Intrapulmonary vessel three-dimensional visualization method based on light sheet fluorescence microscope

Abstract

The invention discloses an intrapulmonary vessel three-dimensional visualization method based on a light sheet fluorescence microscopy, belongs to the technical field of three-dimensional imaging, and solves the problem of low spatial resolution of existing imaging and ultrasonic examination equipment. The method comprises the following steps: performing tracheal intubation administration on a mouse, performing dyeing by adopting a hypodermic injection cy7 solution, and performing ultrasonic imaging on the mouse; the method comprises the following steps: dehydrating mouse lung tissues by using a tetrahydrofuran solution with gradient concentration, degreasing by using dichloromethane to enable a sample to be close to be transparent, and carrying out three-dimensional imaging shooting on the lung tissues by using a light sheet fluorescence microscopic imaging system; according to the invention, through fluorescence excitation and acquisition of infrared two-region wave bands, higher imaging depth can be provided for a vasculature in lung tissue, better axial resolution, signal background ratio and contrast ratio can be provided for deep imaging, multiple two-dimensional image slices can be integrated and reconstructed, and the imaging precision is improved. And a visual and complete intra-pulmonary vessel three-dimensional visual model is formed. By quantifying the three-dimensional model, the change of the microstructure of the vessel in the lung can be more accurately observed.

Description

Technical Field

[0001] The present invention belongs to the technical field of three-dimensional imaging, and particularly relates to a method for three-dimensional visualization of pulmonary vasculature based on a light-sheet fluorescence microscope. Background Art

[0002] Plastic Bronchitis (PB) is a relatively rare and complex clinical syndrome, and its imaging features are mainly manifested as atelectasis. The core feature of this disease is the formation of bronchial tree-like casts in the airways, which may cause local or extensive obstruction, leading to symptoms such as cough, fever, and dyspnea in patients. In severe cases, it may even cause acute lethal respiratory distress. The first report of this disease dates back to the early 20th century, and it was considered to be related to suppurative infections at that time. In terms of histopathological classification, the view that bronchial casts are divided into two types has been successively proposed: Type I is an inflammatory cast, mainly composed of inflammatory cells and fibrin structures, and is common in bronchopulmonary diseases; Type II is a non-cellular cast, and its main component is mucin, which is mostly related to the postoperative conditions of congenital heart diseases. In China, PB is mostly related to infection-related diseases, while foreign reports are more related to the postoperative conditions of congenital heart diseases. With the widespread use of bronchoscopy technology, more and more cases of PB have been diagnosed. However, due to the lack of large-scale epidemiological investigations, the severity of PB is still unclear. Nevertheless, the harm of this disease to children is huge. Its onset is usually critical, and the treatment process of the primary disease is often not smooth, which is prone to misdiagnosis and mistreatment, and may even cause iatrogenic damage. Therefore, early diagnosis and appropriate treatment are of great clinical significance for PB.

[0003] In terms of the pathogenesis, research has mostly focused on the release mechanism of inflammatory factors after PB causes damage to the body, the possible mechanism of hypercoagulable state, and the exploration of related risk factors, while the research on the deep pathogenesis of its cast formation is relatively rare. Some possible pathogenesis have been proposed in the academic circle: Given that foreign reports show that PB is common after congenital heart surgery, especially Fontan surgery may lead to increased pulmonary venous pressure, which in turn causes abnormal reactions of the respiratory epithelium and mucus hypersecretion. Another type of speculation is related to lymphatic dysfunction, including increased intrathoracic lymphatic pressure, bronchial lymphatic fistula, and ultimately lymphatic leakage into the bronchi.

[0004] Clinically, there are many challenges in the diagnosis and treatment of PB. This is mainly because the clinical manifestations of PB lack specificity. Common symptoms include cough and shortness of breath. In more severe cases, patients may experience dyspnea, and some patients can even cough up bronchial cast substances. In terms of treatment, the use of bronchodilators often fails to achieve significant improvement. At the same time, although the use of endotracheal intubation and ventilators can relieve clinical symptoms to a certain extent, the improvement effect is not obvious, and it is not sufficient to explain the condition simply by acute respiratory distress syndrome or lung injury. Currently, the main means of treating PB rely on bronchoscopy technology. Through this technology, the cast substances in the trachea can be removed and pathological tests can be performed to clarify the diagnosis.

[0005] However, in terms of imaging observation, existing means such as imaging and ultrasound examinations have certain limitations. The spatial resolution of these methods is relatively low, making it difficult to provide a clear observation of the microscopic structure of the lungs. Therefore, they are insufficient in providing clear visual evidence for the diagnosis of PB. This further increases the difficulty for clinicians in diagnosing and treating PB. To address the above problems, we propose a three-dimensional visualization method for pulmonary vasculature based on light-sheet fluorescence microscopy. Summary of the Invention

[0006] The purpose of the present invention is to provide a three-dimensional visualization method for pulmonary vasculature based on light-sheet fluorescence microscopy in view of the deficiencies of the prior art, and to solve the problem of low spatial resolution of existing imaging and ultrasound examination equipment.

[0007] The present invention is implemented as follows. A three-dimensional visualization method for pulmonary vasculature based on light-sheet fluorescence microscopy, the three-dimensional visualization method for pulmonary vasculature based on light-sheet fluorescence microscopy includes:

[0008] S10, Select healthy male mice. The male mice are BALB / c mice, 6 weeks old, and weighing 25±1 g;

[0009] S20, Use 7.5% 2-chloroethyl ethyl sulfide (CEES) to administer drugs to the mice through endotracheal intubation;

[0010] S30, Anesthetize the mice 18 h after drug administration, stain them by subcutaneous injection of 1 mg / mL cy7 solution. After 5 min, perfuse the mice's hearts with PBS to wash the blood, and then fix the mouse lung tissues and store them in 4% PFA;

[0011] S40, Dehydrate the mouse lung tissues with gradient concentration tetrahydrofuran solution and degrease them with dichloromethane to make the samples close to transparent, and match the refractive index with dibenzyl ether;

[0012] S50. Take the processed mouse lung tissue and use a light-sheet fluorescence microscopy system to perform three-dimensional imaging of the lung tissue to obtain a three-dimensional visualization model of the blood vessels in the lung.

[0013] When using 7.5% 2-chloroethyl ethyl sulfide (CEES) for tracheal intubation in mice, take a 97% CEES solution and dilute it with absolute ethanol in a fume hood to obtain a 7.5% CEES solution. Subsequently, anesthetize the mice with tribromoethanol and use a micropipette to administer 6 μL of the 7.5% solution through tracheal intubation. After administration, monitor the mice in the cage until they fully recover from anesthesia, record their survival status, and evaluate the respiratory quality, wheezing, and degree of activity inhibition according to the clinical performance scoring table.

[0014] The method of dehydrating the mouse lung tissue with a gradient concentration of tetrahydrofuran solution and defatting with dichloromethane to make the sample nearly transparent includes:

[0015] S401. Fix the obtained mouse lung tissue in 4% formalin solution for 24 hours to complete the fixation of the mouse lung tissue.

[0016] S402. Treat the mouse lung tissue using the tetrahydrofuran concentration gradient method.

[0017] S403. Immerse the mouse lung tissue in 100% dichloromethane solution for 1.5 hours to remove fat.

[0018] S404. Immerse the mouse lung tissue in 100% dibenzyl ether solution for 1.5 hours, and store the processed mouse lung tissue sample in 4°C dibenzyl ether solution.

[0019] When treating the mouse lung tissue using the tetrahydrofuran concentration gradient method, sequentially place the mouse lung tissue into 50%, 70%, 80%, and 100% tetrahydrofuran solutions, and immerse it in each concentration of tetrahydrofuran solution for 1.5 hours.

[0020] The method of the three-dimensional imaging process of the lung tissue using a light-sheet fluorescence microscopy system specifically includes:

[0021] S501. Start the femtosecond pulsed laser and set it to emit single-photon excitation light with a wavelength of 730 nm, and replace the filter at the front end of the light-sheet microscope with a 750&800LP type filter.

[0022] S502. Perform phase modulation using a spatial light modulator, load the phase information, and convert the light beam into two beam forms, where the two beam forms are Gaussian beam and Airy beam.

[0023] S503, controlling the translation stage based on the control software, so that the illumination objective lens and the detection objective lens in the imaging assembly are steadily immersed in a medium having a refractive index that matches the transparentized mouse lung tissue, to ensure that the light beam penetrates the mouse lung tissue without damage and efficiently;

[0024] S504, pouring the agarose solution containing gold clusters into the FEP tube, and placing the FEP tube at a predetermined position in the imaging area, rotating the screw micrometer head of the detection objective lens, and fine-tuning the position of the screw micrometer head until a clear and complete image is presented on the display screen of the camera, completing the focusing process, and after the focus is correct, placing the mouse lung tissue to be tested in a medium with a matching refractive index;

[0025] S505, converting the illumination beam from a Gaussian beam to an Airy beam, and after accurately framing the imaging area, immediately starting the image stack acquisition to complete the shooting;

[0026] S506, performing deconvolution processing on the original image data using the calibrated point spread function PSF to restore the axial resolution of the image and obtain image data after deconvolution processing;

[0027] S507, importing the image data after deconvolution processing into image analysis software to form an intuitive and complete three-dimensional model, and obtaining a three-dimensional visualization model of the intrapulmonary vessels.

[0028] When the image data after deconvolution processing is imported into the image analysis software, the image analysis software is Fiji-image J or Imaris.

[0029] The light sheet fluorescence microscopy imaging system comprises a femtosecond pulse laser, a shooting camera, a filter, a spatial light modulator, an imaging component, a displacement stage, and a detection objective lens.

[0030] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0031] The present invention can image the vascular system in the lung tissue at a higher depth through fluorescence excitation and acquisition in the second infrared band, and has better axial resolution, signal-to-background ratio and contrast in deep imaging. It can skillfully integrate and reconstruct many two-dimensional image slices to form an intuitive and complete three-dimensional visualization model of the pulmonary vessels. By quantifying the three-dimensional model, the microstructural changes of the pulmonary vessels can be observed more accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention provides a light path diagram of the light sheet fluorescence microscopy system.

[0033] Figure 2 A schematic diagram of the fixed lung tissue specimen in Example 1 is shown.

[0034] Figure 3 Shows a schematic diagram of the transparent lung tissue specimen in Example 1.

[0035] Figure 4 Shows a schematic diagram of the three-dimensional imaging of the mouse lung tissue in Example 1.

[0036] Figure 5 Shows a schematic diagram of the cross-sectional imaging of the mouse lung tissue in Example 1.

[0037] Figure 6 Shows a schematic diagram of the three-dimensional imaging of the mouse lung tissue in Example 2.

[0038] Figure 7 Shows a schematic diagram of the cross-sectional imaging of the mouse lung tissue in Example 2. Detailed implementation manners

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs; the terms used in the description 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 description and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the description and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0040] Existing means such as imaging and ultrasound examination have certain limitations. The spatial resolution of these methods is relatively low, and it is difficult to provide a clear observation of the microscopic structure of the lungs. To address the above problems, we propose a three-dimensional visualization method for pulmonary vasculature based on light-sheet fluorescence microscopy. When implementing this method, first, tracheal intubation is performed on the mouse for drug administration, and subcutaneous injection of a 1 mg / mL cy7 solution is used for staining. Then, the mouse lung tissue is dehydrated with a gradient concentration of tetrahydrofuran solution and defatted with dichloromethane to make the sample close to transparent. Finally, a light-sheet fluorescence microscopy imaging system is used to perform three-dimensional imaging of the lung tissue to obtain a three-dimensional visualization model of the pulmonary vasculature. Through fluorescence excitation and acquisition in the second near-infrared region, this invention can achieve a higher imaging depth for the vasculature system inside the lung tissue, and has better axial resolution, signal-to-background ratio, and contrast in deep imaging. It can skillfully integrate and reconstruct numerous two-dimensional image slices to form an intuitive and complete three-dimensional visualization model of the pulmonary vasculature. By quantifying the three-dimensional model, the subtle structural changes of the pulmonary vasculature can be observed more accurately.

[0041] The present invention focuses on three-dimensional imaging of pulmonary vasculature, and labels the vasculature by injecting a fluorescent probe. This process involves fixing the fluorescent probe in the vasculature, and then performing tissue clearing so that the laser can penetrate the entire lung tissue for imaging. This technique can achieve three-dimensional imaging of blood vessels and lymphatic vessels in relatively large lung tissue samples, thereby providing more intuitive and detailed structural information for the study of the mechanism and fine structure of PB.

[0042] Example 1

[0043] The embodiment of the present invention provides a method for three-dimensional visualization of pulmonary vasculature based on a light-sheet fluorescence microscope. The method for three-dimensional visualization of pulmonary vasculature based on a light-sheet fluorescence microscope specifically includes:

[0044] S10, Select healthy male mice. The male mice are BALB / c mice, 6 weeks old, with a body weight of 25 ± 1 g;

[0045] S20, Administer drugs to the mice through tracheal intubation using 7.5% 2-chloroethyl ethyl sulfide (CEES);

[0046] In this embodiment, when administering drugs to the mice through tracheal intubation using 7.5% CEES, take the 97% CEES solution and dilute it with absolute ethanol in a fume hood to obtain a 7.5% CEES solution. Subsequently, the mice are anesthetized with tribromoethanol, and 6 μL of the 7.5% solution is administered through tracheal intubation using a micropipette. After administration, monitor the mice in the cage until they fully recover from anesthesia, record their survival status, and evaluate the degree of respiratory quality, wheezing, and activity inhibition according to the clinical performance scoring table.

[0047] S30, Anesthetize the mice 18 h after drug administration, stain them by subcutaneous injection of a 1 mg / mL cy7 solution, flush the blood by perfusing PBS into the heart of the mice 5 min later, and then fix and store the lung tissue of the mice in 4% PFA;

[0048] It should be noted that when anesthetizing the mice 18 h after drug administration, dissolve cy7 according to the ratio of 1 mL of PBS to 1 mg of cy7, then inject the above dye into the limbs of the mice. Wait for 5 min and then dissect the mice, perfuse about 30 mL of PBS into the heart, cut off the lung tissue of the mice and fix and store it in 4% PFA for 24 hours.

[0049] Among them, the sampling standard is based on monitoring the respiratory status of the mice after drug administration. If the mice show signs of impending death, such as a weight loss of more than 25% or inability to eat or drink, ethical standards require euthanasia to be implemented before ending the planned study. 18 hours after administration of each group of CEES doses, perform terminal anesthesia, and excise the complete lung tissue samples of the mice after staining and labeling.

[0050] S40. The mouse lung tissue was dehydrated with a gradient concentration of tetrahydrofuran solution and defatted with dichloromethane to make the sample nearly transparent, and the refractive index was matched with dibenzyl ether;

[0051] In this embodiment, the method for dehydrating the mouse lung tissue with a gradient concentration of tetrahydrofuran solution and defatting with dichloromethane to make the sample nearly transparent includes:

[0052] S401. The obtained mouse lung tissue was fixed in 4% formalin solution for 24 hours to complete the fixation process of the mouse lung tissue. Among them, the whole process of the stained mouse lung tissue needs to be protected from light. Figure 2 Figure 9 shows a schematic diagram of the fixed lung tissue specimen in Example 1;

[0053] S402. The mouse lung tissue was treated by the tetrahydrofuran concentration gradient method. When the mouse lung tissue was treated by the tetrahydrofuran concentration gradient method, the mouse lung tissue was successively placed in 50%, 70%, 80% and 100% tetrahydrofuran solutions, and soaked in each concentration of tetrahydrofuran solution for 1.5 hours;

[0054] S403. The mouse lung tissue was soaked in 100% dichloromethane solution for 1.5 hours to remove fat;

[0055] S404. The mouse lung tissue was soaked in 100% dibenzyl ether solution for 1.5 hours. The processed mouse lung tissue sample was stored in 4°C dibenzyl ether solution. When transferring the mouse lung tissue from dichloromethane solution to dibenzyl ether solution, the operation needs to be rapid to prevent the refractive index of the sample from matching that of the air, thereby affecting the quality of subsequent imaging. Figure 3 Figure 20 shows a schematic diagram of the cleared lung tissue specimen in Example 1.

[0056] S50. The processed mouse lung tissue was taken, and a light-sheet fluorescence microscopy system was used to perform three-dimensional imaging on the lung tissue to obtain a three-dimensional visualization model of the blood vessels in the lung.

[0057] In this embodiment, a method for performing three-dimensional imaging on lung tissue using a light-sheet fluorescence microscopy system is provided. Figure 4 Figure 28 shows a schematic diagram of the three-dimensional imaging of the mouse lung tissue in Example 1. Figure 5 Figure 30 shows a schematic diagram of the cross-sectional imaging of the mouse lung tissue in Example 1. The method for performing three-dimensional imaging on lung tissue using a light-sheet fluorescence microscopy system includes:

[0058] S501, start the femtosecond pulse laser and set it to emit single-photon excitation light with a wavelength of 730nm, set the vibration frequency of the scanning galvanometer to 300Hz and the amplitude to 4Vpp to form a light sheet for exciting the sample to generate a fluorescence signal, and replace the filter at the front end of the light sheet microscope with a 750&800LP filter, which is a long-pass filter;

[0059] S502, performing phase modulation using a spatial light modulator, loading phase information, and converting the light beam into two light beam forms, wherein the two light beam forms are a Gaussian beam and an Airy beam;

[0060] In this embodiment, specific phase information is loaded by means of a spatial light modulator. Through this phase modulation, the light beam can be converted into two forms: one is a Gaussian beam, which is used for the precise focusing operation in the early stage; the other is an Airy beam, which is used for the subsequent actual imaging stage. The control software precisely controls the translation stage to ensure that the imaging components, that is, the illumination objective lens and the detection objective lens, can be steadily immersed in a medium that matches the refractive index of the transparent sample. This step is crucial, as it ensures that the light beam can penetrate the mouse lung tissue sample non-destructively and efficiently.

[0061] S503, controlling the translation stage based on the control software, so that the illumination objective lens and the detection objective lens in the imaging assembly are steadily immersed in a medium having a refractive index that matches the transparentized mouse lung tissue, to ensure that the light beam penetrates the mouse lung tissue without damage and efficiently;

[0062] S504, pouring the agarose solution containing gold clusters into the FEP tube, and placing the FEP tube at a predetermined position in the imaging area, rotating the screw micrometer head of the detection objective lens, and fine-tuning the position of the screw micrometer head until a clear and complete image is presented on the display screen of the camera, completing the focusing process, and after the focus is correct, placing the mouse lung tissue to be tested in a medium with a matching refractive index;

[0063] S505, converting the illumination beam from a Gaussian beam to an Airy beam, and after accurately framing the imaging area, immediately starting the collection of the image stack to complete the shooting.

[0064] S506, performing noise removal processing on the obtained original image data, performing deconvolution processing on the original image data using a calibrated point spread function PSF, restoring the axial resolution of the image, and obtaining image data after deconvolution processing;

[0065] It should be noted that due to the inherent limitations of the optical imaging system, there is often a certain degree of blurring in the axially collected original image data. To solve this problem, it is necessary to perform deconvolution processing on the original image data with the help of the point spread function (PSF) pre-calibrated by the system. This processing step can effectively restore the axial resolution of the image, enabling us to obtain clearer and more accurate three-dimensional structure information.

[0066] S507. Import the image data after deconvolution processing into image analysis software to form an intuitive and complete three-dimensional model, and obtain a three-dimensional visualization model of the blood vessels in the lung. When importing the image data after deconvolution processing into the image analysis software, the image analysis software is Fiji-image J or imaris.

[0067] It should be noted that the light sheet fluorescence microscopy system includes a femtosecond pulsed laser, a camera, a filter, a spatial light modulator, an imaging component, a displacement stage, and a detection objective lens. Figure 1 The optical path diagram of the light sheet fluorescence microscopy system is shown. Among them, the femtosecond pulsed laser is the light source part of the system, which is used to provide high-energy and ultrashort pulsed laser. The output laser first passes through the filter to select a suitable wavelength for exciting the sample. The role of the filter is to select a specific wavelength of laser to match the excitation spectrum of the fluorescent dye in the sample. The laser after passing through the filter is guided to the spatial light modulator, which is used to perform phase modulation on the laser to generate a specific light field distribution (such as an Airy beam or a Gaussian beam). The modulated beam is focused into a light sheet for illuminating the sample. The camera (such as an sCMOS or EMCCD camera) is located on the imaging path of the detection objective lens and is used to capture the fluorescence signal and convert it into a digital image. The two-dimensional image recorded by the camera is then reconstructed into three dimensions through software.

[0068] The invention mainly includes the following aspects: injection of the probe, selection of a suitable fluorescent dye: select a fluorescent dye with high brightness and good stability, such as cy7, to ensure that a good fluorescence signal can still be maintained in the cleared tissue; in addition, it also includes the step of tissue clearing: this step can remove the opacity of the tissue, enabling the subsequent imaging process to proceed smoothly. For example, the lung tissue after being cleared by the 3DISCO method will become almost transparent, allowing light to penetrate deep into the tissue, providing the possibility for high-resolution imaging; during the staining process, the tissue needs to be properly fixed, and fixation with formalin can ensure the accuracy of fluorescence labeling; debug the light sheet fluorescence microscope and select a suitable filter for imaging. Through these details, more accurate detailed three-dimensional structure information of the blood vessel system in the lung can be obtained, including the distribution, morphology, and interconnection of blood vessels and lymphatic vessels.

[0069] Example 2

[0070] An embodiment of the present invention provides a three-dimensional visualization method for pulmonary vasculature based on a light sheet fluorescence microscope. The three-dimensional visualization method for pulmonary vasculature based on a light sheet fluorescence microscope specifically includes:

[0071] S10. Select healthy male mice, which are BALB / c mice, 6 weeks old and weighing 25 ± 1 g.

[0072] S20. Administer drugs to the mice through tracheal intubation using normal saline.

[0073] When administering drugs to the mice through tracheal intubation using normal saline, the mice are anesthetized with tribromoethanol. After anesthesia, 6 μL of normal saline is injected into the trachea using a micro syringe as a control.

[0074] S30. After 18 h of drug administration, anesthetize the mice, stain them by subcutaneous injection of a 1 mg / mL cy7 solution. After 5 min, perfuse the mice's hearts with PBS to wash out the blood, and then fix and store the mouse lung tissues in 4% PFA.

[0075] It should be noted that after 18 h of drug administration, anesthetize the mice. Cy7 is configured by dissolving it in a ratio of 1 mL of PBS to 1 mg of cy7. Then inject the above dye into the four limbs of the mice. After waiting for 5 min, dissect the mice, perfuse the hearts with about 30 mL of PBS, cut off the mouse lung tissues and fix and store them in 4% PFA for 24 hours.

[0076] S40. Dehydrate the mouse lung tissues with a gradient concentration of tetrahydrofuran solution, degrease them with dichloromethane to make the samples close to transparent, and match the refractive index with dibenzyl ether.

[0077] In this embodiment, the method of dehydrating the mouse lung tissues with a gradient concentration of tetrahydrofuran solution and degreasing them with dichloromethane to make the samples close to transparent includes:

[0078] S401. Fix the obtained mouse lung tissues in a 4% formalin solution for 24 hours to complete the fixation process of the mouse lung tissues. Among them, the whole process of the stained mouse lung tissues needs to be protected from light.

[0079] S402. Treat the mouse lung tissues using the tetrahydrofuran concentration gradient method. When treating the mouse lung tissues using the tetrahydrofuran concentration gradient method, sequentially place the mouse lung tissues into 50%, 70%, 80% and 100% tetrahydrofuran solutions, and soak them in each concentration of tetrahydrofuran solution for 1.5 hours.

[0080] S403. Soak the mouse lung tissues in 100% dichloromethane solution for 1.5 hours to remove fat.

[0081] S404, the mouse lung tissue was immersed in 100% benzyl ether solution for 1.5 hours. The mouse lung tissue samples after treatment were stored in benzyl ether solution at 4°C. When transferring the mouse lung tissue from the dichloromethane solution to the benzyl ether solution, the operation must be quick to prevent the refractive index of the sample from matching that of the air, thereby affecting the quality of subsequent imaging.

[0082] S50, taking the treated mouse lung tissue, and using a light sheet fluorescence microscopy system to perform three-dimensional imaging of the lung tissue to obtain a three-dimensional visualization model of the blood vessels in the lung.

[0083] In this embodiment, a method for performing three-dimensional imaging of lung tissue using a light sheet fluorescence microscopy system is provided. Figure 6 Schematic diagram of three-dimensional imaging of mouse lung tissue in Example 2 is shown. Figure 7 A schematic diagram of cross-sectional imaging of mouse lung tissue in Example 2 is shown, wherein the light sheet fluorescence microscopy system is used to perform three-dimensional imaging of the lung tissue, including:

[0084] S501, start the femtosecond pulse laser and set it to emit single-photon excitation light with a wavelength of 730nm, set the vibration frequency of the scanning galvanometer to 300Hz and the amplitude to 4Vpp to form a light sheet for exciting the sample to generate a fluorescence signal, and replace the filter at the front end of the light sheet microscope with a 750&800LP filter, which is a long-pass filter;

[0085] S502, performing phase modulation using a spatial light modulator, loading phase information, and converting the light beam into two light beam forms, wherein the two light beam forms are a Gaussian beam and an Airy beam;

[0086] S503, controlling the translation stage based on the control software, so that the illumination objective lens and the detection objective lens in the imaging assembly are steadily immersed in a medium having a refractive index that matches the transparentized mouse lung tissue, to ensure that the light beam penetrates the mouse lung tissue without damage and efficiently;

[0087] S504, pouring the agarose solution containing gold clusters into the FEP tube, and placing the FEP tube at a predetermined position in the imaging area, rotating the screw micrometer head of the detection objective lens, and fine-tuning the position of the screw micrometer head until a clear and complete image is presented on the display screen of the camera, completing the focusing process, and after the focus is correct, placing the mouse lung tissue to be tested in a medium with a matching refractive index;

[0088] S505, converting the illumination beam from a Gaussian beam to an Airy beam, and after accurately framing the imaging area, immediately starting the collection of the image stack to complete the shooting.

[0089] S506. Denoise the obtained original image data, and perform deconvolution processing on the original image data using the calibrated point spread function (PSF) to restore the axial resolution of the image, and obtain the image data after deconvolution processing.

[0090] It should be noted that due to the inherent limitations of the optical imaging system, there is often a certain degree of blurring in the axially collected original image data. To solve this problem, it is necessary to perform deconvolution processing on the original image data by means of the point spread function (PSF) calibrated in advance by the system. This processing step can effectively restore the axial resolution of the image, thereby enabling us to obtain clearer and more accurate three-dimensional structure information.

[0091] S507. Import the image data after deconvolution processing into image analysis software to form an intuitive and complete three-dimensional model, and obtain a three-dimensional visualization model of the pulmonary vasculature. When importing the image data after deconvolution processing into the image analysis software, the image analysis software is Fiji-image J or Imaris.

[0092] The present invention uses fluorescence excitation and collection in the second near-infrared (NIR-II) band, which can achieve a higher imaging depth, and has better axial resolution, signal-to-background ratio, and contrast in deep imaging. On the other hand, all reagents of the present invention can be commercially obtained and the operation is simple. On the further hand, the three-dimensional imaging of pulmonary lymphatic vessels and blood vessels based on light sheet microscopy proposed by the present invention not only solves the problem of observing the fine structure of the lungs, but also realizes further application in the field of light sheet microscopy imaging.

[0093] In summary, the present invention provides a method for three-dimensional visualization of pulmonary vasculature based on light sheet fluorescence microscopy. By fluorescence excitation and collection in the second near-infrared (NIR-II) band, the present invention can achieve a higher imaging depth for the vasculature system inside the lung tissue, and has better axial resolution, signal-to-background ratio, and contrast in deep imaging. It can skillfully integrate and reconstruct numerous two-dimensional image slices to form an intuitive and complete three-dimensional visualization model of the pulmonary vasculature. By quantifying the three-dimensional model, the fine structural changes of the pulmonary vasculature can be observed more accurately.

[0094] 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.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting 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 making creative efforts fall within 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 and without making creative efforts, combine, add or delete the features in the embodiments of the present invention according to the circumstances or make other adjustments, so as to obtain different technical solutions that essentially do not deviate from the concept of the present invention, and these technical solutions also fall within the scope of protection of the present invention.

Claims

1. A three-dimensional visualization method of pulmonary vessels based on light sheet fluorescence microscopy, characterized in that: The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy comprises: S10, select healthy male mice, male mice are BALB / c mice, 6 weeks old, weighing 25±1g; S20, mice were intubated with 7.5% 2-chloroethyl ethyl sulfide CEES; S30, 18 h after administration, the mice were anesthetized and stained with 1 mg / mL cy7 solution subcutaneously. 5 min later, the mice were cardiac perfused with PBS to flush the blood, and then the lung tissues were fixed and stored in 4% PFA. S40, mouse lung tissue was dehydrated using gradient tetrahydrofuran solution, defatted using dichloromethane to make the sample nearly transparent, and dibenzyl ether was used to match the refractive index; S50, taking the treated mouse lung tissue, and using a light sheet fluorescence microscopy system to perform three-dimensional imaging of the lung tissue to obtain a three-dimensional visualization model of the blood vessels in the lung.

2. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to claim 1, characterized in that: When mice were intubated and administered with 7.5% 2-chloroethyl ethyl sulfide CEES, 97% CEES solution was diluted with anhydrous ethanol in a fume hood to obtain a 7.5% CEES solution, and then the mice were anesthetized with tribromoethanol and 6 μL of 7.5% solution was administered through the endotracheal cannula using a micropipette. After administration, the mice were monitored in the cage until they fully recovered from anesthesia, their survival status was recorded, and the quality of breathing, panting, and activity inhibition were evaluated according to the clinical performance score sheet.

3. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to claim 1, characterized in that: The method for dehydrating the mouse lung tissue using a gradient concentration tetrahydrofuran solution and defatting with dichloromethane to make the sample nearly transparent comprises: S401, the obtained mouse lung tissue was fixed in 4% formalin solution for 24 hours to complete the mouse lung tissue fixation process; S402, mouse lung tissue was treated with tetrahydrofuran concentration gradient method; S403, mouse lung tissue was immersed in 100% dichloromethane solution for 1.5 h to remove fat; S404, the mouse lung tissue was immersed in 100% benzyl ether solution for 1.5 hours, and the mouse lung tissue samples after treatment were stored in the benzyl ether solution at 4°C.

4. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to claim 3, characterized in that: When the mouse lung tissue was treated with the tetrahydrofuran concentration gradient method, the mouse lung tissue was placed in 50%, 70%, 80% and 100% tetrahydrofuran solutions in sequence, and immersed in each concentration of tetrahydrofuran solution for 1.5 hours.

5. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to any one of claims 2 to 4, characterized in that: The method of using a light sheet fluorescence microscopy system to perform three-dimensional imaging of lung tissue specifically includes: S501, start the femtosecond pulse laser and set it to emit single-photon excitation light with a wavelength of 730nm, and use a 750&800LP filter to replace the filter at the front end of the light sheet microscope; S502, performing phase modulation using a spatial light modulator, loading phase information, and converting the light beam into two light beam forms, wherein the two light beam forms are a Gaussian beam and an Airy beam; S503, controlling the translation stage based on the control software, so that the illumination objective lens and the detection objective lens in the imaging assembly are steadily immersed in a medium having a refractive index that matches the transparentized mouse lung tissue, to ensure that the light beam penetrates the mouse lung tissue without damage and efficiently; S504, pouring the agarose solution containing gold clusters into the FEP tube, and placing the FEP tube at a predetermined position in the imaging area, rotating the screw micrometer head of the detection objective lens, and fine-tuning the position of the screw micrometer head until a clear and complete image is presented on the display screen of the camera, completing the focusing process, and after the focus is correct, placing the mouse lung tissue to be tested in a medium with a matching refractive index; S505, converting the illumination beam from a Gaussian beam to an Airy beam, and after accurately framing the imaging area, immediately starting the collection of the image stack to complete the shooting.

6. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to claim 5, characterized in that: The method of using a light sheet fluorescence microscopy system to perform three-dimensional imaging of lung tissue specifically includes: S506, performing deconvolution processing on the original image data using the calibrated point spread function PSF to restore the axial resolution of the image and obtain image data after deconvolution processing; S507, importing the image data after deconvolution processing into image analysis software to form an intuitive and complete three-dimensional model, and obtaining a three-dimensional visualization model of the intrapulmonary vessels.

7. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to claim 6, characterized in that: When the image data after deconvolution processing is imported into the image analysis software, the image analysis software is Fiji-image J or Imaris.

8. The method for three-dimensional visualization of pulmonary vessels based on light sheet fluorescence microscopy according to claim 7, characterized in that: The light sheet fluorescence microscopy imaging system comprises a femtosecond pulse laser, a shooting camera, a filter, a spatial light modulator, an imaging component, a displacement stage, and a detection objective lens.

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