A Three-Dimensional Visualization Method for Intrapulmonary Vascular System Based on Light-Slide Fluorescence Microscopy

Abstract

This invention discloses a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy, belonging to the field of three-dimensional imaging technology. It solves the problem of low spatial resolution in existing imaging and ultrasound examination equipment. The method includes endotracheal intubation of mice, subcutaneous injection of Cy7 solution for staining, dehydration of mouse lung tissue using gradient concentration tetrahydrofuran solution, and defatting with dichloromethane to make the sample nearly transparent. A light-sheet fluorescence microscopy system is then used to perform three-dimensional imaging of the lung tissue. This invention, through fluorescence excitation and acquisition in the infrared II band, can achieve higher imaging depth of the pulmonary vascular system, and deeper imaging offers better axial resolution, signal-to-background ratio, and contrast. Numerous two-dimensional image slices can be integrated and reconstructed to form an intuitive and complete three-dimensional visualization model of the pulmonary vessels. By quantifying the three-dimensional model, the fine structural changes of the pulmonary vessels can be observed more accurately.

Description

Technical Field

[0001] This invention belongs to the field of three-dimensional imaging technology, specifically relating to a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy. Background Technology

[0002] Plastic bronchitis (PB) is a relatively rare and complex clinical syndrome, primarily characterized by atelectasis on imaging. The core feature of this condition is the formation of bronchial tree-like casts within the airways. These casts can cause localized or widespread obstruction, leading to symptoms such as cough, fever, and dyspnea, and in severe cases, even acute and fatal respiratory distress. The first reports of this disease date back to the early 20th century, when it was thought to be associated with purulent infections. Histopathologically, bronchial plasticity has been classified into two types: Type I, inflammatory casts, mainly composed of inflammatory cells and fibrin structures, commonly seen in bronchopulmonary diseases; and Type II, acellular casts, primarily composed of mucin, often associated with post-congenital heart disease surgery. In China, PB is often associated with infection-related diseases, while international reports are more frequently linked to post-congenital heart disease outcomes. With the widespread use of bronchoscopy, an increasing number of PB cases have been diagnosed. However, due to the lack of large-scale epidemiological surveys, the severity of PB remains unclear. Nevertheless, this disease poses a significant threat to affected children, often presenting with severe symptoms. Treatment of the underlying disease is frequently unsuccessful, leading to misdiagnosis and mistreatment, and potentially even iatrogenic harm. Therefore, early diagnosis and appropriate treatment are of paramount clinical importance for PB.

[0003] Regarding the pathogenesis, research has largely focused on the release mechanisms of inflammatory factors after PB-induced bodily deformation (PB), the possible mechanisms of hypercoagulable states, and the exploration of related high-risk factors. However, research on the deeper pathogenesis of PB formation is relatively rare. The academic community has proposed several possible mechanisms: given that PB is commonly reported after congenital heart disease surgery, especially Fontan surgery which may lead to increased pulmonary venous pressure, resulting in abnormal respiratory epithelial reactions and a state of hypermucus secretion, another hypothesis suggests a connection to lymphatic dysfunction, including increased intrathoracic lymphatic pressure, broncholymphatic fistula, and ultimately, intrabronchial lymphatic leakage.

[0004] Clinically, the diagnosis and treatment of bronchopulmonary bronchitis (PB) present numerous challenges. This is primarily because the clinical manifestations of PB lack specificity; common symptoms include cough and shortness of breath, while in more severe cases, patients may experience respiratory distress, and some may even cough up bronchial plastic material. In terms of treatment, bronchodilators often fail to provide significant improvement. Furthermore, although endotracheal intubation and mechanical ventilation can alleviate clinical symptoms to some extent, their effects are not significant, and simply attributing the condition to acute respiratory distress syndrome or lung injury is insufficient. Currently, the main treatment for PB relies on bronchoscopy, which allows for the removal of bronchial plastic material for pathological examination to confirm the diagnosis.

[0005] However, existing imaging techniques such as radiology and ultrasound have limitations in terms of observation. These methods have relatively low spatial resolution, making it difficult to provide clear observation of the microscopic structures of the lungs, thus failing to provide clear visual evidence for the diagnosis of pulmonary vasculature (PB). This further increases the difficulty for clinicians in diagnosing and treating PB. To address these issues, we propose a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy, thus solving the problem of low spatial resolution in existing imaging and ultrasound examination equipment.

[0007] This invention is implemented as follows: a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy, wherein the three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy includes:

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

[0009] S20 was administered to mice via tracheal intubation using 7.5% 2-chloroethyl ethyl sulfide CEES.

[0010] S30 was administered to mice 18 hours after administration. The mice were stained with 1 mg / mL cy7 solution via subcutaneous injection. Five minutes later, the mice were perfused with PBS to wash away the blood. The mouse lung tissue was then fixed and preserved in 4% PFA.

[0011] S40, mouse lung tissue was dehydrated using a gradient concentration of tetrahydrofuran solution and defatted with dichloromethane to make the sample nearly transparent, and dibenzyl ether was used to match the refractive index;

[0012] S50: Processed mouse lung tissue was taken, and three-dimensional imaging of the lung tissue was performed using a light-film fluorescence microscopy system to obtain a three-dimensional visualization model of the pulmonary vessels.

[0013] When administering 7.5% 2-chloroethyl ethyl sulfide (CEES) to mice via endotracheal intubation, a 97% CEES solution was diluted with anhydrous ethanol in a fume hood to obtain a 7.5% CEES solution. Mice were then anesthetized with tribromoethanol, and 6 μL of the 7.5% solution was administered via endotracheal intubation using a micropipette. Mice were monitored in cages after administration until they fully recovered from anesthesia, and their survival status was recorded. Respiratory quality, wheezing, and activity inhibition were assessed using a clinical performance rating scale.

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

[0015] S401, the obtained mouse lung tissue was fixed in 4% formalin solution for 24 hours to complete the mouse lung tissue fixation process;

[0016] S402, mouse lung tissue was treated using a tetrahydrofuran concentration gradient method;

[0017] S403, mouse lung tissue was immersed in 100% dichloromethane solution for 1.5 hours to remove fat;

[0018] S404, mouse lung tissue was soaked in 100% dibenzyl ether solution for 1.5 hours, and the treated mouse lung tissue samples were stored in dibenzyl ether solution at 4°C.

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

[0020] The method for three-dimensional imaging of 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 730nm, and replace the filter at the front end of the light sheet microscope with a 750&800LP type filter.

[0022] S502 uses a spatial light modulator for phase modulation, loads phase information, and converts the beam into two beam forms, namely a Gaussian beam and an Airy beam.

[0023] S503, based on the control software to manipulate the displacement stage, allows the illumination objective and probe objective in the imaging component to be smoothly immersed in a medium with a refractive index that matches the transparentized mouse lung tissue, ensuring that the beam penetrates the mouse lung tissue without damage and efficiently.

[0024] S504. Agarose solution containing gold clusters is poured into an FEP tube, and the FEP tube is placed at a predetermined position in the imaging area. The micrometer head of the probe lens is rotated and its position is finely adjusted until a clear and complete image is displayed on the camera screen, thus completing the focusing process. After the focus is correct, the lung tissue of the mouse to be tested is placed in a medium with a matching refractive index.

[0025] The S505 converts the illumination beam from a Gaussian beam to an Airy beam, and after accurately defining the imaging area, it immediately starts the image stack acquisition process to complete the image capture.

[0026] S506 uses a calibrated point spread function (PSF) to perform deconvolution processing on the original image data to restore the axial resolution of the image and obtain the deconvolution-processed image data.

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

[0028] When importing the deconvolution-processed image data into image analysis software, the image analysis software is either Fiji-image J or imaris.

[0029] The light-sheet fluorescence microscopy imaging system includes a femtosecond pulsed laser, a camera, a filter, a spatial light modulator, an imaging component, a displacement stage, and a probe lens.

[0030] Compared with the prior art, the embodiments of this application have the following main advantages:

[0031] This invention utilizes fluorescence excitation and acquisition in the infrared second band to achieve higher imaging depth of the vascular system within lung tissue. Deep imaging offers better axial resolution, signal-to-background ratio, and contrast. Numerous two-dimensional image slices can be cleverly integrated and reconstructed to form an intuitive and complete three-dimensional visualization model of the pulmonary vessels. By quantifying this three-dimensional model, the subtle structural changes of the pulmonary vessels can be observed more accurately. Attached Figure Description

[0032] Figure 1 This is the optical path diagram of the light-sheet fluorescence microscopy imaging system provided by the present invention.

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

[0034] Figure 3 A schematic diagram of the transparent lung tissue specimen in Example 1 is shown.

[0035] Figure 4 A three-dimensional imaging schematic diagram of mouse lung tissue in Example 1 is shown.

[0036] Figure 5 A schematic diagram of a cross-sectional image of mouse lung tissue in Example 1 is shown.

[0037] Figure 6 A three-dimensional imaging schematic diagram of mouse lung tissue in Example 2 is shown.

[0038] Figure 7 A schematic diagram of a cross-sectional image of mouse lung tissue in Example 2 is shown. Detailed Implementation

[0039] 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 herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0040] Existing methods such as imaging and ultrasound examinations have certain limitations. These methods have relatively low spatial resolution, making it difficult to provide clear observation of the microscopic structure of the lungs. To address these issues, we propose a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy. In this method, mice are first intubated and stained with 1 mg / mL Cy7 solution via subcutaneous injection. Then, the mouse lung tissue is dehydrated using a gradient concentration of tetrahydrofuran solution and defatted with dichloromethane until the sample is nearly transparent. Finally, a light-sheet fluorescence microscopy system is used to perform three-dimensional imaging of the lung tissue, obtaining a three-dimensional visualization model of the pulmonary vessels. This invention, through fluorescence excitation and acquisition in the infrared II band, can achieve higher imaging depth of the pulmonary vascular system. Deep imaging offers better axial resolution, signal-to-background ratio, and contrast. Numerous two-dimensional image slices can be cleverly integrated and reconstructed to form an intuitive and complete three-dimensional visualization model of the pulmonary vessels. By quantifying the three-dimensional model, the fine structural changes of the pulmonary vessels can be observed more accurately.

[0041] This invention focuses on three-dimensional imaging of pulmonary vessels by labeling them with injected fluorescent probes. This process involves immobilizing the fluorescent probes within the vessels, followed by tissue transparency treatment to allow laser penetration of the entire lung tissue for imaging. This technology enables three-dimensional imaging of blood vessels and lymphatic vessels in larger lung tissue samples, providing more intuitive and detailed structural information for the study of the mechanism and fine structure of pulmonary vasculature (PB).

[0042] Example 1

[0043] This invention provides a method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy. Specifically, this method includes:

[0044] S10. Select healthy male mice. The male mice are BALB / c mice, 6 weeks old, and weigh 25±1g.

[0045] S20 was administered to mice via tracheal intubation using 7.5% 2-chloroethyl ethyl sulfide CEES.

[0046] In this embodiment, when administering 7.5% 2-chloroethyl ethyl sulfide (CEES) to mice via endotracheal intubation, a 97% CEES solution was diluted with anhydrous ethanol in a fume hood to obtain a 7.5% CEES solution. The mice were then anesthetized with tribromoethanol, and 6 μL of the 7.5% solution was administered via endotracheal intubation using a micropipette. Following administration, the mice were monitored in cages until they fully recovered from anesthesia, and their survival status was recorded. Respiratory quality, wheezing, and activity inhibition were assessed using a clinical performance rating scale.

[0047] S30 was administered to mice 18 hours after administration. The mice were stained with 1 mg / mL cy7 solution via subcutaneous injection. Five minutes later, the mice were perfused with PBS to wash away the blood. The mouse lung tissue was then fixed and preserved in 4% PFA.

[0048] It should be noted that the mice were anesthetized 18 hours after administration. The cy7 was prepared by dissolving 1 mg of cy7 in 1 mL of PBS. The dye was then injected into the limbs of the mice. After waiting for 5 minutes, the mice were dissected, and the heart was perfused with about 30 mL of PBS. The lung tissue of the mice was excised and fixed and preserved in 4% PFA for 24 hours.

[0049] The sampling criteria were based on monitoring the respiratory status of mice after drug administration. If mice showed signs of impending death, such as weight loss exceeding 25% or inability to eat or drink, ethical standards required euthanasia before the planned study was terminated. Terminal anesthesia was performed 18 hours after administration of each CEES dose, and complete mouse lung tissue samples were removed after staining and marking.

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

[0051] In this embodiment, the method for dehydrating the mouse lung tissue using a gradient concentration of tetrahydrofuran solution and defatting it 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. The entire process, including staining the mouse lung tissue, required protection from light. Figure 2 A schematic diagram of the fixed lung tissue specimen in Example 1 is shown;

[0053] S402, mouse lung tissue was treated using a tetrahydrofuran concentration gradient method. In this method, mouse lung tissue was placed in 50%, 70%, 80%, and 100% tetrahydrofuran solutions, and each solution was soaked for 1.5 hours.

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

[0055] S404: Mouse lung tissue was immersed in 100% dibenzyl ether solution for 1.5 hours. After treatment, the mouse lung tissue samples were stored in dibenzyl ether solution at 4°C. When transferring mouse lung tissue from dichloromethane solution to dibenzyl ether solution, the operation must be rapid to prevent the refractive index of the sample from matching with that of air, which could affect the quality of subsequent imaging. Figure 3 A schematic diagram of the transparent lung tissue specimen in Example 1 is shown.

[0056] S50: Processed mouse lung tissue was taken, and three-dimensional imaging of the lung tissue was performed using a light-film fluorescence microscopy system to obtain a three-dimensional visualization model of the pulmonary vessels.

[0057] In this embodiment, a method for three-dimensional imaging of lung tissue using a light-sheet fluorescence microscopy system is provided. Figure 4 A three-dimensional imaging schematic diagram of mouse lung tissue in Example 1 is shown. Figure 5 This diagram illustrates a cross-sectional image of mouse lung tissue in Example 1. The method of using a light-sheet fluorescence microscopy system to perform three-dimensional imaging of the lung tissue includes:

[0058] S501, start the femtosecond pulsed 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, which is used to excite the sample to generate a fluorescence signal. Replace the filter at the front end of the light sheet microscope with a 750&800LP type filter. The filter is a long-pass filter.

[0059] S502 uses a spatial light modulator for phase modulation, loads phase information, and converts the beam into two beam forms, namely a Gaussian beam and an Airy beam.

[0060] In this embodiment, a spatial light modulator is used to load specific phase information. Through this phase modulation, the light beam can be converted into two forms: a Gaussian beam for precise focusing in the initial stage, and an Airy beam for subsequent imaging. Precise manipulation of the displacement stage by the control software ensures that the imaging components, namely the illumination and probe objectives, can be smoothly immersed in a medium with a refractive index matching the transparent sample. This step is crucial, as it ensures that the light beam can penetrate the mouse lung tissue sample without damage and efficiently.

[0061] S503, based on the control software to manipulate the displacement stage, allows the illumination objective and probe objective in the imaging component to be smoothly immersed in a medium with a refractive index that matches the transparentized mouse lung tissue, ensuring that the beam penetrates the mouse lung tissue without damage and efficiently.

[0062] S504. Agarose solution containing gold clusters is poured into an FEP tube, and the FEP tube is placed at a predetermined position in the imaging area. The micrometer head of the probe lens is rotated and its position is finely adjusted until a clear and complete image is displayed on the camera screen, thus completing the focusing process. After the focus is correct, the lung tissue of the mouse to be tested is placed in a medium with a matching refractive index.

[0063] The S505 converts the illumination beam from a Gaussian beam to an Airy beam, and after accurately defining the imaging area, it immediately starts the image stack acquisition process to complete the image capture.

[0064] S506, the obtained original image data is processed to remove noise, and the original image data is deconvolved using a calibrated point spread function (PSF) to restore the axial resolution of the image and obtain the deconvolved image data.

[0065] It should be noted that due to the inherent limitations of optical imaging systems, the acquired raw image data often exhibits a certain degree of blurring along the axial direction. To address this issue, it is necessary to use a pre-calibrated point spread function (PSF) to perform deconvolution processing on the raw image data. This processing step effectively restores the axial resolution of the image, thereby enabling us to obtain clearer and more accurate three-dimensional structural information.

[0066] S507 imports the deconvolution-processed image data into image analysis software to form an intuitive and complete 3D model, obtaining a 3D visualization model of the pulmonary vessels. The image analysis software used for importing the deconvolution-processed image data is either Fiji-image J or imaris.

[0067] It should be noted that the light-sheet fluorescence microscopy imaging system includes a femtosecond pulsed laser, a camera, a filter, a spatial light modulator, an imaging assembly, a displacement stage, and a probe objective. Figure 1 The optical path diagram of a light-sheet fluorescence microscopy system is shown. A femtosecond pulsed laser is the system's light source, providing high-energy, ultrashort pulses of laser light. The output laser first passes through a filter to select an appropriate wavelength for exciting the sample. The filter's function is to select a specific wavelength to match the excitation spectrum of the fluorescent dye in the sample. The filtered laser is then guided to a spatial light modulator, which modulates the phase of 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 to illuminate the sample. A camera (such as an sCMOS or EMCCD camera) is positioned in the imaging path of the probe objective to capture the fluorescence signal and convert it into a digital image. The two-dimensional image recorded by the camera is then used for three-dimensional reconstruction using software.

[0068] The invention mainly includes the following aspects: probe injection and selection of appropriate fluorescent dyes: selecting fluorescent dyes with high brightness and good stability, such as Cy7, to ensure that good fluorescence signals are maintained in the tissue after clearing treatment; it also includes a tissue clearing step: this step can remove the opacity of the tissue, allowing the subsequent imaging process to proceed smoothly. For example, lung tissue cleared using the 3DISCO method will become nearly transparent, allowing light to penetrate the tissue deeply, making high-resolution imaging possible; during the staining process, the tissue needs to be properly fixed, and formalin fixation can ensure the accuracy of fluorescent labeling; adjusting the light-sheet fluorescence microscope and selecting appropriate filters for imaging. Through these details, more precise three-dimensional structural information of the pulmonary vascular system can be obtained, including the distribution, morphology, and interconnection of blood vessels and lymphatic vessels.

[0069] Example 2

[0070] This invention provides a method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy. Specifically, this method includes:

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

[0072] S20, administering medication to mice via tracheal intubation using physiological saline;

[0073] When administering medication to mice via tracheal intubation using physiological saline, the mice were anesthetized with tribromoethanol, and after anesthesia, 6 μL of physiological saline was injected into the trachea using a micro-syringe as a control.

[0074] S30 was administered to mice 18 hours after administration. The mice were stained with 1 mg / mL cy7 solution via subcutaneous injection. Five minutes later, the mice were perfused with PBS to wash away the blood. The mouse lung tissue was then fixed and preserved in 4% PFA.

[0075] It should be noted that the mice were anesthetized 18 hours after administration. The cy7 was prepared by dissolving 1 mg of cy7 in 1 mL of PBS. The dye was then injected into the limbs of the mice. After waiting for 5 minutes, the mice were dissected, and the heart was perfused with about 30 mL of PBS. The lung tissue of the mice was excised and fixed and preserved in 4% PFA for 24 hours.

[0076] S40, mouse lung tissue was dehydrated using a gradient concentration of tetrahydrofuran solution and defatted with dichloromethane to make the sample nearly transparent, and dibenzyl ether was used to match the refractive index;

[0077] In this embodiment, the method for dehydrating the mouse lung tissue using a gradient concentration of tetrahydrofuran solution and defatting it with dichloromethane to make the sample nearly transparent includes:

[0078] S401, the obtained mouse lung tissue was fixed in 4% formalin solution for 24 hours to complete the fixation process of mouse lung tissue. The entire process of fixing the mouse lung tissue after staining should be done in the dark.

[0079] S402, mouse lung tissue was treated using a tetrahydrofuran concentration gradient method. In this method, mouse lung tissue was placed in 50%, 70%, 80%, and 100% tetrahydrofuran solutions, and each solution was soaked for 1.5 hours.

[0080] S403, mouse lung tissue was immersed in 100% dichloromethane solution for 1.5 hours to remove fat;

[0081] S404: Mouse lung tissue was immersed in 100% dibenzyl ether solution for 1.5 hours. After processing, the mouse lung tissue samples were stored in dibenzyl ether solution at 4°C. When transferring mouse lung tissue from dichloromethane solution to dibenzyl ether solution, the operation must be rapid to prevent the refractive index of the sample from matching with that of air, which would affect the quality of subsequent imaging.

[0082] S50: Processed mouse lung tissue was taken, and three-dimensional imaging of the lung tissue was performed using a light-film fluorescence microscopy system to obtain a three-dimensional visualization model of the pulmonary vessels.

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

[0084] S501, start the femtosecond pulsed 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, which is used to excite the sample to generate a fluorescence signal. Replace the filter at the front end of the light sheet microscope with a 750&800LP type filter. The filter is a long-pass filter.

[0085] S502 uses a spatial light modulator for phase modulation, loads phase information, and converts the beam into two beam forms, namely a Gaussian beam and an Airy beam.

[0086] S503, based on the control software to manipulate the displacement stage, allows the illumination objective and probe objective in the imaging component to be smoothly immersed in a medium with a refractive index that matches the transparentized mouse lung tissue, ensuring that the beam penetrates the mouse lung tissue without damage and efficiently.

[0087] S504. Agarose solution containing gold clusters is poured into an FEP tube, and the FEP tube is placed at a predetermined position in the imaging area. The micrometer head of the probe lens is rotated and its position is finely adjusted until a clear and complete image is displayed on the camera screen, thus completing the focusing process. After the focus is correct, the lung tissue of the mouse to be tested is placed in a medium with a matching refractive index.

[0088] The S505 converts the illumination beam from a Gaussian beam to an Airy beam, and after accurately defining the imaging area, it immediately starts the image stack acquisition process to complete the image capture.

[0089] S506, the obtained original image data is processed to remove noise, and the original image data is deconvolved using a calibrated point spread function (PSF) to restore the axial resolution of the image and obtain the deconvolved image data.

[0090] It should be noted that due to the inherent limitations of optical imaging systems, the acquired raw image data often exhibits a certain degree of blurring along the axial direction. To address this issue, it is necessary to use a pre-calibrated point spread function (PSF) to perform deconvolution processing on the raw image data. This processing step effectively restores the axial resolution of the image, thereby enabling us to obtain clearer and more accurate three-dimensional structural information.

[0091] S507 imports the deconvolution-processed image data into image analysis software to form an intuitive and complete 3D model, obtaining a 3D visualization model of the pulmonary vessels. The image analysis software used for importing the deconvolution-processed image data is either Fiji-image J or imaris.

[0092] This invention employs fluorescence excitation and acquisition in the infrared second band, achieving higher imaging depth and providing better axial resolution, signal-to-background ratio, and contrast at deeper depths. Furthermore, all reagents used in this invention are commercially available, and the operation is simple. Moreover, the three-dimensional imaging of pulmonary lymphatic vessels and blood vessels based on light-sheet microscopy proposed in this invention not only solves the problem of observing the fine structures of the lungs but also achieves further application in the field of light-sheet microscopy.

[0093] In summary, this invention provides a three-dimensional visualization method for pulmonary vessels based on light-sheet fluorescence microscopy. By utilizing fluorescence excitation and acquisition in the infrared second band, this invention achieves higher imaging depth of the pulmonary vascular system, offering better axial resolution, signal-to-background ratio, and contrast at deeper depths. It also allows for the ingenious integration and reconstruction of numerous two-dimensional image slices to form an intuitive and complete three-dimensional visualization model of the pulmonary vessels. By quantifying this three-dimensional model, the subtle structural changes of the pulmonary vessels can be observed more accurately.

[0094] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are 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 to limit the scope of protection of the invention. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also fall within the scope of protection of the present invention.

Claims

1. A method for three-dimensional visualization 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 includes: S10. Select healthy male mice. The male mice are BALB / c mice, 6 weeks old, and weigh 25±1g. S20 was administered to mice via tracheal intubation using 7.5% 2-chloroethyl ethyl sulfide CEES. S30 was administered to mice 18 hours after administration. The mice were stained with 1 mg / mL cy7 solution via subcutaneous injection. Five minutes later, the mice were perfused with PBS to wash away the blood. The mouse lung tissue was then fixed and preserved in 4% PFA. S40, mouse lung tissue was dehydrated using a gradient concentration of tetrahydrofuran solution and defatted with dichloromethane to make the sample nearly transparent, and dibenzyl ether was used to match the refractive index; S50, processed mouse lung tissue was taken and three-dimensional imaging of the lung tissue was performed using a light-sheet fluorescence microscopy system to obtain a three-dimensional visualization model of the pulmonary vessels. The method for three-dimensional imaging of lung tissue using a light-sheet fluorescence microscopy system specifically includes: S501, start the femtosecond pulsed laser and set it to emit single-photon excitation light with a wavelength of 730nm, and replace the filter at the front end of the light sheet microscope with a 750&800LP type filter. S502 uses a spatial light modulator for phase modulation, loads phase information, and converts the beam into two beam forms, namely a Gaussian beam and an Airy beam. S503, based on the control software to manipulate the displacement stage, allows the illumination objective and probe objective in the imaging component to be smoothly immersed in a medium with a refractive index that matches the transparentized mouse lung tissue, ensuring that the beam penetrates the mouse lung tissue without damage and efficiently. S504. Agarose solution containing gold clusters is poured into an FEP tube, and the FEP tube is placed at a predetermined position in the imaging area. The micrometer head of the probe lens is rotated and its position is finely adjusted until a clear and complete image is displayed on the camera screen, thus completing the focusing process. After the focus is correct, the lung tissue of the mouse to be tested is placed in a medium with a matching refractive index. The S505 converts the illumination beam from a Gaussian beam to an Airy beam, and after accurately defining the imaging area, it immediately starts the image stack acquisition process to complete the image capture. S506 uses a calibrated point spread function (PSF) to perform deconvolution processing on the original image data to restore the axial resolution of the image and obtain the deconvolution-processed image data. S507 imports the deconvolution-processed image data into image analysis software to form an intuitive and complete three-dimensional model, obtaining a three-dimensional visualization model of the pulmonary vessels.

2. The method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy as described in claim 1, characterized in that: When administering 7.5% 2-chloroethyl ethyl sulfide (CEES) to mice via endotracheal intubation, dilute 97% CEES solution with anhydrous ethanol in a fume hood to obtain a 7.5% CEES solution. Subsequently, anesthetize the mice with tribromoethanol and administer 6 μL of the 7.5% solution via endotracheal intubation using a micropipette. Monitor the mice in cages after administration until they fully recover from anesthesia, record their survival status, and assess respiratory quality, wheezing, and activity inhibition using a clinical performance rating scale.

3. The method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy as described in claim 1, characterized in that: The method for dehydrating mouse lung tissue using gradient concentration tetrahydrofuran solution and defatting with dichloromethane to make the sample nearly transparent includes: 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 using a tetrahydrofuran concentration gradient method; S403, mouse lung tissue was immersed in 100% dichloromethane solution for 1.5 hours to remove fat; S404, mouse lung tissue was immersed in 100% dibenzyl ether solution for 1.5 hours, and the treated mouse lung tissue samples were stored in dibenzyl ether solution at 4°C.

4. The method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy as described in claim 3, characterized in that: When treating mouse lung tissue using the tetrahydrofuran concentration gradient method, the mouse lung tissue was placed in 50%, 70%, 80%, and 100% tetrahydrofuran solutions sequentially, and each concentration of tetrahydrofuran solution was soaked for 1.5 hours.

5. The method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy as described in any one of claims 2-4, characterized in that: When importing the deconvolution-processed image data into image analysis software, the image analysis software is either Fiji-image J or imaris.

6. The method for three-dimensional visualization of pulmonary vessels based on light-sheet fluorescence microscopy as described in claim 5, characterized in that: The light-sheet fluorescence microscopy imaging system includes a femtosecond pulsed laser, a camera, a filter, a spatial light modulator, an imaging component, a displacement stage, and a probe lens.

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