CN116840131A - In-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device and method in tissue section - Google Patents
In-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device and method in tissue section Download PDFInfo
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Abstract
The invention relates to an in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device and method in tissue sections, comprising the following steps: and a laser emission unit: coupling two laser beams with different wavelengths on the same point to excite single cells to be detected; sample control unit: driving the sample to realize three-dimensional space movement; transmission type fluorescence imaging acquisition unit: collecting a bicolor fluorescent image of the targeted in-situ single cell in the tissue slice; back-to-back raman spectrum acquisition unit: collecting spontaneous Raman spectra of targeted in-situ single cells in a tissue slice; spectral analysis unit: spectral analysis and automatic classification are realized. The invention captures the fluorescence image and spontaneous Raman spectrum of the in-situ single cell in the tissue slice at the same time, the fluorescence imaging technology can rapidly locate the targeted single cell and obtain the morphological information of the cell, and the Raman scattering technology can detect various biological molecular information of the single cell at the same time; the invention also provides an in-situ single cell spontaneous Raman spectrum analysis and automatic classification method in the tissue section.
Description
Technical Field
The invention relates to a fluorescence imaging and Raman spectrum bimodal detection device and a Raman spectrum bimodal detection method for in-situ single cells in a tissue slice, which can realize fluorescence imaging positioning of in-situ single cells in a tissue slice with a micron-sized thickness, and further realize Raman scattering measurement of in-situ single cells in the tissue, so as to obtain morphology and various biomolecule information of in-situ cells. The invention can be used for analyzing and automatically classifying targeted in-situ single cells in tissue sections, and has important application in the fields of oncology and the like.
Background
Clinical tumor tissue retains almost all in situ information, with nearly complete tumor microenvironment. At present, most of researches on tissue sections and in-situ single cells in tissues analyze the changes of various factors by means of HE staining, immunohistochemistry and the like, and the research technology of the immunohistochemistry requires various specific antibodies in the research process, and one antibody corresponds to one targeting factor, so that the research cost is higher, and the research process is complex.
The fluorescence imaging technology can realize the positioning and morphological analysis of the targeted in-situ single cells in the tissue section, but has great challenges for fluorescent labeling of various lipid, sugar and other substances in the cells in the tissue section, and the simultaneous analysis of various biological macromolecules in the cells needs to solve the problems of light path design, difficulty in simultaneous labeling of various fluorescence and higher research cost. The invention obtains the spectrum information which can be obtained by multicolor dyeing by utilizing the spontaneous Raman scattering technology, and solves the problem of multicolor fluorescent dyeing.
More and more researchers study the changes of various molecules in biological samples through a Raman scattering technology, and analyze the relationship between diseases and material changes from an all-molecular perspective. Raman scattering techniques allow rapid, label-free, non-destructive measurement of chemical fingerprints of samples and show potential for cancer screening, diagnosis and other substance state analysis. The spontaneous Raman spectrum has higher repeatability and more biological information, and is suitable for cancer diagnosis and substance molecular research. If the biomolecule information of the targeted single cells is obtained from the tissue slice only by the Raman scattering technology, the whole tissue needs to be comprehensively scanned, and then the Raman scanning imaging result is analyzed. The realization method has great limitation, and on one hand, the scanning imaging of the whole tissue by the Raman scattering technology takes a long time; on the other hand, there are tens of different cells in the tissue, which presents a great challenge for the determination and study of target single cells. The traditional spontaneous Raman scattering detection obtains global information of tissue sections, and lacks a direct information obtaining means of single cells after positioning.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device in a tissue slice;
the invention also provides an in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection method in the tissue slice;
in order to achieve capture of a specific in-situ single-cell Raman spectrum from a tissue slice, the invention adopts a transmission fluorescent imaging technology as a positioning system of a targeted in-situ single cell in the tissue slice, and obtains a molecular fingerprint spectrum of the targeted in-situ single cell through a Raman scattering technology. The fluorescence microscopy technology can rapidly detect the targeted single cells, and can obtain the morphological and biochemical information of specific organelles of the single cells, while the Raman scattering technology can detect various molecular substances at the same time, and the combination targeting of the two technologies is high and can save more time and cost. The invention can be applied to the morphology of in-situ single cells in cancer tissue sections and the bimodal detection of molecular substances, and can realize the analysis and automatic classification of various substances by combining machine learning.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an in situ single cell fluorescence imaging and raman spectroscopy bimodal detection apparatus in a tissue slice, comprising:
a laser emitting unit for: coupling two laser beams with different wavelengths on the same excitation point, and simultaneously exciting a sample to be detected;
a sample control unit for: driving a sample to be tested to realize three-dimensional space movement;
the transmission type fluorescence imaging acquisition unit is used for: collecting a bicolor fluorescent image of the targeted in-situ single cell in the tissue slice of the sample to be detected, and realizing fluorescent positioning of the targeted in-situ single cell in the tissue slice and acquisition of specific organelle morphology and biochemical information;
the back-to-back Raman spectrum acquisition unit is used for: collecting Raman spectrum of fluorescence-positioned in-situ single cells in a tissue slice of a sample to be detected;
a spectrum analysis unit for: the analysis and automatic classification of the spectrum are realized on the basis of spectrum pretreatment.
According to the invention, the laser emission unit comprises two laser light sources with different wavelengths, a first optical filter, a first microscope objective, a first dichroic mirror, a reflecting mirror, a second dichroic mirror and a second microscope objective;
after passing through the first optical filter and the first micro-objective lens in sequence, the laser beam emitted by the laser light source and the laser beam emitted by the other laser light source respectively penetrate from the two sides of the first dichroic mirror at an angle perpendicular to each other and then are co-beamed, and then the laser beam is focused on the same center point of the sample to be detected through the second micro-objective lens after the propagation direction is controlled through the reflecting mirror and the second dichroic mirror.
It is further preferred that the two laser light sources of different wavelengths comprise a diode semiconductor solid state laser and a diode pumped crystal laser.
According to the invention, the sample control unit comprises a fixer for fixing the tissue slice of the sample to be tested and a triaxial displacement table for driving the sample to be tested to move along the X axis, the Y axis and the Z axis.
According to the invention, the transmission type fluorescent imaging acquisition unit comprises a third micro objective lens, a second optical filter, a third dichroic mirror, a lens barrel lens, a third optical filter and a CMOS detector which are sequentially arranged along an optical path;
and focusing the fluorescence signal of the sample to be detected after the accurate positioning by the third microscope objective, filtering excitation light by the fluorescence signal through the second optical filter and the third dichroic mirror, focusing the bicolor fluorescence image to the plane of the CMOS detector after passing through the lens barrel lens and the third optical filter, and recording the bicolor fluorescence image by the CMOS detector.
According to the invention, the back-facing raman spectrum acquisition unit comprises a second micro objective lens, a second dichroic mirror, a fourth optical filter, a first cemented lens, an aperture, a second cemented lens and a raman spectrometer which are sequentially arranged along an optical path;
and the Raman scattering signal of the sample to be detected is focused by a second microscope objective at the excitation end, excitation light and partial fluorescence are filtered by a second dichroic mirror and a fourth optical filter, then the cover glass and the substrate sheet Raman spectrum at the non-focusing position are removed by a first bonding lens, a small hole and a second bonding lens, and finally the Raman spectrum of the sample to be detected at the focusing position is obtained by a Raman spectrometer.
According to the invention, the spectrum analysis unit comprises a spectrum preprocessing module and a support vector machine automatic classification module;
the spectrum preprocessing module is used for: removing cosmic rays, smoothing, removing fluorescent substrates, removing substrate slice background and normalizing;
the support vector machine automatic classification module is used for: the pretreated Raman spectrum is divided into a training set and a testing set, and the automatic classification of the sample to be detected is realized by combining ten-fold cross validation.
The in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection method in the tissue slice is realized by the Raman spectrum detection device and comprises the following steps:
(1) Starting a laser emission unit, calibrating a light path, determining that the bicolor laser beams are on the same central line, focusing through the center of the second micro objective lens, and recording that focusing light spots of the bicolor laser beams are positioned at the same central point;
(2) Controlling the triaxial displacement table to move the sample to be detected, enabling the sample to be detected fixed on the triaxial displacement table to be positioned on the focal planes of the second microscope objective and the third microscope objective, calibrating the optical path, and triggering the Raman spectrometer to record the Raman signal with the highest intensity of the sample to be detected;
(3) Fixing a sample to be measured, namely a sample to be measured fluorescent staining tissue slice sample, on a triaxial displacement table;
(4) Controlling the triaxial displacement table to move the sample to be detected, enabling the sample to be detected to be positioned on focal planes of the second microscope objective and the third microscope objective, and triggering the CMOS detector to record a bicolor fluorescent image of in-situ cells in the tissue slice;
(5) The single laser beam excites a Raman signal of the targeted in-situ single cell positioned by fluorescence imaging, and a Raman spectrometer is triggered to record the Raman spectrum of the measured point;
(6) And inputting the Raman spectrum acquired by the Raman spectrometer into a spectrum preprocessing module and a support vector machine automatic classification module to perform spectrum preprocessing analysis and classification of the sample.
The beneficial effects of the invention are as follows:
1. the invention adopts the Raman scattering technology to realize the molecular-level substance analysis of biological samples such as cells, and the technology can simultaneously acquire the content relation among various biological molecules of a single-point sample;
2. the acquisition device has two working modes, can simultaneously capture fluorescent images and Raman spectrum signals of a sample, can rapidly position in-situ targeted single cells by a fluorescent imaging technology and obtain the morphological and biochemical information of specific organelles of the single cells, and can simultaneously detect various biological molecular information of the sample to be detected by a Raman scattering technology;
3. the invention is suitable for analyzing targeted in-situ single cells in a micron-sized clinical tissue slice sample, can analyze the change of substances such as biomolecules from a single cell angle, and retains in-situ information of the cells;
4. according to the invention, dual-wavelength laser beam coupling is adopted, and a specific region of a sample is excited at the same time, so that double verification of a targeted single cell is realized;
5. the invention can realize the rapid and automatic classification of single cells in the tissue slices beside the cancer and the cancer, and gets rid of the complicated process of manual film reading;
6. the invention is suitable for the identification and classification of other biological cells and has universal popularization.
Drawings
FIG. 1 is a schematic diagram of the structure of an in situ single cell fluorescence imaging and Raman spectroscopy bimodal detection apparatus in a tissue slice according to the present invention;
FIG. 2 (a) is a schematic diagram showing the relationship between the wavelength and the intensity distribution of the Raman scattering excitation light source;
FIG. 2 (b) is a schematic diagram showing the results of a Raman spectrum test of 75% ethanol provided by the example of the present invention;
FIG. 3 is a schematic view of a spot size analysis image of a Raman scattering excitation light source focused on a sample to be measured;
FIG. 4 (a) is a fluorescence diagram of cell membrane CY3 of CD68+ cells;
FIG. 4 (b) is a nuclear DAPI fluorescence of cells;
FIG. 4 (c) is a simulated laser spot and nucleus plot at a first location;
FIG. 4 (d) is a simulated laser spot and nucleus plot at a second location;
FIG. 4 (e) is a simulated laser spot and nucleus plot at a third location;
FIG. 4 (f) is a simulated laser spot and nucleus plot at a fourth location;
FIG. 4 (g) is a simulated laser spot and nucleus plot at a fifth location;
FIG. 5 (a) is a schematic diagram of the original Raman spectrum of TAMs provided by examples of the present invention;
FIG. 5 (b) is a schematic diagram of Raman spectra after pretreatment of TAMs provided in examples of the present invention;
FIG. 6 is a schematic diagram showing analysis of average Raman spectra and difference spectra of TAMs in clinical liver cancer and paracancestral tissue sections provided by the example of the present invention.
Wherein, 1, a first laser, 2, a first optical filter, 3, a first microscope objective, 4, a second laser, 5, a first dichroic mirror, 6, a reflecting mirror, 7, a second dichroic mirror, 8, a second microscope objective, 9, a triaxial displacement table, 10, a third microscope objective, 11, a second filter, 12, a third dichroic mirror, 13, a tube lens, 14, a third filter, 15, a CMOS detector, 16, a fourth filter, 17, a first cemented lens, 18, an aperture, 19, a second cemented lens, 20, a Raman spectrometer.
Detailed Description
The invention is further described below with reference to the drawings and examples, but is not limited thereto.
Example 1
An in situ single cell fluorescence imaging and raman spectroscopy bimodal detection apparatus in a tissue slice, comprising:
a laser emitting unit for: coupling two laser beams with different wavelengths on the same excitation point, and simultaneously exciting a sample to be detected;
a sample control unit for: driving a sample to be tested to realize three-dimensional space movement;
the transmission type fluorescence imaging acquisition unit is used for: collecting a bicolor fluorescent image of the targeted in-situ single cell in the tissue slice of the sample to be detected, and realizing fluorescent positioning of the targeted in-situ single cell in the tissue slice and acquisition of specific organelle morphology and biochemical information; the method comprises the steps of carrying out a first treatment on the surface of the
The back-to-back Raman spectrum acquisition unit is used for: collecting Raman spectrum of fluorescence-positioned in-situ single cells in a tissue slice of a sample to be detected;
a spectrum analysis unit for: the analysis and automatic classification of the spectrum are realized on the basis of spectrum pretreatment.
The invention adopts a transmission type fluorescence imaging technology as a positioning system of the targeted in-situ single cells in the tissue section, obtains the molecular fingerprint spectrum of the targeted in-situ single cells through a Raman scattering technology, can rapidly position the targeted cells through a fluorescence microscopy technology, further detects various molecular substances in the single cells through the Raman scattering technology, and can realize the morphology of the in-situ single cells in the tissue section and the detection, analysis and automatic classification of various biological molecules. In the Raman spectrum acquisition process, the sample to be detected always maintains the in-situ state of cells, and is not damaged.
Example 2
An in situ single cell fluorescence imaging and raman spectroscopy dual modality detection apparatus in tissue slices according to embodiment 1, which is characterized in that:
as shown in fig. 1, the laser emission unit includes two laser light sources of different wavelengths, a first optical filter 2, a first microscope objective 3, a first dichroic mirror 5, a reflecting mirror 6, a second dichroic mirror 7, and a second microscope objective 8;
after passing through the first optical filter 2 and the first micro objective 3 in sequence, the laser beam emitted by the laser light source and the laser beam emitted by the other laser light source respectively penetrate from the two sides of the first dichroic mirror 5 at an angle perpendicular to each other and then are co-beamed, and after the propagation direction is controlled by the reflecting mirror 6 and the second dichroic mirror 7, the laser beam is focused on the same center point of the sample to be detected by the second micro objective 8.
The two laser light sources of different wavelengths include a diode semiconductor solid state laser and a diode pumped crystal laser. The wavelengths were 532nm and 375nm, respectively, the diameters of the generated laser beams were 1mm and 1.1mm, respectively, and the highest powers of the lasers were 100mW and 60mW, respectively.
The model of the first microscope objective 3 is: olympus, PLN10X, japan; the model of the second microscope objective 8 is: japanese, olympus, LUCPLLN 40X.
The model of the first dichroic mirror 5 is: U.S., thorlabs, DMLP505; the model of the second dichroic mirror 7 is: united states, thorlabs, DMLP550.
The sample control unit comprises a fixer for fixing tissue slices of a sample to be tested and a triaxial displacement table 9 for driving the sample to be tested to move along an X axis, a Y axis and a Z axis.
The triaxial displacement table 9 is composed of a high-precision electric triaxial displacement table and a biaxial displacement table, and the model of the electric triaxial displacement table is as follows: U.S. THORLABS, MAX311D; the model of the two-axis displacement table is as follows: china, zhuo Lihan light, ALB25A-6520SR.
The transmission type fluorescence imaging acquisition unit comprises a third microscope objective 10, a second optical filter 11, a third dichroic mirror 12, a lens barrel lens 13, a third optical filter 14 and a CMOS detector 15 which are sequentially arranged along the light path;
the third microscope objective 10 focuses the fluorescence signal of the sample to be detected after accurate positioning, the fluorescence signal filters the excitation light through the second optical filter 11 and the third dichroic mirror 12, and then focuses the bicolor fluorescence image to the plane of the CMOS detector 15 through the lens barrel lens 13 and the third optical filter 14, and the CMOS detector 15 records the bicolor fluorescence image.
The model of the third microscope objective 10 is: japanese, olympus, LUCPLLN 20X.
The model of the second filter 11 is: U.S. Thorlabs, NF533-17; the model of the third filter 14 is: U.S. Thorlabs, FB580-10.
The back-facing Raman spectrum acquisition unit comprises a second microscope objective 8, a second dichroic mirror 7, a fourth optical filter 16, a first cemented lens 17, a small hole 18, a second cemented lens 19 and a Raman spectrometer 20 which are sequentially arranged along the light path;
the raman scattering signal of the sample to be detected is focused by the second micro objective lens 8 at the excitation end, excitation light and partial fluorescence are filtered by the second dichroic mirror 7 and the fourth optical filter 16, then the cover glass and the substrate raman spectrum at the non-focusing position are removed by the first bonding lens 17, the small hole 18 (circular precise pinhole) and the second bonding lens 19, and finally the raman spectrum of the sample to be detected at the focusing position is obtained by the raman spectrometer 20 (ocean optics, 600 lines/mm).
The fourth filter 16 model is: semrock, BLP01-532R-25, U.S.
The model of the first cemented lens 17 is: U.S. Thorlabs, AC254-030-A-ML; the model of the second cemented lens is: U.S. Pat. No. 4,000,000, TRS254-040-A-ML.
The size of the aperture 18 is: U.S. Thorlabs, P20K.
The spectrum analysis unit comprises a spectrum preprocessing module and an automatic classification module of a support vector machine;
a spectrum preprocessing module for: removing cosmic rays, smoothing, removing fluorescent substrates, removing substrate slice background and normalizing;
removing cosmic rays: cosmic ray removal is performed using the function 'filters' in MATLAB R2021a, where the fill method 'filemethod' for replacing outliers is 'clip' (filled with lower thresholds for elements smaller than the lower threshold determined by 'findmethod'), and for elements larger than the upper threshold determined by 'findmethod', fill with upper thresholds), and the method 'findmethod' for detecting outliers is set to 'mean' (outliers are defined as elements that differ from median by more than three times the converted MAD, which is defined as c media (abs (a-media (a)), where c= -1/(sqrt (2) ×erfcinv (3/2)), a is the input data matrix), and the window length 'window' is set to 7.
Smoothing: the data smoothing was performed using the function ' smoothdata ' in MATLAB R2021a, where the smoothing method ' was set to ' sgolay ' (Savitzky-Golay filter that smoothed according to a quadratic polynomial fit over each window of the data matrix), setting the window length ' window ' to 7.
The fluorogenic substrate: background estimation was performed by a polynomial of order 7 using MATLAB R2021a tool, thereby removing the background.
Background removal of the substrate sheet: and searching a first Raman characteristic peak position P1 of the quartz substrate slice by using a MATLAB R2021a tool, taking the Raman characteristic peak without a sample at the characteristic peak, respectively taking the quartz substrate slice and the Raman spectrum of the sample, taking the average of 3 points at the P1 position, obtaining the divided coefficient at the P1 position, multiplying the Raman spectrum of the quartz substrate slice by the two coefficients, and subtracting the Raman signal of the quartz slice in the divided sample.
Normalization: vector normalization processing was used by MATLAB R2021a tool.
The support vector machine automatic classification module is used for: the pretreated Raman spectrum is divided into a training set and a testing set, and the automatic classification of the sample to be detected is realized by combining ten-fold cross validation.
The invention uses the 'fitecoc' function of MATLAB R2021a to fit multiple models of support vector machine, and adopts 10-fold cross validation algorithm of cross validation function 'cross val' to evaluate the loss. In the constructed support vector machine classification model, the invention adopts Gaussian envelope as kernel function to realize nonlinear mapping of input feature space, selects 'Kernel scale' in kernel scale parameters as 'auto', sets 'Box constraint' as 2, and respectively performs weighted column mean value and standard deviation centering and scaling on each column of prediction data.
The support vector machine classification model which is initially constructed divides the whole preprocessed spectrum data set into ten parts, 9 parts of the spectrum data set are used as training data in turn, and 1 part of the spectrum data set is used as test data for test.
The final classification result comprises specificity, sensitivity and accuracy; 93.9%, 88.4% and 91.2% respectively.
Example 3
The in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection method in the tissue slice is realized by the Raman spectrum detection device in the embodiment 2 and comprises the following steps:
(1) Starting a laser emission unit, calibrating a light path, determining that the bicolor laser beams are on the same central line, and triggering the CMOS detector 15 to record that the focusing light spots of the bicolor laser beams are positioned at the same central point through the center and focusing of the second micro objective lens 8; the method is concretely realized as follows: the laser beam emitted by the first laser 1 is shaped by the first optical filter 2 and the first micro objective lens 3, the propagation direction is adjusted by the first dichroic mirror 5, the laser beam is on the same straight line with the laser beam emitted by the second laser 4, the transmission direction of the two laser beams is adjusted again by the reflecting mirror 6 and the second dichroic mirror 7, the laser beams are focused on the same center point by the second micro objective lens 8, and the focusing light spot size of the laser beam emitted by the second laser 4 is 2.83um.
(2) Controlling the triaxial displacement table 9 to move the sample to be detected, enabling the sample to be detected fixed on the precise triaxial displacement table 9 to be positioned on focal planes of the second micro objective lens 8 and the third micro objective lens 10, calibrating a light path, and triggering the Raman spectrometer 20 to record a Raman signal with the highest intensity of the sample to be detected;
(3) Fixing a sample to be measured, namely a sample to be measured fluorescent staining tissue slice sample, on a triaxial displacement table 9;
(4) The two beams of laser excite sample fluorescence at the same time, the sample to be detected is positioned on the focal plane of the third microscope objective 10 by controlling the triaxial displacement table 9 to move, the excitation light and the stray light are filtered by the second optical filter 11, the third dichroic mirror 12 and the third optical filter 14, and then focused on the CMOS detector 15 through the lens cone lens 13, and the bicolor fluorescence image of the in-situ cells in the tissue slice of the sample to be detected is recorded.
(5) The single laser beam excites the Raman signal of the targeted in-situ single cell positioned by fluorescence imaging, and triggers the Raman spectrometer 20 to record the Raman spectrum of the measured point; the method is concretely realized as follows: the second laser 4 is used for exciting Raman scattering signals of the targeted in-situ single cells positioned through fluorescence imaging, the Raman scattering signals are collected by the second microscope objective 8, the second dichroic mirror 7 and the fourth filter 16 are used for filtering excitation light and partial fluorescence signals, the first bonding lens 17, the small hole 18 and the second bonding lens 19 are used for removing non-focusing noise, and the Raman spectrometer 20 is triggered to record Raman spectrum signals of measured points.
(6) The raman spectrum acquired by the raman spectrometer 20 is input to a spectrum preprocessing module and a support vector machine automatic classification module, and spectrum preprocessing analysis and classification of the sample are performed.
Example 4
In order to verify the matching of the excitation wavelength in the Raman scattering system and the frequency shift conversion of the spectrometer software, ethanol is used for experimental verification and device calibration. In this embodiment, 75% ethanol is selected as a test sample, a raman spectrum thereof is obtained, and an experimental result is compared with peak positions in a raman standard database for analysis, thereby calibrating an experimental system. The specific operation steps comprise:
(1) Introducing a 75% ethanol solution into the constructed sample chamber;
(2) Turning on a laser light source, calibrating a light path, using a diffusely-reflecting paperboard to reflect micro laser, triggering a Raman spectrometer 20 to obtain a spectrum of an excitation light source, wherein the spectrum result is shown in FIG. 2 (a), and the central wavelength of the laser light source is 531.23nm;
(3) Triggering a Raman spectrum display interface of the Raman spectrometer 20, and inputting the central wavelength of laser into 531.23nm;
(4) By adjusting the sample stage to the focal plane of the third microscope objective 10 (40 x, na=0.6), the raman spectrometer 20 is triggered to acquire the raman spectrum of the ethanol solution, consistent with the peak positions in the standard raman spectrum database being within the tolerance limits, as shown in fig. 2 (b).
Example 5
An in-situ single cell fluorescence imaging and Raman spectrum bimodal detection method in tissue section is used for identifying and classifying Tumor-associated macrophages (Tumor-associated macrophages, TAMs) in liver cancer and liver cancer side tissue sections. In this example, the spot size of the stimulated in-situ single-cell raman scattering was adjusted to 2.83um, and as shown in fig. 3, fluorescence imaging and raman spectrum acquisition were performed on a single TAM in tissue sections stained with goat serum diluted fluorescent secondary antibody and DAPI, 30 raman spectra of TAMs were acquired in each tissue section, and 5 spots were acquired on average for each TAM in different positions. Raman spectra of the TAMs in the cancer and paracancel tissue sections of 12 liver cancer patients are obtained in total, and are input into an automatic classification module of a support vector machine after being processed by a spectrum preprocessing module, so as to classify the TAMs in the cancer and paracancel. The specific implementation process comprises the following steps:
(1) Fixing the tissue slice after fluorescent staining on a glass slide, and integrally fixing the tissue slice on a triaxial displacement table 9;
(2) Starting the first laser 1 and the second laser 4, controlling the triaxial displacement table 9 and the fluorescence imaging unit, and obtaining fluorescence images of the nuclei and the cell membranes of the TAMs in a focusing state, as shown in fig. 4 (a) and 4 (b);
(3) Triggering the raman spectrometer 20 to acquire raman spectra that are localized to five different positions of a single TAM by fluorescence imaging, fig. 4 (c) is a simulated laser spot and nuclear map at the first position; FIG. 4 (d) is a simulated laser spot and nucleus plot at a second location; FIG. 4 (e) is a simulated laser spot and nucleus plot at a third location; FIG. 4 (f) is a simulated laser spot and nucleus plot at a fourth location; FIG. 4 (g) is a simulated laser spot and nucleus plot at a fifth location;
(4) Inputting all acquired Raman spectra into a spectrum preprocessing module, respectively performing processing such as removing cosmic rays, removing fluorescent substrates, smoothing, removing substrate slice background, normalizing and the like, wherein FIG. 5 (a) is a schematic diagram of an original Raman spectrum of TAMs provided by the embodiment of the invention; FIG. 5 (b) is a schematic diagram of Raman spectra after pretreatment of TAMs provided in examples of the present invention;
(5) The raman spectra after pretreatment of TAMs in the cancer and the paracancer are averaged respectively and subjected to difference to obtain difference spectra, as shown in fig. 6;
(6) And (3) inputting the Raman spectra after the TAMs pretreatment in the cancer and the cancer side into an automatic classification module of a support vector machine, taking the spectrum waveforms as classification characteristic values of the Raman spectra, executing an SVM algorithm, and constructing a classifier by adopting a 10-fold cross-validation method. The 2919 spectrum data of TAMs in cancer and paracancer are randomly divided into 10 groups of data sets, 292 data are arranged in each data set, 9 groups of data sets are circulated to serve as training sets, the rest group of data sets serve as test sets, and after 10 times of repeated cross validation, the average value of the 10 times of prediction accuracy is taken as the final classification accuracy. Table 1 is a table of the correspondence of species and Raman peaks in TAMs; table 2 shows the SVM classification results of TAMs in tissue sections beside and along with cancer.
TABLE 1
TABLE 2
The results of this example are shown in FIG. 5, in which the substance differences of liver cancer and TAMs in the paracancer are derived from proteins, lipids, saccharides and nucleic acids, the content of part of lipids of TAMs in the tissue sections of cancer is increased relative to the TAMs in the paracancer, the content of most of proteins, saccharides and nucleic acids is decreased, and the SVM classification accuracy by these substance differences is 91.2%.
While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the invention, but rather, it is intended to cover all modifications or variations within the scope of the invention as defined by the claims of the present invention.
Claims (8)
1. An in-situ single-cell fluorescence imaging and raman spectroscopy bimodal detection device in a tissue slice, comprising:
a laser emitting unit for: coupling two laser beams with different wavelengths on the same excitation point, and simultaneously exciting a sample to be detected;
a sample control unit for: driving a sample to be tested to realize three-dimensional space movement;
the transmission type fluorescence imaging acquisition unit is used for: collecting a bicolor fluorescent image of the targeted in-situ single cell in the tissue slice of the sample to be detected, and realizing fluorescent positioning of the targeted in-situ single cell in the tissue slice and acquisition of specific organelle morphology and biochemical information;
the back-to-back Raman spectrum acquisition unit is used for: collecting Raman spectrum of fluorescence-positioned in-situ single cells in a tissue slice of a sample to be detected;
a spectrum analysis unit for: the analysis and automatic classification of the spectrum are realized on the basis of spectrum pretreatment.
2. The in-situ single-cell fluorescence imaging and raman spectroscopy bimodal detection apparatus in a tissue slice according to claim 1, wherein the laser emission unit comprises two laser light sources of different wavelengths, a first optical filter, a first microscope objective, a first dichroic mirror, a reflecting mirror, a second dichroic mirror, and a second microscope objective;
after passing through the first optical filter and the first micro-objective lens in sequence, the laser beam emitted by the laser light source and the laser beam emitted by the other laser light source respectively penetrate from the two sides of the first dichroic mirror at an angle perpendicular to each other and then are co-beamed, and then the laser beam is focused on the same center point of the sample to be detected through the second micro-objective lens after the propagation direction is controlled through the reflecting mirror and the second dichroic mirror.
3. The in situ single cell fluorescence imaging and raman spectroscopy bimodal detection apparatus in tissue section according to claim 2, wherein the two different wavelength laser light sources comprise a diode semiconductor solid state laser and a diode pumped crystal laser.
4. The in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device in a tissue section according to claim 1, wherein the sample control unit comprises a fixer for fixing the tissue section of the sample to be detected and a triaxial displacement table for driving the sample to be detected to move along an X axis, a Y axis and a Z axis.
5. The in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device in tissue section according to claim 1, wherein the transmission type fluorescence imaging acquisition unit comprises a third microscope objective, a second optical filter, a third dichroic mirror, a lens barrel lens, a third optical filter and a CMOS detector which are sequentially arranged along an optical path;
and focusing the fluorescence signal of the sample to be detected after the accurate positioning by the third microscope objective, filtering excitation light by the fluorescence signal through the second optical filter and the third dichroic mirror, focusing the bicolor fluorescence image to the plane of the CMOS detector after passing through the lens barrel lens and the third optical filter, and recording the bicolor fluorescence image by the CMOS detector.
6. The in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device in tissue section according to claim 1, wherein the back-facing Raman spectrum acquisition unit comprises a second micro objective lens, a second dichroic mirror, a fourth optical filter, a first cemented lens, a small hole, a second cemented lens and a Raman spectrometer which are sequentially arranged along an optical path;
and the Raman scattering signal of the sample to be detected is focused by a second microscope objective at the excitation end, excitation light and partial fluorescence are filtered by a second dichroic mirror and a fourth optical filter, then the cover glass and the substrate sheet Raman spectrum at the non-focusing position are removed by a first bonding lens, a small hole and a second bonding lens, and finally the Raman spectrum of the sample to be detected at the focusing position is obtained by a Raman spectrometer.
7. The in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection device in a tissue slice according to any one of claims 1-6, wherein the spectrum analysis unit comprises a spectrum preprocessing module and a support vector machine automatic classification module;
the spectrum preprocessing module is used for: removing cosmic rays, smoothing, removing fluorescent substrates, removing substrate slice background and normalizing;
the support vector machine automatic classification module is used for: the pretreated Raman spectrum is divided into a training set and a testing set, and the automatic classification of the sample to be detected is realized by combining ten-fold cross validation.
8. An in-situ single-cell fluorescence imaging and Raman spectrum bimodal detection method in a tissue slice, which is realized by the fluorescence imaging and Raman spectrum bimodal detection device according to any one of claims 1-7, and is characterized by comprising the following steps:
(1) Starting a laser emission unit, calibrating a light path, determining that the bicolor laser beams are on the same central line, focusing through the center of the second micro objective lens, and recording that focusing light spots of the bicolor laser beams are positioned at the same central point;
(2) Controlling the triaxial displacement table to move the sample to be detected, enabling the sample to be detected fixed on the triaxial displacement table to be positioned on the focal planes of the second microscope objective and the third microscope objective, calibrating the optical path, and triggering the Raman spectrometer to record the Raman signal with the highest intensity of the sample to be detected;
(3) Fixing a sample to be measured, namely a sample to be measured fluorescent staining tissue slice sample, on a triaxial displacement table;
(4) Controlling the triaxial displacement table to move the sample to be detected, enabling the sample to be detected to be positioned on focal planes of the second microscope objective and the third microscope objective, triggering the CMOS detector to record a bicolor fluorescent image of in-situ single cell nuclei and cell membranes in the fluorescent staining tissue slice to be detected;
(5) The single laser beam excites a Raman signal of the targeted in-situ single cell positioned by fluorescence imaging, and a Raman spectrometer is triggered to record the Raman spectrum of the measured point;
(6) And inputting the Raman spectrum acquired by the Raman spectrometer into a spectrum preprocessing module and a support vector machine automatic classification module to perform spectrum preprocessing analysis and classification of the sample.
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