Application of second harmonic imaging in collagen detection

Abstract

The invention relates to an application of second harmonic imaging in collagen detection. The application comprises the following steps: irradiating a sample containing collagen by utilizing near-infrared laser, collecting a second harmonic signal generated by the collagen in the sample, and carrying out imaging and quantitative analysis on collagenous fibers based on the second harmonic signal. A collagen detection scheme based on secondary harmonic imaging is developed, samples are not damaged, low photobleaching and phototoxicity are achieved, the resolution ratio is high, dynamic ultra-long space-time in-vivo imaging can be achieved, a scheme for detecting myocardial fibrosis is further developed, in-situ and in-vivo space-time dynamic imaging on myocardial fibrosis is achieved for the first time, and the method has the advantages of being high in sensitivity, high in sensitivity and the like. An important tool is provided for accurate detection of myocardial fibrosis, and the method can be effectively applied to construction of products for detecting myocardial fibrosis, screening / evaluation of therapeutic drugs and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to application of second harmonic generation imaging in detection of collagen. BACKGROUND

[0002] After myocardial infarction, the structural integrity of the heart wall is damaged, leading to exposure of fibroblasts to an abnormal mechanical stress environment. Under the synergistic action of growth factors and cytokines, especially the key profibrotic factor transforming growth factor-β (TGF-β), fibroblasts are activated and proliferate, migrate to the infarct area, and then differentiate into myocardial fibroblasts (MF). MF does not exist in normal myocardial tissue, but is transformed from cardiac fibroblasts after myocardial infarction, showing a phenotype with characteristics of both fibroblasts and smooth muscle cells. MF has the ability to synthesize and secrete a large amount of extracellular matrix proteins. Among them, type I and type III collagen are the main structural components of myocardial extracellular matrix, which not only provide mechanical support for myocardium, but also participate in the transmission of mechanical signals and help maintain the orderly contraction of the heart. The steady state of collagen metabolism is precisely regulated by matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs). MMPs are responsible for degrading extracellular matrix, while TIMPs inhibit MMPs activity, and both maintain the dynamic balance of collagen synthesis and degradation. After myocardial infarction, TGF-β1 upregulates the expression of TIMPs and significantly inhibits the activity of MMPs, leading to blocked collagen degradation, and thus causing excessive deposition of collagen. During this process, the ratio of type I and type III collagen abnormally increases and arranges in disorder, ultimately promoting myocardial fibrosis and heart failure.

[0003] Second harmonic generation (SHG) technology can label-free image non-centrosymmetric biological tissues, and has become an important means of life science research. Harmonic imaging does not require any fluorescent labeling of the sample to achieve imaging, which maximizes the original state of the sample and avoids the interference and damage that may be caused by the labeling process. This technology has high spatial and temporal resolution, and can accurately capture and analyze the subtle structure and rapid dynamic changes of the sample. In addition, since its imaging principle is based on nonlinear optical effects, the photon energy used is low, thereby greatly reducing the problems of photobleaching and phototoxicity in traditional optical imaging, and enabling long-time dynamic observation of living samples, providing technical support for non-invasive, long-term, high-fidelity in vivo imaging of life processes.

[0004] In conclusion, developing a technical solution for detecting collagen, especially myocardial fibrosis, using second harmonic imaging technology is of great significance for preparing detection products for early myocardial fibrosis and for screening and evaluating therapeutic drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides the application of second harmonic imaging in collagen detection, aiming to achieve efficient and accurate collagen detection and provide high-resolution, high-specificity, label-free, and dynamically repeatable precise visualization and quantitative analysis of early myocardial fibrosis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the application of second harmonic imaging in the detection of collagen, characterized in that the application includes:

[0008] A sample containing collagen is irradiated with a near-infrared laser, and the second harmonic signal generated by the collagen in the sample is collected. Based on the second harmonic signal, the collagen fibers are imaged and quantitatively analyzed.

[0009] This invention develops a collagen detection scheme based on second harmonic imaging, which adopts a completely label-free, endogenous optical detection mechanism that does not damage the sample, has low photobleaching and phototoxicity, and has high resolution, enabling dynamic ultra-long-term spatiotemporal in vivo imaging.

[0010] Optionally, the infrared laser includes an infrared femtosecond laser.

[0011] Optionally, the wavelength of the near-infrared laser is 800~1300 nm, preferably 1000~1100 nm, and more preferably 1040~1050 nm.

[0012] Optionally, a two-photon excitation microscope is used to collect the second harmonic signal generated by collagen in the sample, and to image and quantify the collagen fibers.

[0013] Optionally, the two-photon excitation microscope is equipped with a hybrid detector.

[0014] Optionally, the objective lens of the two-photon excitation microscope is a 20× water mirror; the resolution is 1024×1024 pixels.

[0015] Optionally, the hybrid detector includes a Power HyD NDD hybrid detector.

[0016] Optionally, the quantitative analysis includes:

[0017] The area of ​​myocardial fibrosis was statistically analyzed, and the percentage of SHG fibrosis area was calculated. SHG fibrosis area percentage (%) = myocardial fibrosis area / total heart area;

[0018] Obtain collagen fiber parameters, including length and width (reflecting the characteristics of collagen fibers during the progression of myocardial fibrosis).

[0019] Secondly, the present invention provides a method for detecting myocardial fibrosis for non-disease diagnosis and / or treatment purposes, the method comprising the following steps:

[0020] (1) Irradiate heart tissue samples with near-infrared laser;

[0021] (2) Collect the second harmonic signal generated by collagen in the sample;

[0022] (3) Based on the second harmonic signal, collagen fibers are imaged and quantitatively analyzed to detect and evaluate myocardial fibrosis.

[0023] This invention presents a method specifically designed for detecting myocardial fibrosis. It employs a completely label-free, endogenous optical detection mechanism, combined with the low phototoxicity and high penetration of near-infrared femtosecond lasers. This enables both in vitro and in vivo detection, allowing for long-term, repeatable, and dynamic in vivo observation of collagen deposition and remodeling processes in living animal hearts. It not only provides images but also, through Z-axis tomography and polarization analysis, extracts key morphological parameters of collagen fibers (such as density, diameter, alignment, and infiltration depth) in a three-dimensional and quantitative manner. This provides objective and accurate data for precisely assessing the degree and type of fibrosis, enabling accurate identification of early, diffuse fibrosis. It can be effectively applied to the development of products for detecting myocardial fibrosis and the screening / evaluation of therapeutic drugs.

[0024] Optionally, the heart tissue sample includes a living heart or a slice of ex vivo heart tissue.

[0025] Optionally, the wavelength of the near-infrared laser is 800~1300 nm, preferably 1000~1100 nm, and more preferably 1040~1050 nm.

[0026] Optionally, a two-photon excitation microscope is used to collect the second harmonic signal generated by collagen in the sample, and to image and quantify the collagen fibers.

[0027] Optionally, the two-photon excitation microscope is equipped with a hybrid detector.

[0028] Thirdly, the present invention provides the application of second harmonic imaging in the preparation of products for the diagnosis or monitoring of myocardial fibrosis.

[0029] Optionally, the product may include a complete solution including an imaging system, analysis software, and operating procedure guide.

[0030] Fourthly, the present invention provides a system for detecting myocardial fibrosis, the system comprising:

[0031] (1) Excitation unit, used to emit near-infrared laser;

[0032] (2) Scanning unit, used for focusing near-infrared laser scanning onto the heart sample;

[0033] (3) Detection unit, used to collect the second harmonic signal generated by collagen in heart samples;

[0034] (4) A data processing unit, used to generate an image of collagen fibers based on the second harmonic signal and to quantitatively calculate and evaluate one or more morphological parameters of myocardial fibrosis.

[0035] Optionally, the morphological parameters include at least one of density, diameter, alignment direction, or infiltration depth.

[0036] Optionally, the quantitative calculation and assessment of myocardial fibrosis includes: calculating the myocardial fibrosis area and the percentage of SHG fibrosis area, where SHG fibrosis area percentage (%) = myocardial fibrosis area / total heart area; and extracting collagen fiber parameters, including length, width, and other characteristics of collagen fibers that reflect the progression of myocardial fibrosis.

[0037] Fifthly, the present invention provides the application of the method for detecting myocardial fibrosis described in the second aspect or the system for detecting myocardial fibrosis described in the fourth aspect in evaluating the efficacy of anti-myocardial fibrosis drugs, wherein the evaluation includes comparing changes in the second harmonic signal parameters of collagen fibers before and after drug administration to evaluate drug efficacy.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention develops a collagen detection scheme based on second harmonic imaging, employing a completely label-free, endogenous optical detection mechanism that does not damage the sample, exhibits low photobleaching and phototoxicity, and boasts high resolution, enabling dynamic ultra-long-term spatiotemporal in vivo imaging. Furthermore, it develops a scheme for detecting myocardial fibrosis, achieving for the first time in situ, in vivo, spatiotemporal dynamic imaging of myocardial fibrosis. This provides an important tool for the accurate detection of fibrosis, the differentiation between active and end-stage diseases, and prognostic risk assessment. It can be effectively applied to the development of products for detecting myocardial fibrosis and the screening / evaluation of therapeutic drugs. Attached Figure Description

[0040] Figure 1The images show the results of TGF-β1 stimulation of CFs. Figure A shows the effect of TGF-β1 induction on the expression level of Collagen-I protein in cells after 24 h and 48 h. Figure B shows the immunofluorescence staining and quantitative analysis results of Collagen-I protein in cells after 48 h of TGF-β1 induction.

[0041] Figure 2 To verify the reliability of the second harmonic distortion (HHD) detection at the tissue level, Figure A shows the echocardiograms of the sham-operated group and mice 14 days after myocardial infarction, as well as the left ventricular short-axis shortening rate and left ventricular ejection fraction; Figure B shows the Masson trichrome staining images of transverse sections of the left ventricle in the sham-operated group and mice at 2, 4, 6, 8, and 10 days after myocardial infarction, and the HHD detection results; Figure C shows the quantitative analysis of fibrosis area at 2, 4, 6, 8, and 10 days after myocardial infarction in mice, and the analysis of changes in the length and width of collagen fibers at 4 and 10 days after myocardial infarction in mice.

[0042] Figure 3 The results of the in vivo verification of the reliability of harmonic detection for myocardial fibrosis are shown in Figure A, which is a schematic diagram of second harmonic detection for in vivo mouse cardiac fibrosis; Figure B is the second harmonic detection diagram and 3D diagram of in vivo mouse cardiac fibrosis; Figure C is the second harmonic detection 3D diagram of in vivo mouse cardiac fibrosis at 4, 8 and 14 days after myocardial infarction.

[0043] Figure 4A The image shows the results of detecting type I collagen using second harmonic signals.

[0044] Figure 4B The image shows the results of detecting type III collagen using second harmonic signals.

[0045] Figure 4C The image shows the results of detecting type IV collagen using second harmonic signals.

[0046] Figure 4D The image shows the results of detecting X-type collagen using second harmonic signals.

[0047] Figure 4E The image shows the results of detecting type XII collagen using second harmonic signals.

[0048] Figure 4F The image shows the results of detecting bovine serum albumin using second harmonic signals.

[0049] Figure 4G The image shows the results of exenatide detection using second harmonic signals. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0051] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0052] Example 1

[0053] This embodiment describes the isolation and culture of primary cardiac fibroblasts (CFs).

[0054] Ten 3-day-old SD rats were sacrificed and then soaked in 75% ethanol. The lower edge of the sternum was held by tissue forceps, and tissue scissors were used to cut along the lower edge of the sternum to expose the heart. The ventricles were removed using ophthalmic scissors, and excess vascular tissue was trimmed. After washing with PBS, the heart tissue was transferred to a culture dish containing DMEM and quickly minced using small straight scissors. The heart tissue was transferred to centrifuge tubes, and digestive enzymes were added for repeated digestion. Digestion was stopped when the tissue changed from reddish-white to translucent and the tissue fragments completely disappeared. Undigested tissue fragments and collagen were filtered through a disposable cell filter, and the filtered cell suspension was collected in a sterile centrifuge tube. The cells were centrifuged at 2000 rpm for 8 minutes to pellet the cells. The supernatant was discarded, culture medium was added, and the cells were gently pipetted to prepare a cell suspension. The cell suspension was then seeded into labeled culture flasks. The culture flasks were incubated at 37°C for 1.5–2 hours, and cell lines (CFs) were isolated using the differential adhesion method. Third-generation cells were used in the experiment. CFs in the TGF-β1 stimulation group were treated with culture medium containing TGF-β1 (10 nM) for 24 h and 48 h, respectively. After the intervention, subsequent experiments were carried out.

[0055] Example 2

[0056] This embodiment performs protein blot analysis.

[0057] (1) Extraction of cell proteins: The RIPA lysis buffer, which was at -20°C, was moved to room temperature until completely thawed before use. The thawed RIPA buffer was used to dilute the PMSF stock solution to a final concentration of 1 mmol / L. Ice was prepared in advance, and the items needed for protein extraction were placed on ice. The entire protein extraction process was carried out at low temperature. 200 μL of RIPA lysis buffer was added to the CFs. Lysis was carried out at low temperature for 10 minutes. Cell detachment was observed under a microscope. Cells were scraped off with a cell scraper, and the cell suspension was collected by pipette and transferred to a centrifuge tube. After sonication, the cells were centrifuged at 4°C, 14000 g, for 10 minutes. The cell supernatant was transferred to a new centrifuge tube, and 5×SDS-PAGE protein loading buffer (BL502A, Biosharp) was added. The tube was then boiled in water for 10 minutes. After cooling, the tube was stored at -80°C.

[0058] (2) Electrophoresis: Select an SDS-PAGE gel with a molecular weight matching the protein and load it into the electrophoresis tank. Fill the electrophoresis tank with electrophoresis buffer (PM5060, Collabor). Estimate the sample loading amount based on the protein concentration. Set the voltage to 80V. When the protein reaches the separating gel, adjust the voltage to 120V. Stop electrophoresis when the protein reaches 1 cm from the bottom edge of the gel.

[0059] (3) Transfer: Remove the SDS-PAGE gel after electrophoresis and cut off the gel from the desired protein region. Open the transfer clamps in the following order: black plate (bottom) - black sponge - white filter paper - gel - PVDF membrane - white filter paper - black sponge - white plate (top). Finally, fasten the clips and place the membrane in the transfer tank, filling it with transfer buffer (PM5070, Collabor). Maintain a constant current of 300mA for 1 hour. After the transfer is complete, remove the PVDF membrane and wash it three times in 1×TBST (5 minutes, 10 minutes, and 15 minutes).

[0060] (4) Sealing: Immerse the PVDF membrane in a rapid sealing solution for 2 hours.

[0061] (5) Incubation with primary antibody: Prepare the appropriate concentration of Collagen I antibody (A22090, ABclonal) incubation solution according to the antibody instructions, and incubate overnight at 4°C. After incubation, wash three times in 1×TBST (for 5 minutes, 10 minutes, and 15 minutes).

[0062] (6) Incubation of secondary antibody: Prepare the secondary antibody incubation solution according to the instructions of the primary antibody. Place the PVDF membrane on a horizontal workbench, blot dry the membrane, and spread the incubation solution evenly on the membrane. Cover with the black box and let stand at room temperature for 1 hour. After incubation, wash the membrane three times in 1×TBST (for 5 minutes, 10 minutes, and 15 minutes).

[0063] (7) Development: Prepare the chemiluminescent solution (MA0186-1, Meilunbio) by mixing solution A and solution B in a ratio of 1:1. Immerse the membrane in the chemiluminescent solution, manually adjust the exposure time, observe the imaging results, and save the image.

[0064] Example 3

[0065] In this embodiment, the cells were subjected to immunofluorescence staining.

[0066] Cells were seeded onto coverslips. When the cells reached approximately 80%-90% confluence, they were washed three times with PBS on a shaker and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After washing three times for 5 minutes each time, 200 μL of 0.5% Triton X-100 permeation buffer was added to the cell sample and incubated at room temperature for 30 minutes. After washing three times with PBS for 5 minutes each time, 200 μL of BSA blocking buffer was added to the CFs and the mixture was incubated at room temperature for 1 hour. After washing three times with PBS for 5 minutes each time, the primary antibody (ab138492, recombinant Anti-Collagen I antibody) was diluted with PBS according to the manufacturer's instructions. The coverslips were placed flat in a humidified chamber, and an appropriate amount of primary antibody dilution buffer was added. The mixture was incubated overnight at 4°C. After washing three times with PBS for 5 minutes each time, the secondary antibody was diluted with PBS. The mixture was then transferred to a dark chamber, and 60 μL of fluorescent secondary antibody dilution buffer was added. The mixture was incubated at room temperature in a humidified chamber for 2 hours. Discard the secondary antibody, wash three times with PBS on a shaker for 5 minutes each time, aspirate the PBS, add DAPI staining solution to the coverslip, and react at room temperature in the dark for 5 minutes. Wash the coverslip three times with PBS for 5 minutes each time, and mount with anti-fluorescence quenching mounting solution. Observe the slide and acquire images using a confocal microscope.

[0067] Example 4

[0068] This embodiment constructs a myocardial fibrosis model.

[0069] (1) Preparation: Select several healthy male Kunming mice that are three weeks old and weigh 18-22 g. Anesthetize the mice by intraperitoneal injection of 1.25% aphthylamine (LAT20250416) at a dose of 0.2 mL / 10 g. Remove the hair from the chest of the mice with depilatory cream. Fix the mice on a mouse board and disinfect the surgical site with povidone-iodine.

[0070] (2) Chest ligation: Cut the skin of the mouse's left chest about 1 cm, use a bent hemostat to separate the muscles above the sternum, exposing the third and fourth ribs. Observe the exact location of the heart in the intercostal space, use forceps to separate the intercostal muscles, squeeze out the heart, and use an 8-0 suture to pass through the upper part of the left anterior descending coronary artery and ligate to block blood flow. Cut off the suture end, expel the air from the pleural cavity, and quickly close the pleural cavity. Wait for the mouse to be in good condition, and then close the pleural cavity with a 5-0 suture.

[0071] (3) Mice were fed normally, and models were established after 2, 4, 6, 8, 10 and 14 days for subsequent experiments.

[0072] Example 5

[0073] This embodiment demonstrates ultrasound examination of small animals.

[0074] (1) Small animal ultrasound examinations were performed on Sham group mice and mice with myocardial fibrosis for 14 days. Mice were anesthetized by intraperitoneal injection of 1.25% aphthylamine (LAT20250416) at a dose of 0.2 mL / 10 g. Ultrasound examination was performed after hair removal cream was used to remove hair from the chest of the mice.

[0075] (2) Fix the mouse in a supine position. Connect the leads and adjust the detection frequency and image depth. Detect the long-axis section of the left ventricle. Place the probe on the left chest of the mouse and adjust the angle between the probe and the midline of the sternum according to the image on the monitor. Rotate the probe 90 degrees to acquire the short-axis section of the left ventricle. During the acquisition process, try to keep the heart rate of mice in different groups at around 400 beats / min, and select clear echocardiograms for ultrasound data line analysis.

[0076] (3) Based on the statistical results, plot the results of left ventricular ejection fraction and fractional shortening.

[0077] Example 6

[0078] In this embodiment, the myocardial fibrosis model constructed in Example 4 was subjected to Masson staining.

[0079] (1) Fixation and dehydration: After anesthetizing the model mice, the heart was quickly removed, washed with physiological saline to remove blood, and then immersed in 4% paraformaldehyde tissue fixative (SL1830, Coolaber) for 24 hours. The sutures used to ligate the heart were cut, the heart was placed in an embedding box, and thoroughly washed with running water. The heart was then passed through the following reagents in sequence according to the time requirements: 50% ethanol: 30 minutes; 60% ethanol: 30 minutes; 70% ethanol: 30 minutes; 80% ethanol: 2 hours; 90% ethanol: 2 hours; 95% ethanol: 2 hours; anhydrous ethanol: 2 hours; anhydrous ethanol: 2 hours; 1:1 mixture of anhydrous ethanol and xylene: 15 minutes; xylene: 15 minutes.

[0080] (2) Embedding and sectioning: The heart was placed in a 1:1 mixture of xylene and paraffin for 2 hours, and then soaked in pure paraffin oil for more than 4 hours, or overnight. After cooling, the heart was cut into thin slices with a thickness of 5 μm, and dried in a dryer at 65℃ for 2 hours.

[0081] (3) Dewaxing and hydration: The dried sections were passed through the following reagents in sequence: xylene: 10 minutes; xylene: 10 minutes; anhydrous ethanol: 5 minutes; anhydrous ethanol: 5 minutes; 95% ethanol: 5 minutes; 90% ethanol: 5 minutes; 80% ethanol: 5 minutes; 70% ethanol: 5 minutes; ddH2O: 1 minute.

[0082] (4) Staining: Immerse the sections in nuclear staining solution for 2-5 minutes, rinse with ddH2O for 3-5 seconds, differentiate with 1% hydrochloric acid alcohol for 1 second, and then rinse in tap water for 30 minutes to re-blue. Immerse the sections in eosin staining solution for 2-5 minutes, rinse three times with ddH2O, and blot the water around the sections with filter paper. Immerse the sections in 1% phosphomolybdic acid for 1-3 minutes, and pour off the excess liquid. No water washing is required. Add aniline blue solution and stain for 2-5 minutes. Observe the staining status under a microscope. When the collagen turns blue, rinse with 1% glacial acetic acid solution for a few seconds until no blue tinge appears. Immerse the sections in 95% ethanol for 2-3 seconds, anhydrous ethanol for 5 seconds (3 times) for dehydration, and xylene for 1 minute (3 times) for clearing. Finally, mount the sections.

[0083] (5) Image acquisition: After the resin has dried and fixed, observe the collected sample images under a microscope.

[0084] Example 7

[0085] This embodiment performs second harmonic signal detection.

[0086] Fibrous collagen was imaged in the cardiac scar area following myocardial infarction in mice.

[0087] Second harmonic imaging was acquired using two-photon excitation microscopy, and a Power HyD NDD hybrid detector was added to enhance detection capabilities in the near-infrared (NIR) spectral range (Stellaris 8 Dive, Germany). Using infrared light at a wavelength of 1045 nm, second harmonic generation imaging was performed at the corresponding half-wavelength to capture oscillating signals. The spectral bandwidth of second harmonic imaging was ≤ 10 nm. Myocardial fibrosis sections and in vivo imaging were excited by 1045 nm excitation light, and the second harmonic signals were collected by the hybrid detector (HyD) to detect the signals reflected by collagen.

[0088] Example 8

[0089] This embodiment tests collagen I film, collagen III film, collagen IV film, collagen X film, collagen XII film, bovine serum albumin film, and exenatide film SHG polarization dependence.

[0090] Polarizers were placed at the excitation and acquisition optical paths to align the excitation and acquisition beams horizontally. Tungsten disulfide (WS2), collagen film, bovine serum albumin film, and exenatide film were irradiated with an 800 nm femtosecond laser, respectively. A λ / 2 waveplate was rotated to adjust the polarization direction. Rotation corresponding to the polarization direction ,collection The second harmonic polarization signal is used to calibrate the pattern of the measured sample by measuring the WS2 polarization pattern.

[0091] During inflammation and fibrosis, TGF-β1 strongly promotes collagen production in cells. Western blot analysis of CFs stimulated with TGF-β1 showed that Collagen-I protein expression was upregulated in CFs with increasing TGF-β1 stimulation time. Figure 1 (Figure A). Compared with the control group, the intensity of green fluorescence signal in cells treated with TGF-β1 for 48 hours was significantly enhanced. Further quantitative analysis revealed that the average fluorescence intensity of Collagen-I in the TGF-β1-treated group was statistically significantly higher than that in the control group, indicating that the expression of Collagen-I protein in cells was upregulated after TGF-β1 induction. Figure 1 (Figure B in the middle)

[0092] Fourteen days after myocardial infarction in the model mice, compared with the sham-operated control group (Sham), echocardiography in the myocardial infarction model group (MI) showed decreased cardiac function. Left ventricular ejection fraction (EF) and fractional shortening (FS) are the main indicators for evaluating left ventricular systolic function. Compared with the sham-operated group, EF and FS were decreased in the myocardial infarction model group, indicating weakened cardiac pumping capacity and decreased myocardial contractility. Figure 2 (Figure A). The Masson trichrome staining patterns of left ventricular transverse sections from the sham-operated group and mice at 2, 4, 6, 8, and 10 days after myocardial infarction were largely consistent with the results of second harmonic generation (SHG) detection. Figure 2 (Figure B) Quantitative analysis showed that SHG could detect fibrosis sites after myocardial infarction, corresponding to the fibrosis area in Masson staining results. Furthermore, with prolonged time, the morphology of collagen fibers changed significantly, with increased length and width. Figure 2 Figure C shows that early SHG detection is reliable at the tissue level.

[0093] Figure 3Figure A shows the process of SHG detection of cardiac fibrosis in live mice. The suction cup is precisely placed on the surface of the mouse heart. The suction cup is connected to a suction device that can precisely control the negative pressure through a tube. Blood is continuously and gently aspirated, keeping the surface of the heart clearly visible, which is conducive to subsequent SHG detection operations. Figure 3 Image B shows SHG imaging of the fibrotic areas of the heart in three different mice 14 days after myocardial infarction. Collagen is clearly visible in the fibrotic areas of the heart, indicating that the fibrotic areas of the heart can be detected in vivo by SHG. Figure 3 The middle image (C) shows SHG imaging of the fibrotic areas of the heart in Sham mice and mice 4, 8, and 14 days after myocardial infarction. As time progresses, collagen fibers increase, which verifies the reliability of SHG in detecting myocardial fibrosis at the in vivo level.

[0094] To further investigate whether SHG can specifically detect and distinguish biological samples with different molecular structures and degrees of order, polarization second harmonic detection was performed on collagen I, collagen III, collagen IV, collagen X, collagen XII, bovine serum albumin, and exenatide (peptide chain). Collagen I, collagen III, collagen IV, collagen X, and collagen XII generate SHG signals due to their highly ordered non-centrosymmetric structures. Figures 4A-4E Bovine serum albumin (BSA) is a globulin whose molecular structure is centrosymmetric in three-dimensional space and is usually in a random and disordered state in solution or tissue. It cannot meet the non-centrosymmetric condition necessary for generating a strong SHG signal and therefore cannot produce an effective SHG signal. Figure 4F Exendin-4 is a disordered short peptide with a molecular conformation lacking a stable, non-centrosymmetric arrangement. It cannot self-assemble into a highly ordered supramolecular structure like fibrous collagen, nor does it generate an effective SHG signal. Figure 4G The results showed that SHG can detect collagen with high specificity.

[0095] In summary, this invention develops a collagen detection scheme based on second harmonic imaging, employing a completely label-free, endogenous optical detection mechanism that does not damage the sample, exhibits low photobleaching and phototoxicity, and boasts high resolution, enabling dynamic ultra-long-term spatiotemporal in vivo imaging. Furthermore, it develops a system for detecting myocardial fibrosis, achieving for the first time in situ, in vivo, spatiotemporal dynamic imaging of myocardial fibrosis. This provides an important tool for the accurate detection of fibrosis, the differentiation between active and end-stage diseases, and prognostic risk assessment. It can be effectively applied to the development of products for detecting myocardial fibrosis and the screening / evaluation of therapeutic drugs.

[0096] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The application of second harmonic imaging in collagen detection, characterized in that, The applications include: A sample containing collagen is irradiated with a near-infrared laser, and the second harmonic signal generated by the collagen in the sample is collected. Based on the second harmonic signal, the collagen fibers are imaged and quantitatively analyzed.

2. The application according to claim 1, characterized in that, The wavelength of the near-infrared laser is 800~1300 nm, preferably 1000~1100 nm, and more preferably 1040~1050 nm.

3. The application according to claim 1 or 2, characterized in that, Two-photon excitation microscopy was used to collect the second harmonic signal generated by collagen in the sample, and the collagen fibers were imaged and quantitatively analyzed. Optionally, the two-photon excitation microscope is equipped with a hybrid detector.

4. The application according to any one of claims 1-3, characterized in that, The quantitative analysis includes: Calculate the area of ​​fibrosis; Obtain collagen fiber parameters, including width and length.

5. A method for detecting myocardial fibrosis for non-disease diagnosis and / or treatment purposes, characterized in that, The method includes the following steps: (1) Irradiate heart tissue samples with near-infrared laser; (2) Collect the second harmonic signal generated by collagen in the sample; (3) Based on the second harmonic signal, collagen fibers are imaged and quantitatively analyzed to detect and evaluate myocardial fibrosis.

6. The method for detecting myocardial fibrosis for non-disease diagnosis and / or treatment purposes according to claim 5, characterized in that, The heart tissue sample includes a living heart or a slice of ex vivo heart tissue. Optionally, the wavelength of the near-infrared laser is 800~1300 nm, preferably 1000~1100 nm, and more preferably 1040~1050 nm; Optionally, a two-photon excitation microscope is used to collect the second harmonic signal generated by collagen in the sample, and to image and quantitatively analyze the collagen fibers. Optionally, the two-photon excitation microscope is equipped with a hybrid detector.

7. Application of second harmonic imaging in the preparation of products for the diagnosis or monitoring of myocardial fibrosis.

8. A system for detecting myocardial fibrosis, characterized in that, The system includes: (1) Excitation unit, used to emit near-infrared laser; (2) Scanning unit, used for focusing near-infrared laser scanning onto the heart sample; (3) Detection unit, used to collect the second harmonic signal generated by collagen in heart samples; (4) A data processing unit, used to generate an image of collagen fibers based on the second harmonic signal and to quantitatively calculate and evaluate one or more morphological parameters of myocardial fibrosis.

9. The system for detecting myocardial fibrosis according to claim 8, characterized in that, The morphological parameters include at least one of density, diameter, alignment direction, or infiltration depth.

10. The application of the method for detecting myocardial fibrosis according to claim 5 or 6, or the system for detecting myocardial fibrosis according to claim 8, in evaluating the efficacy of anti-myocardial fibrosis drugs, characterized in that... The evaluation includes comparing changes in the second harmonic signal parameters of collagen fibers before and after medication to assess efficacy.

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