Intrahepatic blood vessel three-dimensional imaging method based on non-diffracting Airy beam

Abstract

The invention discloses an intrahepatic blood vessel three-dimensional imaging method based on a non-diffracting Airy beam, belongs to the technical field of intrahepatic blood vessel three-dimensional imaging, and solves the problem that an existing detection method is insufficient in accuracy and detection sensibility. The method comprises the steps that liver tissue is obtained through liver transplantation or biopsy operation, the liver tissue is subjected to fixing treatment through 4% PFA, and the detection sensitivity is high. The method comprises the following steps: bleaching liver tissues by adopting an H2O2 solution, performing antigen repair on the liver tissues by adopting a FLASH solution, and performing closing treatment on the liver tissues subjected to antigen repair by utilizing goat serum closing liquid; the method comprises the following steps: performing immunostaining treatment on liver tissues, performing dehydration treatment on the liver tissues, and shooting and imaging the treated liver tissues based on a non-diffracting Airy beam and a high-flux microscope to obtain a three-dimensional imaging picture of the liver tissues; according to the invention, the liver tissue is subjected to immunofluorescence staining and transparentizing treatment, so that laser can smoothly penetrate through the tissue to carry out three-dimensional imaging, and single-time large-range image capture can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of three-dimensional imaging of intrahepatic blood vessels, and particularly relates to a method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams. Background Art

[0002] Liver cancer is a malignant tumor with insidious onset and no obvious early symptoms. Most patients are in the middle and late stages at the time of diagnosis. If early diagnosis can be achieved through advanced imaging techniques, patients will have a greater chance of receiving effective treatment, thus significantly improving the survival rate. Currently, there are deficiencies in the accuracy and detection sensitivity of imaging diagnosis for liver diseases. Especially in the diagnosis of early liver diseases, it is often difficult to accurately identify lesions. Commonly used imaging examination methods include ultrasound, CT, and MRI, etc. Although ultrasound examination is non-invasive and has low cost, its resolution is limited, and it is difficult to provide clear images for small liver disease lesions. Although CT and MRI have higher resolution, there are still certain limitations, especially in differentiating early liver diseases from normal tissues.

[0003] Liver biopsy is considered the gold standard for the diagnosis of liver diseases and the determination of the nature of liver nodules. Through liver biopsy, doctors can take a small tissue sample from the liver and then conduct detailed pathological analysis under a microscope. However, there are also some problems with liver biopsy. First, it is an invasive operation and may cause certain pain and risks to patients. Second, histological staining techniques can only provide limited planar image information, and different pathologists may draw different diagnostic results when interpreting these images. This subjectivity increases the uncertainty of diagnosis, especially in complex cases, which may lead to misdiagnosis or missed diagnosis.

[0004] Therefore, there is an urgent need for a technology that can quickly and with high resolution perform three-dimensional imaging of liver tissue to improve the accuracy and detection sensitivity of three-dimensional imaging of intrahepatic blood vessels. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams in view of the deficiencies of the prior art, and solve the problems of insufficient accuracy and detection sensitivity of existing detection methods.

[0006] The present invention is implemented as follows. A method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams, the method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams includes:

[0007] S10, obtaining liver tissue through liver transplantation or biopsy surgery, fixing the liver tissue with 4% PFA, trimming the size of the liver tissue to 2×2×5 mm 3 , and washing the liver tissue with PBS;

[0008] S20, the liver tissue is bleached with H 2 O 2 solution and washed with PBS;

[0009] S30, the liver tissue is subjected to antigen retrieval with FLASH solution. After the retrieval is completed, it is washed with PBT solution;

[0010] S40, the liver tissue after antigen retrieval is blocked with goat serum blocking solution;

[0011] S50, the liver tissue is subjected to immunostaining. During immunostaining, the liver tissue is incubated with the primary antibody at 10 °C, and after washing, it is incubated with the secondary antibody under the same conditions. The primary antibody is the vascular marker CD31, and the secondary antibody is the anti-rabbit IgG H&L antibody conjugated with gold cluster fluorescence label;

[0012] S60, the liver tissue is dehydrated. The liver tissue is dehydrated with gradient concentration of tetrahydrofuran solution and defatted with dichloromethane to make the sample close to transparent, and the refractive index is matched with dibenzyl ether;

[0013] S70, imaging of the processed liver tissue is taken based on non-diffracting Airy beam and high-throughput microscope to obtain three-dimensional imaging pictures of the liver tissue.

[0014] When the liver tissue is fixed with 4% PFA, the liver tissue obtained by surgery is placed in 4% formalin solution for 24 hours. After fixation is completed, the tissue is trimmed into a size of 2×2×5 mm 3 and immersed in PBS solution, and washed three times on a shaker, each time for 30 minutes.

[0015] When the liver tissue is bleached with H 2 O 2 solution, the washed tissue is put into a solution prepared by mixing hydrogen peroxide, dimethyl sulfoxide and PBS in a ratio of 2:1:4 for bleaching. After bleaching, it is washed with PBS three times again, each time for 30 minutes.

[0016] When the liver tissue is subjected to antigen retrieval with FLASH solution, the washed liver tissue is first soaked in FLASH antigen retrieval solution at room temperature for 1 hour, then processed on a shaker at 45 °C for 6 hours, and then allowed to return to room temperature. The FLASH antigen retrieval solution consists of 20 mM boric acid and 4% SDS, and the pH is adjusted to 7.0 with NaOH. PBT consists of PBS and Triton.

[0017] When using goat serum blocking solution to block the liver tissue after antigen repair, soak the liver tissue in the goat serum blocking solution at room temperature for 8 hours. The goat serum blocking solution consists of 10% fetal bovine serum, 1% goat albumin, 5% dimethyl sulfoxide, and 0.5% PC-300, and is filtered through a 0.22-micron filter.

[0018] When performing immunostaining on the liver tissue, use rabbit anti-human CD31 antibody as the primary antibody, and the dilution ratio of the primary antibody is 1:100 or 1:200. The secondary antibody is goat anti-rabbit IgG H&L, conjugated with gold cluster fluorophore, and the dilution ratio is 1:200. Both the primary antibody and the secondary antibody are diluted with the newly prepared blocking solution and incubated on a shaker at 10°C for 3 - 4 days. After incubation, wash the specimens with PBST.

[0019] The method for dehydrating the liver tissue includes:

[0020] S601, dehydrate the liver tissue through the concentration gradient of tetrahydrofuran, soak and dehydrate for 1.5 hours each time, and the concentration gradients of tetrahydrofuran are 50%, 70%, 80%, and 100% tetrahydrofuran solutions;

[0021] S602, soak the liver tissue in 100% dichloromethane solution for 1.5 hours to degrease;

[0022] S603, soak the tissue in 100% dibenzyl ether solution for 1.5 hours, and the storage temperature is 4°C.

[0023] The method for imaging the processed liver tissue based on non-diffracting Airy beam and high-throughput microscope specifically includes:

[0024] S701, start the femtosecond pulsed laser and set the single-photon excitation light with a wavelength of 730 nm;

[0025] S702, replace the filter in front of the camera with a 750&800LP model filter;

[0026] S703, load the phase information through the spatial light modulator and modulate the phase information into Gaussian beam and Airy beam respectively;

[0027] S704, control the displacement stage to immerse the illumination objective and detection objective of the imaging component into the medium matching the refractive index of the cleared sample;

[0028] S705, load the gold cluster agarose solution into an FEP tube and place it in the imaging area. Adjust its position by rotating the micrometer head of the detection objective until a clear and complete image is obtained in the camera to complete the focusing process;

[0029] After focusing is completed at S706, the sample is placed in a refractive index matching medium. By switching the phase, the illumination beam is transformed from a Gaussian beam into an Airy beam for actual imaging work;

[0030] After determining the image range at S707, an image stack is acquired to obtain at least one set of image slices arranged along the Z-axis direction;

[0031] At S708, the image slices are deconvolved by combining the point spread function (PSF) pre-calibrated by the system to restore the axial resolution of the image and obtain clearer three-dimensional structure information. The processed image data is imported into image analysis software such as Fiji - image J and Imaris, and the two-dimensional image slices are integrated into a complete three-dimensional model to obtain a three-dimensional imaging picture of the liver tissue.

[0032] Compared with the prior art, the embodiments of the present application mainly have the following beneficial effects:

[0033] The present invention is mainly applicable to the imaging of intrahepatic blood vessels. By performing immunofluorescent staining and clearing treatment on liver tissue, the laser can penetrate the tissue smoothly for three-dimensional imaging, enabling single-shot large-range image capture, meeting the higher requirements for imaging in modern biology, and facilitating the pathological analysis and physiological mechanism exploration of liver disease models. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shows the imaging optical path diagram of the processed liver tissue captured by a high-throughput microscope.

[0035] Figure 2 Shows a schematic diagram of the fixed liver tissue specimen in Example 1.

[0036] Figure 3 Shows a schematic diagram of the cleared liver tissue specimen.

[0037] Figure 4 Shows the three-dimensional imaging picture of the liver tissue in Example 1.

[0038] Figure 5 Shows the cross-sectional imaging picture of the liver tissue in Example 1.

[0039] Figure 6 Shows the three-dimensional imaging picture of the liver tissue in Example 2.

[0040] Figure 7 Shows the cross-sectional imaging picture of the liver tissue in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0042] When the prior art uses ultrasound, CT, and MRI for imaging diagnosis and detection of liver diseases, there are deficiencies in accuracy and detection sensitivity. To address the above problems, we propose a three-dimensional imaging method for intrahepatic blood vessels based on non-diffracting Airy beams. Briefly, when implementing the method, the liver tissue is first fixed with 4% PFA, and the liver tissue is bleached with H 2 O 2 solution, antigen repair of the liver tissue is performed with FLASH solution, and then the liver tissue after antigen repair is blocked with goat serum blocking solution; the liver tissue is immunostained, the liver tissue is dehydrated, and finally, based on non-diffracting Airy beams and a high-throughput microscope, the processed liver tissue is imaged to obtain a three-dimensional imaging picture of the liver tissue; the present invention is mainly applicable to the imaging of intrahepatic blood vessels. By performing immunofluorescence staining and clearing treatment on the liver tissue, the laser can penetrate the tissue smoothly for three-dimensional imaging, enabling single-shot large-range image capture, meeting the higher requirements for imaging in modern biology, facilitating the pathological analysis and physiological mechanism exploration of liver disease models, and overcoming the deficiencies in accuracy and detection sensitivity when the prior art uses ultrasound, CT, and MRI for imaging diagnosis and detection of liver diseases.

[0043] Before implementing Examples 1-2, liver tissues were obtained through liver transplantation or biopsy surgery. In the present invention, there are two groups of liver tissues, which are respectively used for three-dimensional imaging in Example 1 and Example 2. The present invention mainly includes the following aspects: improving the immunofluorescence experimental method, selecting appropriate antibodies and fluorescent dyes: selecting antibodies with high specificity and strong affinity to identify specific markers of intrahepatic blood vessels, such as the vascular endothelial cell marker CD31. At the same time, selecting fluorescent dyes with high brightness and good stability, such as gold nanoclusters, to ensure good fluorescence signals in the cleared tissues; in addition, it also includes optimizing the staining steps: during the staining process, the tissues need to be properly fixed, permeated and blocked to improve the permeability and specific binding of antibodies, which helps to ensure the accuracy and consistency of fluorescence labeling; exploring appropriate clearing methods for liver tissues to keep the liver tissues with fluorescence signals and achieve the clearing effect; debugging the light-sheet fluorescence microscope and selecting appropriate filter plates for imaging. This method is based on a spatial light modulator and uses non-diffracting Airy beams to excite fluorescent groups. This imaging system has significant advantages in the three-dimensional analysis of liver diseases. Compared with traditional two-dimensional imaging techniques, three-dimensional imaging techniques can provide more abundant structural information to help doctors more accurately identify and analyze lesions in liver tissues. In addition, three-dimensional imaging techniques can also be combined with other diagnostic means to improve the comprehensive ability of liver disease diagnosis. For example, by combining with hematological marker detection, multi-dimensional analysis of liver diseases can be achieved. Hematological markers such as alpha-fetoprotein (AFP) are commonly used diagnostic indicators for liver cancer, but their sensitivity and specificity are limited. Combining three-dimensional imaging techniques can provide more detailed lesion information in imaging, thus making up for the deficiencies of hematological marker detection.

[0044] Example 1

[0045] An embodiment of the present invention provides a method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams includes:

[0046] S10, obtaining liver tissues through liver transplantation or biopsy surgery, fixing the liver tissues with 4% PFA, trimming the liver tissues to a size of 2×2×5 mm 3 , and washing the liver tissues with PBS;

[0047] Among them, when the liver tissues are fixed with 4% PFA, the liver tissues obtained by surgery are placed in a 4% formalin solution for 24 hours. After fixation, the tissues are trimmed to a size of 2×2×5 mm 3 , soaked in PBS solution, and washed three times on a shaker, each time lasting 30 minutes. Figure 2 Figure 18 shows a schematic diagram of the fixed liver tissue specimen in Example 1.

[0048] S20, the liver tissue is bleached with H 2 O 2 solution and washed with PBS;

[0049] It should be noted that when the liver tissue is bleached with H 2 O 2 solution, the washed tissue is placed in a solution prepared by mixing hydrogen peroxide, dimethyl sulfoxide and PBS in a ratio of 2:1:4 for bleaching. After bleaching, it is washed with PBS three times again, 30 minutes each time.

[0050] S30, the liver tissue is subjected to antigen retrieval with FLASH solution. After the retrieval is completed, it is washed with PBT solution;

[0051] When the liver tissue is subjected to antigen retrieval with FLASH solution, the washed liver tissue is first soaked in the FLASH antigen retrieval solution at room temperature for 1 hour, then processed on a shaker at 45 °C for 6 hours, and then allowed to return to room temperature. Among them, the FLASH antigen retrieval solution consists of 20 mM boric acid and 4% SDS, and the pH is adjusted to 7.0 with NaOH. PBT consists of PBS and Triton.

[0052] S40, the liver tissue after antigen retrieval is blocked with goat serum blocking solution;

[0053] When the liver tissue after antigen retrieval is blocked with goat serum blocking solution, the liver tissue is soaked in the goat serum blocking solution at room temperature for 8 hours. The goat serum blocking solution consists of 10% fetal bovine serum, 1% goat albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered through a 0.22 μm filter.

[0054] S50, the liver tissue is subjected to immunostaining. Among them, during immunostaining, the liver tissue is incubated with the primary antibody at 10 °C, and after washing, it is incubated with the secondary antibody under the same conditions. Among them, the primary antibody is the vascular marker CD31, and the secondary antibody is the anti-rabbit IgG H&L antibody conjugated with gold cluster fluorescence label;

[0055] In this embodiment, when the liver tissue is subjected to immunostaining, the primary antibody used is rabbit anti-human CD31 antibody, the dilution ratio of the primary antibody is 1:100 or 1:200, the secondary antibody is goat anti-rabbit IgG H&L, conjugated with a gold cluster fluorophore, and the dilution ratio is 1:200. Both the primary antibody and the secondary antibody are diluted with freshly prepared blocking solution and incubated on a shaker at 10 °C for 3 - 4 days. After incubation, the specimens are washed with PBST. It should be noted that the secondary antibody incubation process and subsequent operations need to be carried out under light-proof conditions.

[0056] S60. Dehydration treatment of liver tissue. The liver tissue is dehydrated using a gradient concentration of tetrahydrofuran solution, defatted with dichloromethane to make the sample nearly transparent, and the refractive index is matched using dibenzyl ether. Figure 3 Shows a schematic diagram of the liver tissue specimen after clearing.

[0057] Among them, the method for dehydrating the liver tissue includes:

[0058] S601. Dehydrate the liver tissue through the concentration gradient of tetrahydrofuran. Soak and dehydrate for 1.5 hours each time. The concentration gradients of tetrahydrofuran are 50%, 70%, 80% and 100% tetrahydrofuran solutions.

[0059] S602. Immerse the liver tissue in 100% dichloromethane solution for 1.5 hours to defat.

[0060] S603. Immerse the tissue in 100% dibenzyl ether solution for 1.5 hours, and the storage temperature is 4°C.

[0061] It should be noted that the liver tissue sample can be stored in dibenzyl ether at 4°C, and the imaging effect is the best within 3 days. During the process of transferring from dichloromethane solution to dibenzyl ether solution, the operation should be rapid to prevent the sample from matching the refractive index of air, thereby affecting the imaging quality.

[0062] S70. Based on the non-diffracting Airy beam and high-throughput microscope, image the processed liver tissue to obtain a three-dimensional imaging picture of the liver tissue.

[0063] In this embodiment, a method for imaging the processed liver tissue based on the non-diffracting Airy beam and high-throughput microscope is provided. Figure 1 Shows the imaging optical path diagram of the processed liver tissue by the high-throughput microscope. The method for imaging the processed liver tissue based on the non-diffracting Airy beam and high-throughput microscope specifically includes:

[0064] S701. Start the femtosecond pulsed laser and set the single-photon excitation light with a wavelength of 730 nm.

[0065] S702. Replace the filter in front of the camera with a 750&800LP model filter.

[0066] S703. Load the phase information through the spatial light modulator and modulate the phase information into Gaussian beam and Airy beam respectively.

[0067] S704. Control the displacement stage to immerse the illumination objective lens and detection objective lens of the imaging component into the medium that matches the refractive index of the sample after clearing.

[0068] S705. Place the gold cluster agarose solution in an FEP tube and position it within the imaging area. Adjust its position by rotating the micrometer head of the detection objective until a clear and complete image is obtained in the camera to complete the focusing process.

[0069] S706. After focusing is completed, place the sample into the refractive index matching medium. By switching the phase, the illumination beam is transformed from a Gaussian beam to an Airy beam for actual imaging work.

[0070] S707. After determining the image range, acquire an image stack to obtain at least one set of image slices arranged along the Z-axis direction.

[0071] S708. Perform deconvolution processing on the image slices in combination with the point spread function (PSF) pre-calibrated by the system. Due to the limitations of the optical system, there may be certain blurring in the axial direction of these original image data. Therefore, it is necessary to perform deconvolution processing in combination with the pre-calibrated point spread function (PSF) of the system to restore the axial resolution of the image and obtain clearer three-dimensional structure information. Import the processed image data into the image analysis software Fiji-image J and Imaris, and integrate the two-dimensional image slices into a complete three-dimensional model to visually display the microscopic structure inside the sample and obtain the three-dimensional imaging picture of the liver tissue. Figure 4 shows the three-dimensional imaging picture of the liver tissue in Example 1, while Figure 5 shows the cross-sectional imaging picture of the liver tissue in Example 1.

[0072] The present invention focuses on the three-dimensional imaging of intrahepatic blood vessels and improves the immunofluorescence staining technique to label vascular endothelial cells. This process involves combining a fluorescent dye with vascular endothelial cells and then performing tissue clearing so that the laser can penetrate the entire tissue for imaging. This technique can achieve three-dimensional imaging of blood vessels in a relatively large liver tissue sample, thereby providing more intuitive and detailed vascular structure information for the research and diagnosis of liver diseases.

[0073] It should be emphasized that the present invention is not directly used for the diagnosis or treatment of diseases, but provides a method for three-dimensional imaging of intrahepatic blood vessels. This method performs a series of treatments on liver tissue (such as fixation, bleaching, immunostaining, etc.) and uses non-diffracting Airy beams and high-throughput microscopes for imaging to finally obtain the three-dimensional structure information of the liver tissue, which meets the requirements of the patent protection object and does not belong to a disease diagnosis method. It is an innovative and practical imaging technique that can be widely applied in multiple fields.

[0074] The present invention is mainly applicable to liver blood vessels. By performing immunofluorescence staining and clearing treatment on liver tissues, laser can smoothly penetrate the tissues for bile duct imaging. On the other hand, all reagents of the present invention can be commercially obtained and the operation is simple. On the other hand, there is currently a lack of effective means for clinical three-dimensional fine structure pathological diagnosis of liver diseases. The method provided by the present invention can provide a methodological basis for clinical diagnosis of liver diseases.

[0075] Example 2

[0076] An embodiment of the present invention provides a three-dimensional imaging method for intrahepatic blood vessels based on non-diffracting Airy beams. The three-dimensional imaging method for intrahepatic blood vessels based on non-diffracting Airy beams includes:

[0077] S10, obtaining liver tissues through liver transplantation or biopsy surgery, fixing the liver tissues with 4% PFA, trimming the size of the liver tissues to 2×2×5 mm 3 , and washing the liver tissues with PBS;

[0078] Among them, when the liver tissues are fixed with 4% PFA, the liver tissues obtained by surgery are placed in a 4% formalin solution for 24 hours. After fixation, the tissues are trimmed into a size of 2×2×5 mm 3 , and immersed in PBS solution, and washed three times on a shaker, each time for 30 minutes.

[0079] S20, bleaching the liver tissues with H 2 O 2 solution and washing with PBS;

[0080] It should be noted that when the liver tissues are bleached with H 2 O 2 solution, the washed tissues are put into a solution prepared by mixing hydrogen peroxide, dimethyl sulfoxide and PBS in a ratio of 2:1:4 for bleaching. After bleaching, they are washed with PBS three times again, each time for 30 minutes.

[0081] S30, performing antigen repair on the liver tissues with FLASH solution, and washing with PBT solution after the repair;

[0082] When performing antigen repair on the liver tissues with FLASH solution, the washed liver tissues are first immersed in the FLASH antigen repair solution at room temperature for 1 hour, then continue to be treated on a shaker at 45°C for 6 hours, and then the liver tissues are allowed to return to room temperature. Among them, the FLASH antigen repair solution is composed of 20 mM boric acid and 4% SDS, and the pH is adjusted to 7.0 with NaOH. PBT is composed of PBS and Triton.

[0083] S40, performing blocking treatment on the liver tissues after antigen repair with goat serum blocking solution;

[0084] When using goat serum blocking solution to block the liver tissue after antigen repair, soak the liver tissue in the goat serum blocking solution at room temperature for 8 hours. The goat serum blocking solution is composed of 10% fetal bovine serum, 1% goat albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered through a 0.22-micron filter.

[0085] S50, perform immunostaining on the liver tissue. Among them, during immunostaining, the liver tissue is incubated with the primary antibody at 10°C, and after washing, it is incubated with the secondary antibody under the same conditions. Among them, the primary antibody is the vascular marker CD31, and the secondary antibody is the anti-rabbit IgG H&L antibody conjugated with gold cluster fluorescence labeling;

[0086] In this embodiment, when performing immunostaining on the liver tissue, the primary antibody uses rabbit anti-human CD31 antibody, and the dilution ratio of the primary antibody is 1:100 or 1:200. The secondary antibody is goat anti-rabbit IgG H&L, conjugated with a gold cluster fluorophore, and the dilution ratio is 1:200. Both the primary antibody and the secondary antibody are diluted with freshly prepared blocking solution and incubated on a shaker at 10°C for 3 - 4 days. After incubation, wash the specimens with PBST.

[0087] S60, dehydrate the liver tissue. The liver tissue is dehydrated with a gradient concentration of tetrahydrofuran solution, defatted with dichloromethane to make the sample close to transparent, and the refractive index is matched with dibenzyl ether;

[0088] Among them, the method for dehydrating the liver tissue includes:

[0089] S601, dehydrate the liver tissue through the concentration gradient of tetrahydrofuran. Soak and dehydrate each time for 1.5 hours. The concentration gradients of tetrahydrofuran are 50%, 70%, 80% and 100% tetrahydrofuran solutions;

[0090] S602, soak the liver tissue in 100% dichloromethane solution for 1.5 hours to defat;

[0091] S603, soak the tissue in 100% dibenzyl ether solution for 1.5 hours, and the storage temperature is 4°C.

[0092] S70, based on the non-diffracting Airy beam and the high-throughput microscope, take images of the processed liver tissue to obtain three-dimensional imaging pictures of the liver tissue.

[0093] In this embodiment, a method for taking images of the processed liver tissue based on the non-diffracting Airy beam and the high-throughput microscope is provided, Figure 6 Show the three-dimensional imaging pictures of the liver tissue in Example 2. Figure 7The cross-sectional imaging picture of the liver tissue in Example 2 is shown. The method for imaging the processed liver tissue based on the non-diffracting Airy beam and the high-throughput microscope specifically includes:

[0094] S701, Start the femtosecond pulsed laser and set the single-photon excitation light with a wavelength of 730 nm;

[0095] S702, Replace the filter at the front end of the camera with a 750&800LP model filter;

[0096] S703, Load the phase information through the spatial light modulator and modulate the phase information into Gaussian beam and Airy beam respectively.

[0097] S704, Control the displacement stage to immerse the illumination objective lens and the detection objective lens of the imaging assembly into the medium that matches the refractive index of the sample after clearing treatment;

[0098] S705, Load the gold cluster agarose solution into the FEP tube and place it in the imaging area. Adjust its position by rotating the micrometer head of the detection objective lens until a clear and complete image is obtained in the camera to complete the focusing process;

[0099] S706, After focusing is completed, place the sample into the refractive index matching medium. By switching the phase, the illumination beam changes from Gaussian beam to Airy beam for actual imaging work;

[0100] S707, After determining the image range, obtain the image stack to get at least one set of image slices arranged along the Z-axis direction;

[0101] S708, Perform deconvolution processing on the image slices in combination with the point spread function PSF pre-calibrated by the system to restore the axial resolution of the image, obtain clearer three-dimensional structure information, import the processed image data into the image analysis software Fiji-image J and imaris, integrate the two-dimensional image slices into a complete three-dimensional model, and obtain the three-dimensional imaging picture of the liver tissue.

[0102] In the present invention, by combining the high-throughput microscope and the automated focusing technology, the imaging process can be quickly completed, the image slices arranged along the Z-axis direction can be obtained, and integrated into a complete three-dimensional model. Compared with the traditional imaging method, this technology can significantly shorten the imaging time, improve the work efficiency, is applicable to the analysis of large-scale samples, uses the non-diffracting Airy beam and the spatial light modulator to load the phase information, realizes the switching from Gaussian beam to Airy beam, and provides a new optical means for imaging. The self-accelerating and non-diffracting characteristics of the Airy beam make it have unique advantages in three-dimensional imaging and can provide more stable imaging effects.

[0103] In summary, the present invention provides a three-dimensional imaging method for intrahepatic blood vessels based on non-diffracting Airy beams. The present invention is mainly applicable to the imaging of intrahepatic blood vessels. By performing immunofluorescence staining and clearing treatment on liver tissues, laser can penetrate the tissues smoothly for three-dimensional imaging, enabling single-shot large-range image capture, meeting the higher requirements for imaging in modern biology, and facilitating the pathological analysis and physiological mechanism exploration of liver disease models.

[0104] It should be noted that for the foregoing embodiments, for the sake of simple description, they are all expressed as a series of action combinations. However, those skilled in the art should know that the present invention is not limited by the described action sequence, because according to the present invention, certain steps may be performed in other sequences or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the protection scope of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still, without conflict, make combinations, additions, deletions, or other adjustments to the features in the embodiments of the present invention according to the situation without creative efforts, so as to obtain different technical solutions that essentially do not deviate from the concept of the present invention, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams, characterized in that: The intrahepatic vascular three-dimensional imaging method based on non-diffraction Airy beam comprises: S10, liver tissue was obtained by liver transplantation or biopsy, liver tissue was fixed with 4% PFA, and the size of liver tissue was trimmed to 2×2×5 mm 3 , liver tissue was washed with PBS; S20, liver tissue was bleached with H2O2 solution and washed with PBS; S30, antigen repair of liver tissue was performed using FLASH solution, and after repair, it was washed with PBT solution; S40, blocking treatment of liver tissue after antigen retrieval using goat serum blocking solution; S50, immunostaining of liver tissue, wherein during immunostaining, the liver tissue was incubated with a primary antibody at 10°C, washed, and incubated with a secondary antibody under the same conditions, wherein the primary antibody was a vascular marker CD31, and the secondary antibody was an anti-rabbit IgG H&L antibody linked to a gold cluster fluorescent marker; S60, liver tissue dehydration treatment, liver tissue was dehydrated using gradient concentration tetrahydrofuran solution, defatted with dichloromethane to make the sample nearly transparent, and dibenzyl ether was used to match the refractive index; S70, based on the diffraction-free Airy beam and high-throughput microscopy, the processed liver tissue is imaged to obtain a three-dimensional imaging picture of the liver tissue.

2. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams according to claim 1, characterized in that: When the liver tissue is fixed with 4% PFA, the liver tissue obtained by surgery is placed in a 4% formalin solution for 24 hours. After fixation, the tissue is trimmed into 2×2×5mm 3 The size of the cells was determined and they were immersed in PBS solution and washed three times on a shaker, each time for 30 min.

3. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams as claimed in claim 2, characterized in that: When liver tissue was bleached with H2O2 solution, the washed tissue was placed in a solution prepared with hydrogen peroxide, dimethyl sulfoxide and PBS in a ratio of 2:1:4 for bleaching. After bleaching, the tissue was washed again with PBS three times, each time for 30 minutes.

4. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams according to claim 2, characterized in that: When using FLASH solution to perform antigen repair on liver tissue, the washed liver tissue is first immersed in FLASH antigen repair solution at room temperature for 1 hour, and then continued to be treated on a shaker at 45°C for 6 hours, and then the liver tissue is allowed to return to room temperature. The FLASH antigen repair solution is composed of 20mM boric acid, 4% SDS, and the pH is adjusted to 7.0 with NaOH. PBT is composed of PBS and Triton.

5. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams as claimed in claim 4, characterized in that: When the liver tissue after antigen repair is blocked with goat serum blocking solution, the liver tissue is immersed in goat serum blocking solution at room temperature for 8 hours. The goat serum blocking solution consists of 10% fetal bovine serum, 1% goat albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered through a 0.22 micron filter.

6. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams according to claim 1, characterized in that: When the liver tissue was immunostained, the primary antibody used was rabbit anti-human CD31 antibody, the primary antibody dilution ratio was 1:100 or 1:200, the secondary antibody was goat anti-rabbit IgG H&L, which was connected to a gold cluster fluorescent group, and the dilution ratio was 1:

200. Both the primary antibody and the secondary antibody were diluted with a freshly prepared blocking solution and incubated on a shaker at 10°C for 3-4 days. After incubation, the specimen was washed with PBST.

7. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams according to claim 6, characterized in that: The method for dehydrating liver tissue comprises: S601, dehydrating the liver tissue by tetrahydrofuran concentration gradient, each immersion dehydration for 1.5 hours, the tetrahydrofuran concentration gradient is 50%, 70%, 80% and 100% tetrahydrofuran solution respectively; S602, immerse the liver tissue in 100% dichloromethane solution for 1.5 h to defat; S603, immerse the tissue in 100% benzyl ether solution for 1.5 hours and store at 4°C.

8. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams according to claim 1, characterized in that: The method for imaging the treated liver tissue based on a non-diffraction Airy beam and a high-throughput microscope specifically comprises: S701, start the femtosecond pulse laser and set the single-photon excitation light with a wavelength of 730nm; S702, use 750&800LP filter to replace the filter on the front end of the camera; S703, loading phase information through a spatial light modulator, and modulating the phase information into a Gaussian beam and an Airy beam respectively.

9. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffraction Airy beams as claimed in claim 8, characterized in that: The method for imaging the treated liver tissue based on a non-diffraction Airy beam and a high-throughput microscope specifically includes: S704, controlling the translation stage so that the illumination objective lens and the detection objective lens of the imaging component are immersed in a medium that matches the refractive index of the sample after the transparency treatment; S705, placing the gold cluster agarose solution in an FEP tube in the imaging area, and adjusting the position of the detection objective lens by rotating the screw micrometer head until a clear and complete image is obtained in the camera, thus completing the focusing process; S706, after focusing is completed, the sample is placed in a refractive index matching medium, and the illumination beam is transformed from a Gaussian beam to an Airy beam by switching the phase, so as to perform actual imaging work; S707, after determining the image range, acquiring an image stack to obtain at least one set of image slices arranged along the Z-axis direction; S708, deconvolution processing is performed on the image slices in combination with the point spread function PSF pre-calibrated by the system to restore the axial resolution of the image and obtain clearer three-dimensional structural information. The processed image data is imported into the image analysis software Fiji-image J and Imaris, and the two-dimensional image slices are integrated into a complete three-dimensional model to obtain a three-dimensional imaging picture of the liver tissue.

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