WO2023066158A1 - Establishment of non-marking quantification method for detecting lung tissue collagen - Google Patents
Establishment of non-marking quantification method for detecting lung tissue collagen Download PDFInfo
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- the invention belongs to the field of analytical chemistry detection, in particular to a non-labeled quantitative method capable of detecting lung tissue collagen.
- Quartz dust and coal dust produced in industrial production are one of the most serious occupational hazards in my country. Long-term inhalation of large amounts of quartz dust during production can cause silicosis, and long-term inhalation of coal dust can cause coal worker pneumoconiosis. Dust deposited in the lungs can cause structural damage to lung tissue, persistent inflammation, and fibrosis due to collagen deposition in the lungs. The diagnosis of pulmonary fibrosis mainly includes lung function tests, lung CT and auxiliary laboratory tests.
- collagen not only plays a scaffolding role in tissue morphology, but also plays an important role in tissue damage repair.
- dust particles silicon dioxide particles and coal dust particles, etc.
- enter the lung tissue it will lead to acute inflammatory injury reaction of the lung tissue, resulting in congestion, edema and tissue hyperplasia, various pro-inflammatory and anti-inflammatory factors and tissue cell apoptosis. death increased.
- Increased collagen content causes lung tissue consolidation, causing fibrosis.
- the present invention uses a confocal Raman microspectrometer to collect Raman spectra in specific areas of lung tissue slices, uses LabSpec6 to preprocess the data, and performs baseline calibration, cosmic ray removal and area normalization processing on all spectra. Not only the accurate determination of collagen content in lung tissue is realized, but also the precise imaging of different components of tissue can be performed, which provides certain technical support and theory for the determination of collagen content in lung tissue and the pathogenic mechanism of pulmonary fibrosis in accordance with.
- the purpose of the present invention is to provide a non-labeled quantitative method capable of detecting lung tissue collagen, which can solve the problems raised in the background technology, and the method has the following characteristics: 1, can accurately determine the collagen content of lung tissue To measure. 2. Through the normalized analysis of the spectral peaks collected in different regions, various information of collagen in different tissue regions can be obtained simply and quickly at low cost. 3. The Raman spectral imaging system can quickly and large-area scan imaging to obtain accurate information of different components of the tissue. 4. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
- the establishment of a non-labeled quantitative method for the detection of lung tissue collagen mainly includes the preparation of tissue frozen sections, the acquisition of confocal Raman microspectroscopy spectra and curve fitting, the rapid scanning imaging of specific regions, and the correlation between specific collagen peaks and Comparison of traditional methods.
- the establishment of a non-labeled quantitative method for detecting lung tissue collagen uses a conventional frozen section tissue preparation method, and the OCT frozen section embedding agent used has no effect on the experimental results after baseline calibration and background removal .
- This method is also applicable to the quantitative detection of liver fibrosis and myocardial collagen.
- the acquisition parameters of the spectrometer are: objective lens: 100 ⁇ ; laser wavelength: 532nm; grating: 600g/mm; spectrometer center: 2300cm-1; Acquisition range: 191-3946cm-1; laser power: 20mW; integration time: 8s.
- the collagen characteristic peaks 1248cm-1 and 1488cm-1 are determined, and the high-power laser beam is expanded by using surface imaging technology.
- the Gaussian distributed laser is shaped into a uniformly distributed flat-top laser, which is evenly irradiated on the entire sample. After filtering out the reflected laser, all the excited Raman spectra are imaged on the area array CCD as a whole, and hundreds of thousands of The measurement of the group Raman spectrum data is completed, and the peak intensity value of the collagen peak is calculated at the same time, which is displayed using the mean ⁇ standard deviation.
- the lung tissue sections of silicosis model mice and normal mice are subjected to Masson staining and Sirius red staining according to the standardized procedure of the kit, The area after staining was analyzed and processed by Image J software to obtain the area of collagen, and the two methods were compared and analyzed.
- the present invention has the following beneficial effects: the establishment of a non-labeled quantitative method for detecting lung tissue collagen involved in the present invention can accurately and quickly measure the collagen content of lung tissue without causing damage to tissue sections, Slices can be used for follow-up experiments; the Raman spectroscopy imaging system can quickly scan a large area to obtain accurate information on different components of the tissue; compared with traditional chemical staining methods, it can remove the specific fluorescence produced by particles in lung tissue interference.
- the information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
- Fig. 1 is a schematic diagram of the collagen content obtained by Raman testing the frozen sections of mouse lungs of the control group and the silicosis group using a confocal Raman microspectrometer.
- Figure 2 shows the spatial distribution information of collagen in tissues obtained by Raman scanning imaging of frozen sections of mouse lungs in the control group and the silicosis group using a confocal Raman microspectrometer.
- Step S1 Put the lung tissue of the silicosis model mouse and the lung tissue of the normal mouse into 30% sucrose for sugar precipitation treatment, use a frozen microtome to embed, and then routinely perform frozen section processing;
- Step S2 Use the WITec- ⁇ 300 Raman microspectrometer to detect lung tissue cells in frozen sections. Repeatedly collect 150-200 Raman spectra at different positions for each group of samples, and perform baseline calibration, cosmic ray removal and area uniformly on all spectra normalized processing;
- Step S3 After determining the collagen characteristic peaks 1248cm-1 and 1488cm-1, the peak intensity values were statistically analyzed to obtain the collagen content of the lung tissue slices in the control group and the silicosis group.
- Step S1 Use the WITec- ⁇ 300 Raman microspectrometer to detect lung tissue cells in frozen sections. Repeatedly collect 150-200 Raman spectra at different positions for each group of samples, and perform baseline calibration, cosmic ray removal and area uniformly on all spectra normalized processing;
- Step S2 Scan and image the 1248cm-1 and 1488cm-1 peaks in a large area through the Raman spectral imaging system to obtain the collagen content in this area of the tissue. It can be seen from Figure 2 that the imaging area of the silicosis group is significantly higher than that of the control group .
- Table 1 shows the measurement of collagen content using confocal Raman microscopy and the evaluation of collagen content using Masson staining and Sirius red staining.
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Abstract
A non-marking quantification method for detecting lung tissue collagen, comprising the following steps: step 1: placing mouse lung tissue of a silicosis model and lung tissue of a normal mouse into 30% sucrose for sugar precipitation, embedding same in a cryostat, and then carrying out a conventional frozen slice operation treatment; step 2: detecting lung tissue cells in the frozen slices by using a WITec-α300 Raman microspectrometer, repeatedly collecting 150-200 Raman spectra at different positions for each group of samples, and uniformly carrying out baseline calibration, cosmic ray removal, and area normalization processing on all of the spectra; and step 3: determining feature peaks of collagen at 1248 cm-1 and 1488 cm-1, and then carrying out statistical analysis on peak intensity values to obtain the collagen content of lung tissue slices of a control group and a silicosis group.
Accurate information of different components of tissue is obtained by rapid large-area scanning imaging. Compared to a conventional chemical dyeing method, interference of specific fluorescence generated by particles in lung tissue may be removed. Obtained information has high light-passing efficiency, high sensitivity and good repeatability.
Description
本发明属于分析化学检测领域，具体是一种能够检测肺组织胶原的非标记定量方法。The invention belongs to the field of analytical chemistry detection, in particular to a non-labeled quantitative method capable of detecting lung tissue collagen.
工业生产中产生的石英粉尘和煤尘是我国最严重的职业有害因素之一，生产过程中若长期吸入大量石英粉尘可以引起矽肺，长期吸入煤尘可引起煤工尘肺。肺内沉着的粉尘可以引起肺组织的结构破坏、持续性炎症和肺部胶原沉积引起纤维化。肺纤维化的诊断主要有肺功能检测、肺部CT以及辅助的实验室检查。Quartz dust and coal dust produced in industrial production are one of the most serious occupational hazards in my country. Long-term inhalation of large amounts of quartz dust during production can cause silicosis, and long-term inhalation of coal dust can cause coal worker pneumoconiosis. Dust deposited in the lungs can cause structural damage to lung tissue, persistent inflammation, and fibrosis due to collagen deposition in the lungs. The diagnosis of pulmonary fibrosis mainly includes lung function tests, lung CT and auxiliary laboratory tests.
胶原作为细胞外基质的主要成分之一，不仅在组织形态中起支架作用，而且在组织损伤修复中起着重要作用。粉尘颗粒(二氧化硅颗粒和煤尘颗粒等)进入肺组织后，导致肺组织发生急性炎症损伤反应致充血、水肿并伴有组织增生，各种促炎、抗炎因子和引起组织细胞的凋亡增加。胶原蛋白含量增加引起肺组织实变，引起纤维化。As one of the main components of the extracellular matrix, collagen not only plays a scaffolding role in tissue morphology, but also plays an important role in tissue damage repair. After dust particles (silicon dioxide particles and coal dust particles, etc.) enter the lung tissue, it will lead to acute inflammatory injury reaction of the lung tissue, resulting in congestion, edema and tissue hyperplasia, various pro-inflammatory and anti-inflammatory factors and tissue cell apoptosis. death increased. Increased collagen content causes lung tissue consolidation, causing fibrosis.
现有的胶原蛋白含量的检测方法主要包括紫外分光光度法、羟脯氨酸比色法、高效液相色谱法、天狼星红法和马松染色法。紫外分光光度法、羟脯氨酸比色法和高效液相色谱法都需要将组织进行破坏性处理，天狼星红法和马松染色法需要对组织切片进行相应的化学染色，无法迅速获取胶原的结果。所以，发明一种能够快速进行评估胶原蛋白含量而又不破坏组织形态的方法是非常有必要的。Existing detection methods for collagen content mainly include ultraviolet spectrophotometry, hydroxyproline colorimetry, high performance liquid chromatography, Sirius red method and Masson staining method. Ultraviolet spectrophotometry, hydroxyproline colorimetry, and high-performance liquid chromatography all require destructive treatment of the tissue, and the Sirius red method and Masson staining method require corresponding chemical staining of tissue sections, which cannot quickly obtain collagen content. result. Therefore, it is very necessary to develop a method that can quickly evaluate the collagen content without destroying the tissue morphology.
本发明通过共聚焦拉曼显微光谱仪，对肺组织切片特定区域采集拉曼光谱，使用LabSpec6对数据进行前处理，对所有图谱统一进行基线校准、去宇宙射线和面积归一化处理。不仅实现了将肺组织进行胶原蛋白含量的准确测定，而且还可以进行组织不同组分的精确成像，为肺组织胶原含量的测定和肺部纤维化的致病机理提供了一定的技术支持和理论依据。The present invention uses a confocal Raman microspectrometer to collect Raman spectra in specific areas of lung tissue slices, uses LabSpec6 to preprocess the data, and performs baseline calibration, cosmic ray removal and area normalization processing on all spectra. Not only the accurate determination of collagen content in lung tissue is realized, but also the precise imaging of different components of tissue can be performed, which provides certain technical support and theory for the determination of collagen content in lung tissue and the pathogenic mechanism of pulmonary fibrosis in accordance with.
发明内容Contents of the invention
本发明的目的是提供一种能够检测肺组织胶原的非标记定量的方法，该方法能够解决背景技术中所提出的问题，并且该方法具有如下特点：1、可以准确对肺组织的胶原蛋白含量进行测定。2、通过对收集不同区域的光谱峰值进行归一化分析，可以达到低成本简单快速地获取不同组织区域的胶原蛋白的各种信息。3、通过拉曼光谱成像系统可以快速大面积扫面成像以获得组织不同组分的精确信息。4、光谱获得的信息具有通光效率高、灵敏度高、重复性好的优点。The purpose of the present invention is to provide a non-labeled quantitative method capable of detecting lung tissue collagen, which can solve the problems raised in the background technology, and the method has the following characteristics: 1, can accurately determine the collagen content of lung tissue To measure. 2. Through the normalized analysis of the spectral peaks collected in different regions, various information of collagen in different tissue regions can be obtained simply and quickly at low cost. 3. The Raman spectral imaging system can quickly and large-area scan imaging to obtain accurate information of different components of the tissue. 4. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
本发明实现发明目的采用如下技术方案：The present invention realizes the purpose of the invention and adopts the following technical solutions:
一种检测肺组织胶原的非标记定量方法的建立，主要包括组织冰冻切片的制备、共聚焦拉曼显微光谱仪光谱的获取以及曲线的拟合、特定区域的快速扫描成像和特定胶原谱峰与传统方法的比较。The establishment of a non-labeled quantitative method for the detection of lung tissue collagen mainly includes the preparation of tissue frozen sections, the acquisition of confocal Raman microspectroscopy spectra and curve fitting, the rapid scanning imaging of specific regions, and the correlation between specific collagen peaks and Comparison of traditional methods.
S1：将矽肺模型的小鼠肺组织和正常小鼠的肺组织放入30％蔗糖进行沉糖处理，使用冰冻切片机进行包埋，然后常规行冰冻切片操作处理；S1: Put the lung tissues of silicosis model mice and normal mice into 30% sucrose for sugar precipitation, use a frozen microtome for embedding, and then routinely perform frozen section operations;
S2：使用WITec-α300拉曼显微光谱仪(WITec,UIm,德国)检测冰冻切片中的肺组织细胞，每组样本重复采集150个拉曼图谱，对所有图谱统一进行基线校准、去宇宙射线和面积归一化处理。S2: Use a WITec-α300 Raman microspectrometer (WITec, UIm, Germany) to detect lung tissue cells in frozen sections, collect 150 Raman spectra repeatedly for each group of samples, and perform baseline calibration, cosmic ray and Area normalization.
S3：确定胶原特征峰1248cm-1和1488cm-1。通过可调滤波器为主的高光谱成像组件，整体成像在面阵电荷耦合元件(CCD)上，形成光谱成像信息。S3: Determine the collagen characteristic peaks 1248cm-1 and 1488cm-1. Through the hyperspectral imaging component based on the tunable filter, the overall image is formed on the area charge-coupled device (CCD) to form spectral imaging information.
S4：将1248cm-1和1488cm-1的胶原蛋白峰强度进行收集并统计分析，与传统染色(马松染色和天狼星红染色)进行比较分析。S4: The collagen peak intensities at 1248cm-1 and 1488cm-1 were collected and statistically analyzed, and compared with traditional staining (Masson's staining and Sirius red staining).
作为优选，本发明提供的一种检测肺组织胶原的非标记定量方法的建立，使用常规的冰冻切片组织制备方法，使用的OCT冰冻切片包埋剂经过基线校准、去除背景后对实验结果无影响。此方法在肝脏纤维化、心肌胶原的定量检测中同样适用。As a preference, the establishment of a non-labeled quantitative method for detecting lung tissue collagen provided by the present invention uses a conventional frozen section tissue preparation method, and the OCT frozen section embedding agent used has no effect on the experimental results after baseline calibration and background removal . This method is also applicable to the quantitative detection of liver fibrosis and myocardial collagen.
作为优选，本发明提供的一种检测肺组织胶原的非标记定量方法的建立，光谱仪的采集参数为物镜：100×；激光波长：532nm；光栅：600g/mm；光谱仪中心：2300cm-1；图谱采集范围：191-3946cm-1；激光功率：20mW；积分时间：8s。As a preference, for the establishment of a non-labeled quantitative method for detecting lung tissue collagen provided by the present invention, the acquisition parameters of the spectrometer are: objective lens: 100×; laser wavelength: 532nm; grating: 600g/mm; spectrometer center: 2300cm-1; Acquisition range: 191-3946cm-1; laser power: 20mW; integration time: 8s.
作为优选，本发明提供的一种检测肺组织胶原的非标记定量方法的建立，确定胶原特征峰1248cm-1和1488cm-1，使用面成像技术，将高功率激光扩束后，将扩束后的高斯分布的激光整形成均匀分布的平顶激光，均匀的照射在整个样品上，滤除反射的激光后，所有激发的拉曼光谱整体成像在面阵CCD上，短时间内将数十万组拉曼光谱数据测量完成，同时计算在胶原峰的峰强度值，使用均数±标准差显示。As a preference, in the establishment of a non-labeled quantitative method for detecting lung tissue collagen provided by the present invention, the collagen characteristic peaks 1248cm-1 and 1488cm-1 are determined, and the high-power laser beam is expanded by using surface imaging technology. The Gaussian distributed laser is shaped into a uniformly distributed flat-top laser, which is evenly irradiated on the entire sample. After filtering out the reflected laser, all the excited Raman spectra are imaged on the area array CCD as a whole, and hundreds of thousands of The measurement of the group Raman spectrum data is completed, and the peak intensity value of the collagen peak is calculated at the same time, which is displayed using the mean ± standard deviation.
作为优选，本发明提供的一种检测肺组织胶原的非标记定量方法的建立，将矽肺模型的小鼠和正常小鼠的肺组织切片按试剂盒进行标准化程序进行马松染色和天狼星红染色，使用Image J软件对染色后的面积进行分析处理，得到胶原蛋白面积，将两种方法进行比较分析。As a preference, in the establishment of a non-labeled quantitative method for detecting lung tissue collagen provided by the present invention, the lung tissue sections of silicosis model mice and normal mice are subjected to Masson staining and Sirius red staining according to the standardized procedure of the kit, The area after staining was analyzed and processed by Image J software to obtain the area of collagen, and the two methods were compared and analyzed.
相对于现有技术，本发明具有以下有益效果：本发明所涉及的一种检测肺组织胶原的非标记定量方法的建立可以准确快速对肺组织的胶原蛋白含量进行测定，对组织切片无伤害，切片可以进行后续实验；拉曼光谱成像系统可以快速大面积扫面成像以获得组织不同组分的精确信息；与传统的化学染色方法相比，可以去除颗粒在肺组织中产生的特异性荧光的干扰。光谱获得的信息具有通光效率高、灵敏度高、重复性好的优点。Compared with the prior art, the present invention has the following beneficial effects: the establishment of a non-labeled quantitative method for detecting lung tissue collagen involved in the present invention can accurately and quickly measure the collagen content of lung tissue without causing damage to tissue sections, Slices can be used for follow-up experiments; the Raman spectroscopy imaging system can quickly scan a large area to obtain accurate information on different components of the tissue; compared with traditional chemical staining methods, it can remove the specific fluorescence produced by particles in lung tissue interference. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
图1是使用共聚焦拉曼显微光谱仪对对照组和矽肺组小鼠肺冰冻切片进行拉曼测试获得胶原蛋白含量的示意图。Fig. 1 is a schematic diagram of the collagen content obtained by Raman testing the frozen sections of mouse lungs of the control group and the silicosis group using a confocal Raman microspectrometer.
图2是使用共聚焦拉曼显微光谱仪对对照组和矽肺组小鼠肺冰冻切片进行拉曼扫面成像获得胶原在组织中的空间分布信息。Figure 2 shows the spatial distribution information of collagen in tissues obtained by Raman scanning imaging of frozen sections of mouse lungs in the control group and the silicosis group using a confocal Raman microspectrometer.
以下通过具体实施例对本发明做进一步解释说明。The present invention will be further explained by specific examples below.
如图1所示使用共聚焦拉曼显微光谱仪对对照组和矽肺组小鼠肺冰冻切片进行拉曼测试获得胶原蛋白含量：As shown in Figure 1, use a confocal Raman microspectrometer to conduct Raman tests on frozen sections of mouse lungs in the control group and silicosis group to obtain collagen content:
步骤S1：将矽肺模型的小鼠肺组织和正常小鼠的肺组织放入30％蔗糖进行沉糖处理，使用冰冻切片机进行包埋，然后常规行冰冻切片操作处理；Step S1: Put the lung tissue of the silicosis model mouse and the lung tissue of the normal mouse into 30% sucrose for sugar precipitation treatment, use a frozen microtome to embed, and then routinely perform frozen section processing;
步骤S2：使用WITec-α300拉曼显微光谱仪检测冰冻切片中的肺组织细胞，每组样本在不同位置重复采集150-200个拉曼图谱，对所有图谱统一进行基线校准、去宇宙射线和面积归一化处理；Step S2: Use the WITec-α300 Raman microspectrometer to detect lung tissue cells in frozen sections. Repeatedly collect 150-200 Raman spectra at different positions for each group of samples, and perform baseline calibration, cosmic ray removal and area uniformly on all spectra normalized processing;
步骤S3：确定胶原特征峰1248cm-1和1488cm-1后将峰强度值进行统计分析，得出对照组和矽肺组肺组织切片胶原蛋白含量。Step S3: After determining the collagen characteristic peaks 1248cm-1 and 1488cm-1, the peak intensity values were statistically analyzed to obtain the collagen content of the lung tissue slices in the control group and the silicosis group.
如图2所示，使用共聚焦拉曼显微光谱仪对对照组和矽肺组小鼠肺冰冻切片进行拉曼扫面成像获得胶原在组织中的空间分布信息：As shown in Figure 2, using a confocal Raman microspectrometer to perform Raman scanning imaging on the frozen sections of mouse lungs in the control group and the silicosis group to obtain information on the spatial distribution of collagen in the tissue:
步骤S1：使用WITec-α300拉曼显微光谱仪检测冰冻切片中的肺组织细胞，每组样本在不同位置重复采集150-200个拉曼图谱，对所有图谱统一进行基线校准、去宇宙射线和面积归一化处理；Step S1: Use the WITec-α300 Raman microspectrometer to detect lung tissue cells in frozen sections. Repeatedly collect 150-200 Raman spectra at different positions for each group of samples, and perform baseline calibration, cosmic ray removal and area uniformly on all spectra normalized processing;
步骤S2：通过拉曼光谱成像系统将1248cm-1和1488cm-1峰进行大面积扫面成像以获得组织该区域的胶原含量，通过图2可以看出，矽肺组的成像区域明显高于对照组。Step S2: Scan and image the 1248cm-1 and 1488cm-1 peaks in a large area through the Raman spectral imaging system to obtain the collagen content in this area of the tissue. It can be seen from Figure 2 that the imaging area of the silicosis group is significantly higher than that of the control group .
下表1显示使用共聚焦显微拉曼光谱测量胶原含量和使用马松染色和天狼星红染色对胶原含量的评估。Table 1 below shows the measurement of collagen content using confocal Raman microscopy and the evaluation of collagen content using Masson staining and Sirius red staining.
表1Table 1
| the | 矽肺组Silicosis group | 对照组control group | 比值(矽肺/对照)Ratio (Silicosis/Control) |
| 拉曼光谱法(1248cm -1) Raman spectroscopy (1248cm -1 ) | 0.460.46 | 0.150.15 | 3.073.07 |
| 拉曼光谱法(1488cm -1) Raman spectroscopy (1488cm -1 ) | 0.410.41 | 0.110.11 | 3.733.73 |
| 马松染色Masson staining | 0.530.53 | 0.160.16 | 3.313.31 |
| 天狼星红染色Sirius red stain | 0.710.71 | 0.170.17 | 4.174.17 |
如表1所示，评估肺组织胶原的三种方法显示，拉曼光谱法与马松染色结果相一致，与天狼星红的结果有一定的差异。原因可能是天狼星红染色结果的评估需要偏振光，而二氧化硅颗粒在肺组织中的存在会产生自发荧光干扰了相应的结果，所以天狼星红染色并不适用于评价矽肺肺组织中的胶原蛋白含量。与传统的化学染色方法相比，拉曼光谱法可以去除颗粒在肺组织中产生的特异性荧光的干扰。光谱获得的信息具有通光效率高、灵敏度高、重复性好的优点。As shown in Table 1, the three methods for evaluating lung tissue collagen showed that Raman spectroscopy was consistent with the results of Masson staining, and there was some difference with the results of Sirius red. The reason may be that the evaluation of Sirius red staining results requires polarized light, and the presence of silica particles in lung tissue will produce autofluorescence that interferes with the corresponding results, so Sirius red staining is not suitable for evaluating collagen in silicosis lung tissue content. Compared with traditional chemical staining methods, Raman spectroscopy can remove the interference of specific fluorescence produced by particles in lung tissue. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。In addition, it should be understood that although this specification is described according to implementation modes, not each implementation mode only contains an independent technical solution, and this description in the specification is only for clarity, and those skilled in the art should take the specification as a whole , the technical solutions in the various embodiments can also be properly combined to form other implementations that can be understood by those skilled in the art.
Claims (5)
- 一种检测肺组织胶原的非标记定量方法的建立，其特征在于：首先，可以准确对肺组织的胶原蛋白含量进行测定；其次，通过对收集不同区域的光谱峰值进行归一化分析，可以达到低成本简单快速地获取不同组织区域的胶原蛋白的各种信息；再次，通过拉曼光谱成像系统可以快速大面积扫面成像以获得组织不同组分的精确信息；最后，光谱获得的信息具有通光效率高、灵敏度高、重复性好的优点。The establishment of a non-labeled quantitative method for detecting lung tissue collagen is characterized in that: firstly, the collagen content of lung tissue can be accurately measured; secondly, by normalizing the spectral peaks collected in different regions, it can achieve It is easy and fast to obtain various information of collagen in different tissue regions at low cost; thirdly, the Raman spectral imaging system can quickly and large-area scan imaging to obtain accurate information on different components of the tissue; finally, the information obtained by the spectrum has a general High light efficiency, high sensitivity and good repeatability.
- 根据权利要求1所述的一种检测肺组织胶原的非标记定量方法的建立，其特征在于：使用常规的冰冻切片组织制备方法，使用的OCT冰冻切片包埋剂经过基线校准、去除背景后对实验结果无影响。此方法在肝脏纤维化、心肌胶原的定量检测中同样适用。According to the establishment of a non-labeled quantitative method for detecting lung tissue collagen according to claim 1, it is characterized in that: using a conventional frozen section tissue preparation method, the OCT frozen section embedding agent used is subjected to baseline calibration and background removal. Experimental results have no effect. This method is also applicable to the quantitative detection of liver fibrosis and myocardial collagen.
- 根据权利要求1所述的一种检测肺组织胶原的非标记定量方法的建立，其特征在于：光谱仪的采集参数为物镜：100×；激光波长：532nm；光栅：600g/mm；光谱仪中心：2300cm-1；图谱采集范围：191-3946cm-1；激光功率：20mW；积分时间：8s。According to the establishment of a non-labeled quantitative method for detecting lung tissue collagen according to claim 1, it is characterized in that: the acquisition parameters of the spectrometer are objective lens: 100×; laser wavelength: 532nm; grating: 600g/mm; spectrometer center: 2300cm -1; spectrum acquisition range: 191-3946cm-1; laser power: 20mW; integration time: 8s.
- 根据权利要求1所述的一种检测肺组织胶原的非标记定量方法的建立，其特征在于：确定胶原特征峰1248cm-1和1488cm-1，使用面成像技术，将高功率激光扩束后，将扩束后的高斯分布的激光整形成均匀分布的平顶激光，均匀的照射在整个样品上，滤除反射的激光后，所有激发的拉曼光谱可以整体成像在面阵CCD上，短时间内可以完成数十万组拉曼光谱数据测量，同时可以显示胶原峰的峰强度值。According to the establishment of a non-labeled quantitative method for detecting lung tissue collagen according to claim 1, it is characterized in that: determine the collagen characteristic peaks 1248cm-1 and 1488cm-1, use the area imaging technology, after expanding the high-power laser beam, The expanded Gaussian distributed laser is shaped into a uniformly distributed flat-top laser, which is evenly irradiated on the entire sample. After filtering out the reflected laser, all excited Raman spectra can be imaged on the area array CCD as a whole. Hundreds of thousands of sets of Raman spectrum data measurements can be completed within one day, and the peak intensity value of the collagen peak can be displayed at the same time.
- 根据权利要求1所述的一种检测肺组织胶原的非标记定量方法的建立，其特征在于：将矽肺模型的小鼠和正常小鼠的肺组织切片按试剂盒进行标准化程序进行马松染色和天狼星红染色，使用Image J软件对染色后的面积进行分析处理，得到胶原蛋白面积，将两种方法进行比较分析认为拉曼方法检测胶原的准确性优于传统染色方法。The establishment of a non-labeled quantitative method for detecting lung tissue collagen according to claim 1, wherein the lung tissue sections of silicosis model mice and normal mice are carried out according to the standardization procedure of the kit for Masson staining and Sirius red staining, using Image J software to analyze and process the stained area to obtain the collagen area, and comparing the two methods, it is concluded that the Raman method is more accurate in detecting collagen than the traditional staining method.
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