A method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting airy beam

Abstract

The application discloses a kind of based on non-diffracting airy beam intrahepatic blood vessel three-dimensional imaging method, belong to intrahepatic blood vessel three-dimensional imaging technical field, solve the problem of insufficient accuracy and detection sensitivity of existing detection method, method includes by liver transplantation or biopsy surgery obtains liver tissue, liver tissue is fixed using 4%PFA processing, liver tissue is bleached using H2O2Solution, antigen repair is carried out to liver tissue using FLASH solution, using goat serum blocking solution to the liver tissue after antigen repair blocking treatment;Liver tissue immunostaining treatment, liver tissue dehydration treatment, based on non-diffracting airy beam and high-throughput microscope to the liver tissue after processing imaging, obtain the three-dimensional imaging picture of liver tissue;The application is immunofluorescence staining and transparent treatment to liver tissue, so that laser can smoothly penetrate tissue and carry out three-dimensional imaging, can realize single wide-range image capture.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of three-dimensional imaging of intrahepatic blood vessels, and particularly relates to a three-dimensional imaging method of intrahepatic blood vessels based on a non-diffracting Airy beam. BACKGROUND

[0002] Liver cancer is a malignant tumor with insidious onset, and early symptoms are not obvious. Most patients are in the middle and late stages when diagnosed. If early diagnosis can be achieved through advanced imaging technology, patients will have a greater chance of receiving effective treatment, thereby significantly improving survival rate. At present, there is a lack of accuracy and detection sensitivity in the diagnosis of liver disease in imaging. In particular, in the diagnosis of early liver disease, it is often difficult to accurately identify lesions. Common 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 lesions. CT and MRI have higher resolution, but still have certain limitations, especially in distinguishing early liver disease from normal tissue.

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

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

[0005] The purpose of the present application is to solve the problem of insufficient accuracy and detection sensitivity of existing detection methods by providing a three-dimensional imaging method of intrahepatic blood vessels based on a non-diffracting Airy beam.

[0006] The present application is implemented as follows: a three-dimensional imaging method of intrahepatic blood vessels based on a non-diffracting Airy beam, the three-dimensional imaging method of intrahepatic blood vessels based on a non-diffracting Airy beam comprises:

[0007] S10, obtaining liver tissue through liver transplantation or biopsy surgery, fixing the liver tissue with 4% PFA, and trimming the liver tissue to 2x2x5mm 3 , washing the liver tissue with PBS;

[0008] S20, the liver tissue is bleached with H2O2 solution, and washed with PBS;

[0009] S30, the liver tissue is subjected to antigen repair with a FLASH solution, and after the repair is completed, the liver tissue is washed with a PBT solution;

[0010] S40, the liver tissue after the antigen repair is subjected to blocking treatment with a goat serum blocking solution;

[0011] S50, the liver tissue is subjected to immunostaining treatment, wherein during the immunostaining, the liver tissue is incubated at 10°C with a primary antibody, and after washing, the liver tissue is incubated under the same conditions with a secondary antibody, wherein the primary antibody is a vascular marker CD31, and the secondary antibody is a gold cluster fluorescence-labeled anti-rabbit IgG H&L antibody;

[0012] S60, the liver tissue is subjected to dehydration treatment, the liver tissue is dehydrated with a gradient concentration of tetrahydrofuran solution, defatted with dichloromethane to make the sample close to transparent, and matched with a refractive index by using dibenzyl ether;

[0013] S70, the liver tissue after the treatment is imaged based on a non-diffracting Airy beam and a high-throughput microscope, and a three-dimensional imaging picture of the liver tissue is obtained.

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

[0015] When the liver tissue is bleached with H2O2 solution, the washed tissue is placed in a solution prepared by mixing hydrogen peroxide, dimethyl sulfoxide and PBS at a ratio of 2:1:4 for bleaching, and after bleaching, the tissue is washed with PBS again for three times, each time for 30 minutes.

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

[0017] When the liver tissue after the antigen repair is subjected to blocking treatment with a goat serum blocking solution, the liver tissue is soaked in a goat serum blocking solution at room temperature for 8 hours, the goat serum blocking solution is composed of 10% fetal bovine serum, 1% goat clear albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered by a 0.22 micron filter.

[0018] The first antibody is rabbit anti-human CD31 antibody, the dilution ratio of the first antibody is 1:100 or 1:200, the second antibody is goat anti-rabbit IgG H&L connected with a gold cluster fluorescent group, the dilution ratio is 1:200, the first antibody and the second antibody are diluted with freshly prepared blocking solution, and are incubated on a 10℃ shaking table for 3-4 days, and after incubation, the specimen is washed with PBST.

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

[0020] S601, dehydrate the liver tissue by a concentration gradient of tetrahydrofuran, each time for 1.5 hours, and the concentration gradient of tetrahydrofuran is 50%, 70%, 80% and 100% tetrahydrofuran solution respectively;

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

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

[0023] The method for imaging the treated liver tissue based on the non-diffracting Airy beam and high-throughput microscopy comprises:

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

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

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

[0027] S704, control the displacement table so that the imaging assembly illumination objective and the detection objective are immersed in a medium matching the refractive index of the sample after the transparent treatment;

[0028] S705, place the gold cluster agarose solution in the FEP tube in the imaging area, adjust the position by rotating the screw micrometer head of the detection objective until a clear and complete image is obtained in the camera, and complete the focusing process;

[0029] S706, after focusing, place the sample in the refractive index matching medium, switch the phase, and change the illumination beam from a Gaussian beam to an Airy beam for actual imaging work;

[0030] S707, after determining the image range, obtain the image stack to obtain at least one group of image slices arranged along the Z-axis direction.

[0031] S708, the image slice is deconvoluted in combination with the point spread function PSF pre-calibrated by the system to restore the axial resolution of the image, to obtain clearer three-dimensional structure information, and the processed image data is imported into the image analysis software Fiji-image J and imaris, the two-dimensional image slice is integrated into a complete three-dimensional model, and a three-dimensional imaging picture of the liver tissue is obtained.

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

[0033] The present application is mainly suitable for imaging of intrahepatic blood vessels, and through immunofluorescence staining and transparent treatment of the liver tissue, laser can smoothly penetrate the tissue for three-dimensional imaging, single large-scale image capture can be realized, higher requirements of modern biology on imaging are met, and pathological analysis and physiological mechanism exploration of the liver disease model are facilitated. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 A high-throughput microscope is shown to take an imaging light path diagram of the processed liver tissue.

[0035] Figure 2 A fixed liver tissue specimen schematic diagram of embodiment 1 is shown.

[0036] Figure 3 A transparentized liver tissue specimen schematic diagram is shown.

[0037] Figure 4 A three-dimensional imaging picture of the liver tissue in embodiment 1 is shown.

[0038] Figure 5 A cross-section imaging picture of the liver tissue in embodiment 1 is shown.

[0039] Figure 6 A three-dimensional imaging picture of the liver tissue in embodiment 2 is shown.

[0040] Figure 7 A cross-section imaging picture of the liver tissue in embodiment 2 is shown. DETAILED DESCRIPTION

[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 art to which this application belongs; the terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting upon the application; the terms "comprising," "including," and "having," and variations thereof, as used in enrolling and claims herein, are intended to be open-ended and to mean including, but not limited to; the terms "first," "second," and the like, as used in the description herein, are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order.

[0042] The prior art adopts ultrasound, CT and MRI for imaging diagnosis and detection of liver diseases, which has the problems of insufficient accuracy and detection sensitivity. In view of the above problems, a three-dimensional imaging method of intrahepatic blood vessels based on non-diffracting Airy beams is proposed. In short, when the method is implemented, the liver tissue is first fixed and treated with 4% PFA, the liver tissue is bleached with H2O2 solution, the liver tissue is antigen repaired with FLASH solution, and then the liver tissue after antigen repair is blocked with goat serum blocking solution. The liver tissue is immunostained, dehydrated, and finally imaged based on non-diffracting Airy beams and high-throughput microscopy to obtain three-dimensional imaging of the liver tissue. The present application is mainly applicable to the imaging of intrahepatic blood vessels. By immunofluorescence staining and transparentization of liver tissue, laser can smoothly penetrate the tissue for three-dimensional imaging, which can realize single large-scale image capture, meet the higher requirements of modern biology on imaging, help the pathological analysis and physiological mechanism exploration of liver disease model, and overcome the problems of insufficient accuracy and detection sensitivity of the prior art using ultrasound, CT and MRI for imaging diagnosis and detection of liver diseases.

[0043] Before the implementation of examples 1-2, liver tissue is obtained through liver transplantation or biopsy surgery, and in this application, the liver tissue is divided into two groups. They are used for three-dimensional imaging in example 1 and example 2 respectively. The application mainly includes the following aspects: improving the immunofluorescence experiment method, selecting appropriate antibodies and fluorescent dyes: selecting antibodies with high specificity and strong affinity to recognize specific markers of blood vessels in the liver, such as vascular endothelial cell marker CD31. At the same time, choose fluorescent dyes with high brightness and good stability, such as gold nanoclusters, to ensure that the fluorescent signal can still be maintained in the transparentized tissue; In addition, it also includes optimizing the staining steps: during the staining process, the tissue needs to be properly fixed, permeated and blocked to improve the permeability and specific binding of the antibody, which helps to ensure the accuracy and consistency of the fluorescent labeling; Explore the appropriate liver tissue transparentization method, so that the liver tissue can maintain the fluorescent signal and achieve the effect of transparentization; Adjust the light sheet fluorescence microscope, select appropriate filters for imaging, this method is based on spatial light modulator, uses non-diffractive airy beam to excite fluorescent groups, this imaging system has obvious advantages in three-dimensional analysis of liver diseases. Compared with traditional two-dimensional imaging technology, three-dimensional imaging technology can provide more abundant structural information, helping doctors to more accurately identify and analyze the lesions in the liver tissue. In addition, three-dimensional imaging technology can also be combined with other diagnostic methods to improve the comprehensive ability of liver disease diagnosis. For example, by combining with hematology marker detection, multi-dimensional analysis of liver disease can be realized. Hematology markers such as alpha-fetoprotein (AFP) are commonly used indicators for liver cancer diagnosis, but their sensitivity and specificity are limited. Combined with three-dimensional imaging technology, more detailed lesion information can be provided in imaging, thereby making up for the shortcomings of hematology marker detection.

[0044] Example 1

[0045] The embodiment of the application provides a liver intravascular three-dimensional imaging method based on non-diffractive airy beam, and the liver intravascular three-dimensional imaging method based on non-diffractive airy beam comprises the following steps:

[0046] S10, liver tissue is obtained through liver transplantation or biopsy surgery, and the liver tissue is fixed by using 4% PFA; the size of the liver tissue is trimmed to 2*2*5mm 3 , and the liver tissue is washed by using PBS;

[0047] When the liver tissue is fixed by using 4% PFA, the liver tissue obtained by surgery is placed in a 4% formalin solution for 24 hours; after the fixation is completed, the tissue is trimmed to a size of 2*2*5mm 3 , and is soaked in a PBS solution; the tissue is washed on a shaking bed for three times, and each time lasts for 30 minutes, Figure 2 Fig. 1 shows a schematic diagram of the fixed liver tissue specimen of example 1.

[0048] S20, the liver tissue is bleached with H2O2 solution, and then washed with PBS;

[0049] It should be noted that when the liver tissue is bleached with H2O2 solution, the washed tissue is placed in a solution prepared by mixing hydrogen peroxide, dimethyl sulfoxide and PBS at a ratio of 2:1:4 for bleaching. After bleaching, the liver tissue is washed with PBS again for three times, each time for 30 minutes.

[0050] S30, the liver tissue is subjected to antigen repair with a FLASH solution, and then washed with a PBT solution after the repair is completed;

[0051] When the liver tissue is subjected to antigen repair with a FLASH solution, the washed liver tissue is first soaked in a FLASH antigen repair solution at room temperature for 1 hour, and then treated on a 45℃ shaking table for 6 hours. Then, the liver tissue is restored to room temperature. The FLASH antigen repair solution is composed of 20mM boric acid and 4% SDS, and the pH is adjusted to 7.0 with NaOH. The PBT is composed of PBS and Triton.

[0052] S40, the liver tissue subjected to antigen repair is subjected to blocking treatment with a goat serum blocking solution;

[0053] When the liver tissue subjected to antigen repair is subjected to blocking treatment with a goat serum blocking solution, the liver tissue is soaked in a goat serum blocking solution at room temperature for 8 hours. The goat serum blocking solution is composed of 10% fetal bovine serum, 1% goat clear albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered through a 0.22 micron filter.

[0054] S50, the liver tissue is subjected to immunostaining treatment. When immunostaining, the liver tissue is incubated with a primary antibody at 10℃, and then incubated with a secondary antibody under the same conditions after washing. The primary antibody is a blood vessel marker CD31, and the secondary antibody is a gold cluster fluorescently labeled anti-rabbit IgG H&L antibody.

[0055] In this embodiment, when the liver tissue is subjected to immunostaining treatment, the primary antibody is a rabbit anti-human CD31 antibody, the dilution ratio of the primary antibody is 1:100 or 1:200, the secondary antibody is a goat anti-rabbit IgG H&L, which is connected with a gold cluster fluorescent group, and the dilution ratio is 1:200. The primary antibody and the secondary antibody are both diluted with freshly prepared blocking solution, and are incubated on a 10℃ shaking table for 3-4 days. After incubation, the specimen is washed with PBST. It should be noted that the secondary antibody incubation process and the subsequent operation are carried out in the dark.

[0056] S60, the liver tissue is subjected to dehydration treatment. The liver tissue is dehydrated with a gradient concentration of tetrahydrofuran solution, defatted with dichloromethane to make the sample nearly transparent, and matched with a refractive index with dibenzyl ether, Figure 3A schematic diagram of a liver tissue sample after transparentization is shown;

[0057] The method for dehydrating the liver tissue comprises:

[0058] S601, dehydrate the liver tissue by a concentration gradient of tetrahydrofuran, each time for 1.5 hours, and the concentration gradient of tetrahydrofuran is 50%, 70%, 80% and 100% tetrahydrofuran solution respectively;

[0059] S602, soak the liver tissue in 100% dichloromethane solution for 1.5 hours for defatting;

[0060] S603, soak the tissue in 100% benzyl 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 benzyl ether at 4°C, and the imaging effect is best within 3 days. During the transfer from the dichloromethane solution to the benzyl ether solution, the operation should be quick to prevent the sample from matching the refractive index of the air, thereby affecting the imaging quality.

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

[0063] In this embodiment, a method for taking an image of the treated liver tissue based on the non-diffracting Airy beam and the high-throughput microscope is provided, Figure 1 A light path diagram for taking an image of the treated liver tissue by the high-throughput microscope is shown, and the method for taking an image of the treated liver tissue based on the non-diffracting Airy beam and the high-throughput microscope specifically comprises:

[0064] S701, start the femtosecond pulse laser, and set the single-photon excitation light at a wavelength of 730 nm;

[0065] S702, replace the filter in front of the camera with a 750&800LP type filter;

[0066] S703, load phase information through a spatial light modulator, and modulate the phase information into a Gaussian beam and an Airy beam respectively.

[0067] S704, control the displacement table so that the imaging assembly illumination objective and the detection objective are immersed in a medium matching the refractive index of the sample after transparentization treatment;

[0068] S705, place the gold cluster agarose solution in the FEP tube in the imaging area, adjust the position by rotating the screw micrometer head of the detection objective until a clear and complete image is obtained in the camera, and complete the focusing process;

[0069] S706, after focusing is completed, the sample is placed in the refractive index matching medium, the illumination beam is converted from a Gaussian beam to an Airy beam by switching the phase, so as to carry out actual imaging work;

[0070] S707, after the image range is determined, an image stack is obtained, and at least one group of image slices arranged along the Z-axis direction is obtained;

[0071] S708, the image slices are deconvoluted in combination with the system pre-calibrated point spread function (PSF), due to the limitation of the optical system, the original image data may have certain blurring in the axial direction, therefore, the deconvolution processing is required in combination with the system pre-calibrated point spread function (PSF), so as to restore the axial resolution of the image, obtain clearer three-dimensional structure information, and 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, thereby intuitively displaying the microscopic nodes inside the sample, and obtaining the three-dimensional imaging picture of the liver tissue, Figure 4 The three-dimensional imaging picture of the liver tissue in Example 1 is shown. Figure 5 The cross-sectional imaging picture of the liver tissue in Example 1 is shown.

[0072] The present application focuses on the three-dimensional imaging of intrahepatic blood vessels, and the vascular endothelial cells are labeled by improving the immunofluorescence staining technology. This process involves the combination of fluorescent dyes with vascular endothelial cells, and then the tissue is transparentized, so that the laser can penetrate the entire tissue and be imaged. This technology can realize the three-dimensional imaging of blood vessels in larger liver tissue samples, 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 application is not directly used for the diagnosis or treatment of diseases, but provides a three-dimensional imaging method of intrahepatic blood vessels. This method processes the liver tissue through a series of processes (such as fixation, bleaching, immunostaining, etc.), and uses non-diffractive Airy beams and high-throughput microscopy for imaging, finally obtains the three-dimensional structure information of the liver tissue, which meets the object requirements of patent protection, and does not belong to the diagnosis method of diseases. It is an innovative and practical imaging technology that can be widely used in many fields.

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

[0075] Example 2

[0076] The embodiment of the present application provides a liver-in blood vessel three-dimensional imaging method based on a non-diffracting Airy beam, and the liver-in blood vessel three-dimensional imaging method based on the non-diffracting Airy beam comprises the following steps:

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

[0078] When the liver tissue is fixed by using 4% PFA, the liver tissue obtained through surgery is placed in a 4% formaldehyde solution for fixation for 24 hours, after fixation is completed, the tissue is trimmed to a size of 2*2*5mm 3 , and is soaked in a PBS solution, and is washed on a shaking table for three times, each time for 30 minutes.

[0079] S20, bleaching the liver tissue by using an H2O2 solution, and washing by using PBS;

[0080] It should be noted that when the liver tissue is bleached by using the H2O2 solution, the washed tissue is placed in a solution prepared by hydrogen peroxide, dimethyl sulfoxide and PBS in a proportion of 2:1:4 for bleaching, and after bleaching, the tissue is washed by using PBS for three times, each time for 30 minutes.

[0081] S30, repairing the antigen of the liver tissue by using a FLASH solution, and washing the liver tissue by using a PBT solution after the repairing is completed;

[0082] When the FLASH solution is used to repair the antigen of the liver tissue, the washed liver tissue is first soaked in the FLASH antigen repair solution at room temperature for 1 hour, and then is continuously treated on a 45℃ shaking table for 6 hours, and then the liver tissue is restored to room temperature, wherein the FLASH antigen repair solution is composed of 20mM boric acid and 4% SDS, and the pH is adjusted to 7.0 by using NaOH, and the PBT is composed of PBS and Triton.

[0083] S40, blocking the liver tissue after the antigen repair by using a goat serum blocking solution;

[0084] When the goat serum blocking solution is used to block the liver tissue after the antigen repair, the liver tissue is soaked 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 clear albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered by using a 0.22-micron filter.

[0085] S50, the liver tissue is immunostained, wherein the liver tissue is incubated with a primary antibody at 10 DEG C, and then incubated with a secondary antibody under the same conditions after washing, wherein the primary antibody is a vascular marker CD31, and the secondary antibody is a gold cluster fluorescence-labeled anti-rabbit IgG H&L antibody;

[0086] In this embodiment, when the liver tissue is immunostained, the primary antibody is a rabbit anti-human CD31 antibody, the dilution ratio of the primary antibody is 1:100 or 1:200, the secondary antibody is a goat anti-rabbit IgG H&L antibody, and the dilution ratio of the secondary antibody is 1:200. Both the primary antibody and the secondary antibody are diluted with freshly prepared blocking solution and incubated on a 10 DEG C shaker for 3-4 days. After incubation, the specimen is washed with PBST.

[0087] S60, the liver tissue is dehydrated, 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;

[0088] The method for dehydrating the liver tissue comprises:

[0089] S601, the liver tissue is dehydrated by a concentration gradient of tetrahydrofuran, each time for 1.5 hours, and the concentration gradient of tetrahydrofuran is 50%, 70%, 80% and 100% tetrahydrofuran solution;

[0090] S602, the liver tissue is soaked in 100% dichloromethane solution for 1.5 hours for defatting;

[0091] S603, the tissue is soaked in 100% dibenzyl ether solution for 1.5 hours at a storage temperature of 4 DEG C.

[0092] S70, the treated liver tissue is imaged based on a non-diffracting Airy beam and a high-throughput microscope to obtain a three-dimensional imaging picture of the liver tissue.

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

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

[0095] S702, replace the filter in front of the camera with a 750&800LP filter;

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

[0097] S704, control the displacement table, so that the imaging assembly illumination objective and the detection objective are immersed into a medium matching the refractive index of the sample after the transparent treatment.

[0098] S705, the gold cluster agarose solution is placed in the FEP tube in the imaging area, the position of the detection objective is adjusted by rotating the screw micrometer head until a clear and complete image is obtained in the camera, and the focusing process is completed.

[0099] S706, after the focusing is completed, the sample is placed into the refractive index matching medium, the illumination beam is converted from the Gaussian beam to the Airy beam by switching the phase, so that the actual imaging work is carried out.

[0100] S707, after the image range is determined, the image stack is obtained, and at least one group of image slices arranged along the Z axis direction is obtained.

[0101] S708, the image slices are deconvoluted by combining the point spread function (PSF) calibrated by the system in advance, so that the axial resolution of the image is restored, clearer three-dimensional structure information is obtained, the processed image data is imported into the image analysis software Fiji-image J and imaris, the two-dimensional image slices are integrated into a complete three-dimensional model, and the three-dimensional imaging picture of the liver tissue is obtained.

[0102] In the application, the high-throughput microscope and the automatic focusing technology are combined, the imaging process can be quickly completed, the image slices arranged along the Z axis direction are obtained, and the complete three-dimensional model is integrated. Compared with the traditional imaging method, the imaging time can be significantly shortened, the work efficiency can be improved, the method is suitable for large-scale sample analysis, the non-diffracting Airy beam and the spatial light modulator are used to load the phase information, the switching from the Gaussian beam to the Airy beam is realized, and a new optical means for imaging is provided. The self-acceleration characteristics and the non-diffraction characteristics of the Airy beam make it have unique advantages in three-dimensional imaging, and more stable imaging effect can be provided.

[0103] In summary, the application provides a three-dimensional imaging method of intrahepatic blood vessels based on a non-diffracting Airy beam, and the application is mainly suitable for imaging of intrahepatic blood vessels. The liver tissue is subjected to immunofluorescence staining and transparent treatment, so that the laser can smoothly penetrate the tissue for three-dimensional imaging, single large-range image capture can be realized, the higher requirements of modern biology on imaging are met, and the pathological analysis and physiological mechanism exploration of the liver disease model are facilitated.

[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, but those skilled in the art should know that the present application is not limited by the order of the described actions, because according to the present application, certain steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should 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 application.

[0105] The above embodiments are only used to illustrate the technical solutions of the present application, and not to limit the protection scope of the application. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still combine, add or delete or make other adjustments to the features of the embodiments of the present application according to the circumstances without conflict and without creative labor, so as to obtain different other technical solutions which do not deviate from the concept of the present application in essence. These technical solutions also belong to the scope of the present application.

Claims

1. A method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams, characterized in that, The liver intravascular three-dimensional imaging method based on the non-diffracting Airy beam comprises the following steps: S10, the liver tissue is fixed with 4% PFA, and the size of the liver tissue is trimmed to 2x2x5mm 3 The liver tissue is washed with PBS; S20, the liver tissue is bleached by using an H2O2 solution, and the liver tissue is washed by using PBS; S30, the liver tissue is subjected to antigen repair by using a FLASH solution, and after the repair is completed, the liver tissue is washed by using a PBT solution; S40, the liver tissue subjected to the antigen repair is subjected to blocking treatment by using a goat serum blocking solution; S50, the liver tissue is subjected to immunostaining treatment, wherein during the immunostaining, the liver tissue is incubated by using a primary antibody at 10 DEG C, and after washing, the liver tissue is incubated by using a secondary antibody under the same condition, wherein the primary antibody is a blood vessel marker CD31, and the secondary antibody is an anti-rabbit IgG H&L antibody connected with a gold cluster fluorescent label; S60, the liver tissue is subjected to dehydration treatment, the liver tissue is dehydrated by using a gradient concentration tetrahydrofuran solution, is defatted by using dichloromethane to make the sample close to transparent, and is matched with a refractive index by using dibenzyl ether; S70, the liver tissue subjected to the treatment is imaged by using a non-diffracting Airy beam and a high-throughput microscope, and a three-dimensional imaging picture of the liver tissue is obtained; During the immunostaining treatment of the liver tissue, a rabbit anti-human CD31 antibody is used as the primary antibody, the dilution ratio of the primary antibody is 1:100 or 1:200, the secondary antibody is a goat anti-rabbit IgG H&L connected with a gold cluster fluorescent group, and the dilution ratio is 1:200, the primary antibody and the secondary antibody are both diluted by using a newly prepared blocking solution, and are incubated on a 10 DEG C shaking table for 3-4 days, and after the incubation, the sample is washed by using PBST; The method for dehydrating the liver tissue comprises the following steps: S601, the liver tissue is dehydrated by using a concentration gradient of tetrahydrofuran, and each time of soaking dehydration is 1.5 hours, and the concentration gradient of the tetrahydrofuran is 50%, 70%, 80% and 100% tetrahydrofuran solution respectively; S602, the liver tissue is soaked in 100% dichloromethane solution for 1.5 hours for defatting; S603, the tissue is soaked in 100% dibenzyl ether solution for 1.5 hours, and the storage temperature is 4 DEG C.

2. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams according to claim 1, characterized in that: For liver tissue fixation with 4% PFA, liver tissue was placed in 4% formaldehyde solution for 24 hours. After fixation, the tissue was trimmed to 2 x 2 x 5 mm in size and soaked in PBS solution, washed three times on a shaker for 30 minutes each time. 3 For liver tissue fixation with 4% PFA, liver tissue was placed in 4% formaldehyde solution for 24 hours. After fixation, the tissue was trimmed to 2 x 2 x 5 mm in size and soaked in PBS solution, washed three times on a shaker for 30 minutes each time.

3. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams according to claim 2, characterized in that: When the liver tissue is bleached by using an H2O2 solution, the washed tissue is placed in a solution prepared by hydrogen peroxide, dimethyl sulfoxide and PBS in a ratio of 2:1:4 for bleaching, and after the bleaching, the liver tissue is washed by using PBS again for three times, each time for 30 minutes.

4. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams according to claim 2, characterized in that: When the liver tissue is subjected to antigen repair by using a FLASH solution, the washed liver tissue is first soaked in the FLASH antigen repair solution at room temperature for 1 hour, then is continuously treated on a 45 DEG C shaking table for 6 hours, and then the liver tissue is restored to room temperature, wherein the FLASH antigen repair solution is composed of 20 mM boric acid and 4% SDS, and the pH is adjusted to 7.0 by using NaOH, and the PBT is composed of PBS and Triton.

5. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams according to claim 4, characterized in that: When the liver tissue subjected to the antigen repair is subjected to blocking treatment by using a 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 is composed of 10% fetal bovine serum, 1% goat clear albumin, 5% dimethyl sulfoxide and 0.5% PC-300, and is filtered by using a 0.22-micron filter.

6. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams according to claim 1, characterized in that: The method for imaging the liver tissue subjected to the treatment by using a non-diffracting Airy beam and a high-throughput microscope comprises the following steps: S701, start femtosecond pulse laser, set 730nm wavelength single photon excitation light; S702, replace the filter in front of the camera with a 750&800LP filter; S703, load phase information through a spatial light modulator, and modulate the phase information into a Gaussian beam and an Airy beam respectively.

7. The method for three-dimensional imaging of intrahepatic blood vessels based on non-diffracting Airy beams according to claim 6, characterized in that: The method for imaging the processed liver tissue based on the non-diffracting Airy beam and the high-throughput microscope specifically further comprises: S704, control the displacement table so that the imaging assembly illumination objective and the detection objective are immersed in a medium matching the refractive index of the sample after the transparent treatment; S705, place the gold cluster agarose solution in the FEP tube in the imaging area, adjust the position by rotating the screw micrometer head of the detection objective until a clear and complete image is obtained in the camera, and complete the focusing process; S706, after focusing, place the sample in the refractive index matching medium, switch the phase, and change the illumination beam from a Gaussian beam to an Airy beam for actual imaging work; S707, after determining the image range, obtain an image stack to obtain at least one group of image slices arranged along the Z-axis direction; S708, combine the point spread function PSF pre-calibrated by the system to perform deconvolution processing on the image slices 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 a three-dimensional imaging picture of the liver tissue.

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