt angle, so that the excitation light plane can coincide with the imaging plane of the collecting objective lens 3, thus avoiding aberrations.

[0067] An imaging buffer solution is placed in the liquid chamber 4.

[0068] S3. Sample Transparency Treatment:

[0069] Mouse brain tissue sections (0.2 mm thick) were used as samples and cleared using Scale reagent: the tissue sections were immersed in 0.1 mg / mL PBS-acx solution overnight at room temperature, followed by washing twice with different PBS solutions. The samples were then incubated in freshly prepared 4°C hydrogel solution for 1 h, followed by incubation at 37°C for 3 h. After gelation, the hydrogel containing the sample was cut off with a blade and placed in a digestion solution at 37°C for 8 h. After digestion, the samples were transferred to imaging buffer solution (10 mM Tris-HCl solution), with the solution changed 5 times until the tissue was completely clear (refractive index 1.333).

[0070] S4, 3D scanning imaging execution:

[0071] An imaging buffer solution and a collecting objective 3 with a refractive index matching the transparent sample are selected. The sample is placed in a liquid chamber 4 containing the imaging buffer solution, and a hydrogel imaging buffer solution with a refractive index of 1.333 is injected. The laser source is activated, and a secondary focusing excitation plate excites the sample surface. The collecting objective 3 simultaneously collects the fluorescence signal emitted from this surface. The camera 5 and the displacement stage are moved along the z-axis (step size 0.35 μm) by program control. The camera 5 synchronously acquires the fluorescence signal of each layer, acquiring a total of 1000 layers of images, covering an imaging depth of 0.35 mm, to achieve high-resolution, deep-field three-dimensional scanning imaging of the sample.

[0072] S5. Image Data Processing:

[0073] The 1000-layer image was processed using ImageJ software: first, Gaussian filtering was used to eliminate noise; then, the "TransformJ affine" plugin was used to restore the image to the scaling of the real sample space; finally, the "3D Viewer" plugin was used to perform three-dimensional reconstruction to generate a three-dimensional structural image of mouse brain tissue; at the same time, high-resolution single images of each layer (resolution up to 200nm) were output for microstructure analysis.

[0074] Implementation effect verification:

[0075] The imaging performance of the microscope system was first verified by imaging fluorescent nanospheres embedded in agarose. After the above image acquisition and data processing operations, a three-dimensional reconstructed image of the fluorescent nanospheres can be obtained. By analyzing the intensity distribution of the fluorescent microspheres in the image, the point spread function (PSF) of the system can be obtained. For the 60× objective lens, the experimentally measured full width at half maximum (FWHM) values ​​of the PSF along the three principal axes are 369.93±2.57nm, 375.82±2.41nm, and 1312.77±28.43nm (n=134), respectively; for the 40× objective lens, the values ​​are 514.53±4.60nm, 496.60±3.86nm, and 2537.37±45.58nm (n=158), respectively. These values ​​are close to the theoretical values, and the standard deviation is about 2%, indicating that the imaging system has excellent performance and good system stability. The three-dimensional reconstructed image obtained in this embodiment can clearly observe the neuronal distribution of mouse brain tissue (high resolution), and the imaging depth covers a complete 0.2mm tissue section (large depth).

[0076] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for super-resolution, large-depth three-dimensional scanning light-sheet microscopy, characterized in that: Includes the following steps: S1. Generation of secondary converging excitation light sheet: The excitation laser beam is focused before the back focal plane of the excitation objective by the action of the cylindrical lens. The focused beam formed by the excitation objective will generate a primary light plate at the focal plane of the excitation objective, and at the same time, a secondary focused excitation light plate will be generated outside the focal plane. S2. Optical path setup for collecting objective lens: The collecting objective is placed at a small tilt angle and paired with an optically transparent liquid chamber with the same tilt angle, so that the excitation plane can coincide with the imaging plane of the collecting objective. S3. Sample Transparency Treatment: The sample to be observed undergoes tissue transparency treatment to achieve optical transparency by unifying the refractive index; S4, 3D scanning imaging execution: By selecting an imaging buffer solution and a collecting objective that match the refractive index of the transparent sample, the sample is placed in the liquid chamber and the imaging buffer solution is injected. A secondary converging excitation sheet excites the sample surface, and the collecting objective simultaneously collects the fluorescence signal emitted from the sample plane. The camera and the displacement stage are controlled to achieve high-resolution, deep three-dimensional scanning imaging of the sample. S5. Image Data Processing: The image data acquired in step S4 is processed to obtain a three-dimensional reconstructed image and a high-resolution single image of the sample. In step S1, the excitation distance of the secondary converging excitation plate is 20~100mm; In step S2, the small tilt angle is 5°~30°; In step S5, the resolution of the high-resolution sample single image is 100~300nm.

Images