CN113933285A - Establishment of non-labeling quantitative method for detecting lung tissue collagen - Google Patents
Establishment of non-labeling quantitative method for detecting lung tissue collagen Download PDFInfo
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- CN113933285A CN113933285A CN202111207977.7A CN202111207977A CN113933285A CN 113933285 A CN113933285 A CN 113933285A CN 202111207977 A CN202111207977 A CN 202111207977A CN 113933285 A CN113933285 A CN 113933285A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
Abstract
The invention relates to establishment of a non-labeling quantitative method for detecting lung tissue collagen, which comprises the following steps: step 1: putting the lung tissues of a mouse of a silicosis model and the lung tissues of a normal mouse into 30% sucrose for sugar precipitation treatment, embedding in a frozen microtome, and then performing frozen microtome operation treatment by a conventional method; step 2: detecting lung tissue cells in the frozen section by using a WITec-alpha 300 Raman microscopy spectrometer, repeatedly collecting 150-plus-200 Raman spectra from each group of samples at different positions, and uniformly performing baseline calibration, cosmic ray removal and area normalization processing on all the spectra; and step 3: determining the characteristic peak 1248cm of collagen‑1And 1488cm‑1And then carrying out statistical analysis on the peak intensity values to obtain the collagen content of the lung tissue sections of the control group and the silicosis group. The invention relates to a testThe establishment of the non-labeling quantitative method for lung tissue collagen can accurately and rapidly measure the collagen content of lung tissue, has no harm to tissue slices, and can carry out subsequent experiments on the slices; the Raman spectrum imaging system can rapidly image a large area of a scan to obtain accurate information of different components of tissues; in contrast to conventional chemical staining methods, interference of the specific fluorescence generated by the particles in the lung tissue can be removed. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
Description
Technical Field
The invention belongs to the field of analytical chemistry detection, and particularly relates to a non-labeling quantitative method capable of detecting lung tissue collagen.
Background
Quartz dust and coal dust generated in industrial production are one of the most serious occupational harmful factors in China, silicosis can be caused if a large amount of quartz dust is inhaled for a long time in the production process, and coal dust lung can be caused if coal dust is inhaled for a long time. The deposition of dust in the lungs can cause structural destruction of lung tissue, persistent inflammation and fibrosis due to collagen deposition in the lungs. The diagnosis of pulmonary fibrosis mainly comprises pulmonary function detection, pulmonary CT and auxiliary laboratory examination.
Collagen, one of the major components of the extracellular matrix, plays an important role not only in the organization of the tissue morphology but also in the repair of tissue damage. After the dust particles (silica particles, coal dust particles and the like) enter lung tissues, acute inflammatory injury reaction of the lung tissues is caused to cause congestion and edema and tissue hyperplasia, and various proinflammatory and anti-inflammatory factors and apoptosis of tissue cells are increased. Increased collagen content causes consolidation of lung tissue, causing fibrosis.
The existing methods for detecting the content of collagen mainly comprise an ultraviolet spectrophotometry method, a hydroxyproline colorimetric method, a high performance liquid chromatography method, a sirius red method and a masson dyeing method. The ultraviolet spectrophotometry, hydroxyproline colorimetry and high performance liquid chromatography all need to perform destructive treatment on tissues, and the sirius red method and the masson dyeing method need to perform corresponding chemical dyeing on tissue slices and cannot rapidly obtain the result of collagen. Therefore, it is very necessary to develop a method for rapidly evaluating the collagen content without destroying the tissue morphology.
According to the invention, a confocal Raman micro spectrometer is used for collecting Raman spectra in a specific area of a lung tissue section, LabSpec6 is used for preprocessing data, and all spectra are subjected to baseline calibration, cosmic ray removal and area normalization treatment in a unified manner. Not only the accurate determination of the collagen content of the lung tissue is realized, but also the accurate imaging of different components of the tissue can be carried out, and certain technical support and theoretical basis are provided for the determination of the collagen content of the lung tissue and the pathogenesis of the pulmonary fibrosis.
Disclosure of Invention
The object of the present invention is to provide a method for the non-labeled quantification of collagen in lung tissue, which method solves the problems mentioned in the background and has the following features: 1. the collagen content of the lung tissue can be accurately measured. 2. By carrying out normalization analysis on collected spectrum peaks in different regions, various information of collagen in different tissue regions can be simply and quickly acquired at low cost. 3. By the Raman spectrum imaging system, large-area scanning imaging can be rapidly carried out to obtain accurate information of different components of tissues. 4. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
The invention adopts the following technical scheme for realizing the purpose:
the establishment of non-labeling quantitative method for detecting lung tissue collagen mainly includes preparation of tissue frozen section, acquisition of spectrum of confocal Raman micro spectrometer and fitting of curve, fast scanning imaging of specific area and comparison of specific collagen spectral peak with traditional method.
S1: putting the lung tissues of a mouse of a silicosis model and the lung tissues of a normal mouse into 30% sucrose for sugar precipitation treatment, embedding by using a freezing microtome, and then performing conventional freezing section operation treatment;
s2: lung tissue cells in the frozen sections were examined using a WITec- α 300 raman microspectrometer (WITec, UIm, germany), 150 raman spectra were collected for each set of samples, and baseline calibration, cosmic ray removal, and area normalization processing were performed uniformly on all spectra.
S3: determining the characteristic peak 1248cm of collagen-1And 1488cm-1. Integrally imaging on a Charge Coupled Device (CCD) of an area array by a hyperspectral imaging component mainly comprising a tunable filterSpectral imaging information.
S4: will be 1248cm-1And 1488cm-1The peak collagen intensities of (a) were collected and statistically analyzed, compared to the traditional staining (masson staining and sirius red staining).
Preferably, the establishment of the non-labeling quantitative method for detecting the lung tissue collagen provided by the invention uses a conventional frozen section tissue preparation method, and the OCT frozen section embedding medium used has no influence on the experimental result after baseline calibration and background removal. The method is also suitable for quantitative detection of liver fibrosis and myocardial collagen.
Preferably, the invention provides a method for detecting lung tissue collagen by non-labeling quantification, and the acquisition parameters of the spectrometer are as follows: 100X; laser wavelength: 532 nm; grating: 600 g/mm; the center of the spectrometer: 2300cm < -1 >; the spectrum acquisition range is as follows: 191-3946 cm-1; laser power: 20 mW; integration time: and 8 s.
Preferably, the establishment of the non-labeling quantitative method for detecting the lung tissue collagen provided by the invention determines the characteristic peak 1248cm of the collagen-1And 1488cm-1After high-power laser is expanded by using an area imaging technology, laser with Gaussian distribution after expansion is shaped into flat-top laser with uniform distribution, the flat-top laser is uniformly irradiated on a whole sample, reflected laser is filtered, all excited Raman spectrums are integrally imaged on an area array CCD, hundreds of thousands of groups of Raman spectrum data are measured in a short time, meanwhile, the peak intensity value of a collagen peak is calculated, and the average +/-standard deviation is used for displaying.
Preferably, the invention provides a method for establishing a non-labeled quantitative method for detecting lung tissue collagen, which comprises the steps of performing masson staining and sirius red staining on lung tissue sections of mice and normal mice of a silicosis model according to a standardized program of a kit, analyzing the stained areas by using Image J software to obtain the collagen areas, and comparing and analyzing the two methods.
Has the advantages that:
compared with the prior art, the invention has the following beneficial effects: the establishment of the non-labeling quantitative method for detecting the lung tissue collagen can accurately and quickly measure the collagen content of the lung tissue, has no harm to tissue slices, and can carry out subsequent experiments on the slices; the Raman spectrum imaging system can rapidly image a large area of a scan to obtain accurate information of different components of tissues; in contrast to conventional chemical staining methods, interference of the specific fluorescence generated by the particles in the lung tissue can be removed. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
Drawings
FIG. 1 is a graph showing the content of collagen in a control group and a silicosis group of mice obtained by Raman measurement of frozen lung sections using a confocal Raman microscopy spectrometer.
FIG. 2 is a confocal Raman microscopy spectrometer used for Raman scanning imaging of frozen lung sections of mice in a control group and a silicosis group to obtain the spatial distribution information of collagen in tissues.
Detailed Description
The invention is further illustrated by the following specific examples.
As shown in fig. 1, the collagen content was obtained by raman test using a confocal raman microscopy spectrometer on frozen sections of the lungs of mice in the control group and the silicosis group:
step S1: putting the lung tissues of a mouse of a silicosis model and the lung tissues of a normal mouse into 30% sucrose for sugar precipitation treatment, embedding by using a freezing microtome, and then performing conventional freezing section operation treatment;
step S2: detecting lung tissue cells in the frozen section by using a WITec-alpha 300 Raman microscopy spectrometer, repeatedly collecting 150-plus-200 Raman spectra from each group of samples at different positions, and uniformly performing baseline calibration, cosmic ray removal and area normalization processing on all the spectra;
step S3: determining the characteristic peak 1248cm of collagen-1And 1488cm-1And then carrying out statistical analysis on the peak intensity values to obtain the collagen content of the lung tissue sections of the control group and the silicosis group.
As shown in fig. 2, the spatial distribution information of collagen in the tissues is obtained by raman scanning of the frozen sections of the lungs of mice in the control group and the silicosis group by using a confocal raman microscopy spectrometer:
step S1: detecting lung tissue cells in the frozen section by using a WITec-alpha 300 Raman microscopy spectrometer, repeatedly collecting 150-plus-200 Raman spectra from each group of samples at different positions, and uniformly performing baseline calibration, cosmic ray removal and area normalization processing on all the spectra;
step S2: 1248cm by Raman spectral imaging system-1And 1488cm-1The peak was imaged over a large scan to obtain the collagen content of this region of tissue, as can be seen from fig. 2, the imaged region of the silicosis group was significantly higher than the control group.
Table 1 below shows the measurement of collagen content using confocal micro-raman spectroscopy and the assessment of collagen content using masson staining and sirius red staining.
TABLE 1
As shown in table 1, three methods for evaluating lung tissue collagen showed that raman spectroscopy was consistent with masson staining results, with some differences from sirius red results. The reason may be that sirius red staining is not suitable for evaluating collagen content in silicosis lung tissue because the assessment of the results requires polarized light and the presence of silica particles in lung tissue produces autofluorescence interfering with the corresponding results. In contrast to traditional chemical staining methods, raman spectroscopy can remove interference from the specific fluorescence generated by particles in lung tissue. The information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. The establishment of a non-labeling quantitative method for detecting lung tissue collagen is characterized in that: firstly, the collagen content of the lung tissue can be accurately measured; secondly, various information of collagen in different tissue areas can be simply and quickly acquired at low cost by carrying out normalization analysis on collected spectrum peaks in different areas; thirdly, a large-area scanning imaging can be rapidly carried out through a Raman spectrum imaging system so as to obtain accurate information of different components of the tissue; finally, the information obtained by the spectrum has the advantages of high light transmission efficiency, high sensitivity and good repeatability.
2. The establishment of a non-labeling quantitative method for detecting lung tissue collagen according to claim 1, wherein: the OCT frozen section embedding medium used by the conventional frozen section tissue preparation method has no influence on experimental results after baseline calibration and background removal. The method is also suitable for quantitative detection of liver fibrosis and myocardial collagen.
3. The establishment of a non-labeling quantitative method for detecting lung tissue collagen according to claim 1, wherein: the acquisition parameters of the spectrometer are objective: 100X; laser wavelength: 532 nm; grating: 600 g/mm; the center of the spectrometer: 2300cm < -1 >; the spectrum acquisition range is as follows: 191-3946 cm-1; laser power: 20 mW; integration time: and 8 s.
4. The establishment of a non-labeling quantitative method for detecting lung tissue collagen according to claim 1, wherein: determining the characteristic peak 1248cm of collagen-1And 1488cm-1After the high-power laser is expanded by using the surface imaging technology, the expanded Gaussian distributed laser is shaped into uniformly distributed flat-top laser, the flat-top laser is uniformly irradiated on the whole sample, and after the reflected laser is filtered, all the excited Raman spectrums can be integrally imaged on the surface array CCD, so that hundreds of thousands of groups of Raman spectrum data can be measured in a short time, and the peak intensity value of a collagen peak can be displayed.
5. The establishment of a non-labeling quantitative method for detecting lung tissue collagen according to claim 1, wherein: the lung tissue sections of a mouse of a silicosis model and a normal mouse are subjected to Pinus massoniana staining and sirius red staining according to a standardized procedure of a kit, the stained area is analyzed by using Image J software to obtain the area of collagen, and the two methods are compared and analyzed to judge that the accuracy of the collagen detection by the Raman method is superior to that of the traditional staining method.
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| CN202111207977.7A CN113933285A (en) | 2021-10-18 | 2021-10-18 | Establishment of non-labeling quantitative method for detecting lung tissue collagen |
| PCT/CN2022/125393 WO2023066158A1 (en) | 2021-10-18 | 2022-10-14 | Establishment of non-marking quantification method for detecting lung tissue collagen |
| ZA2023/05116A ZA202305116B (en) | 2021-10-18 | 2023-05-09 | Establishment of unlabeled quantitative method for detecting collagen in lung tissue |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023066158A1 (en) * | 2021-10-18 | 2023-04-27 | 安徽神东生物科技开发有限责任公司 | Establishment of non-marking quantification method for detecting lung tissue collagen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023066158A1 (en) * | 2021-10-18 | 2023-04-27 | 安徽神东生物科技开发有限责任公司 | Establishment of non-marking quantification method for detecting lung tissue collagen |
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