CN113433108B - Stomach peeping biopsy histopathology imaging method based on stimulated Raman scattering - Google Patents
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Abstract
The invention belongs to the technical field of nonlinear optical imaging, and particularly relates to an imaging method of entogastric endoscopic biopsy histopathology based on stimulated Raman scattering. The method of the invention can acquire histopathology image information of gastric biopsy in a short time by utilizing the rapid, treatment-free and marking-free properties of stimulated Raman scattering microscopic imaging. The invention firstly uses the stimulated Raman scattering microscopy in the gastroscope endoscopic biopsy, and compared with the prior traditional histopathology technology, the invention has the advantages that: the imaging speed is fast, and the imaging quality is high, need not to carry out the preliminary treatment, does not have the original tissue of noninvasive retention to can image to each plane in certain degree of depth.
Description
Technical Field
The invention belongs to the technical field of nonlinear optical imaging, and particularly relates to an imaging method of entogastric endoscopic biopsy histopathology based on stimulated Raman scattering.
Background
Gastric cancer is a serious disease affecting human health. According to the latest annual report of national tumor registration centers, the number of new diseases of gastric cancer exceeds 68 ten thousand per year, about 50 ten thousand of new diseases die, and the new diseases become the second most lethal tumor in China. The key point of the prevention and control of the gastric cancer lies in early diagnosis and early treatment. Early cancer is found in time, precancerous lesion is delayed and reversed, and the incidence rate and the fatality rate of gastric cancer can be effectively reduced. Gastric cancer is developed through various steps such as chronic inflammation, precancerous lesion, early cancer, invasive cancer and the like. The development of gastric cancer usually takes decades of development and accumulation of events throughout the process. Even if early cancer has already occurred, it usually takes about 44 months to progress to late stage. Therefore, the precancerous lesion and the precancerous lesion of the gastric cancer are used as key nodes for preventing and controlling the gastric cancer, necessary intervention and treatment are timely given, the morbidity and the fatality rate of the gastric cancer are effectively reduced, and the harm of the gastric cancer is relieved. However, the early gastric cancer and precancerous lesion lack obvious morphological characteristics, and the performance cannot be clarified by only depending on an endoscope, so that the diagnosis of histopathology must be relied on. The latter requires multiple steps of dehydration, embedding, sectioning, staining, scoring, etc., with significant time lag. Thus, it is important to obtain histopathological images quickly intraoperatively, without being time-and labor-intensive.
The Raman spectrum technology can detect the molecular information of pathological changes rapidly, accurately and noninvasively. The Raman spectrum is a nondestructive analysis technology, is based on the principle of inelastic scattering effect of incident light and substances, and can describe the characteristic vibration of chemical bonds/groups in the molecules of a sample to be detected under the incident light. Thus, the raman spectrum contains fingerprint information of the molecule. The method has the important advantages that the species of the substance can be directly distinguished according to the Raman fingerprint characteristics of the sample molecules, and particularly, the method has obvious advantages for fingerprint identification of disease molecules in the field of medical diseases. Compared with other detection methods, such as fluorescence detection, the Raman detection has the obvious advantages of molecular fingerprint identification. Compared with other detection methods, such as fluorescence detection, raman detection not only has the characteristic of molecular fingerprint identification, but also has the advantages of no need of sample treatment, insensitivity to water (water-containing medical biological detection is particularly useful, and cannot be realized by infrared spectroscopy), micro-area micro detection and the like, which are incomparable with other detection technologies at present. A second advantage of raman spectroscopy is "speed". The Raman spectrum collection can be completed within a few seconds, the detection cost is low, and the sample is only required to be placed in a Raman check point during actual detection, so that the detection can be rapidly judged after the detection is finished.
However, the conventional spontaneous raman spectroscopy cannot realize spatial imaging of a sample, and biological samples often have strong spatial heterogeneity, such as nonuniform lesion range distribution, different weights, and the like. The stimulated Raman scattering combines the Raman scattering and the Einstein stimulated emission principle, the gains of Raman effects of 103-105 can be realized, and high-speed imaging of video speed is achieved. The stimulated Raman spectrum inherits the molecular fingerprint characteristics of the spontaneous Raman spectrum, has good molecular specificity, and can express different molecules in a form similar to pathological images according to the characteristic vibration spectrum of the different molecules. Therefore, the method has the main advantages that the spatial imaging of the sample can be realized, if the suspicious region to be detected clinically can be positioned and screened, the Raman spectra of different sites of the sample can be accurately obtained by combining the rapid imaging technology, and the method is more favorable for improving the accuracy and the targeting property of diagnosis.
Disclosure of Invention
The invention aims to provide a histopathology imaging method for endoscopic biopsy of stomach, which has high imaging speed and high imaging quality and solves the problems of complex sample pretreatment, intensive processing process labor, obvious time lag and the like in the prior art.
The invention provides a histopathology imaging method for gastric endoscopic biopsy, which is based on a stimulated Raman scattering technology and comprises the following specific steps:
s1, aiming at biochemical components existing in stomach tissues, selecting proper biomolecules as a substance to be detected according to characteristics concerned in histopathology, and detecting a standard sample corresponding to the substance to be detected by using a stimulated Raman scattering microscopic imaging system to obtain the optimal state of specific parameters in the stimulated Raman imaging system; the parameters include: pump and stokes light wavelengths, relative time delays between pump and stokes light;
s2, setting experiment parameters according to the result of the S1, carrying out region selection on a substance to be detected in the stomach biopsy tissue, and then carrying out rapid microscopic imaging on each region: after scanning of each frame of image is finished, moving the sample stage according to the designed position of the original region, then scanning the next frame of image, repeating the steps until the whole selected region is completely scanned to obtain a blocked image of the field of view of the whole region, and finally splicing the blocked image into a large-size image through a splicing algorithm;
when a plurality of substances to be detected such as lipid, protein, collagen and the like need to be imaged, after each frame of image is scanned, experimental parameters are automatically switched to be adjusted to scan another substance channel to be detected, and then the sample stage is translated; meanwhile, other substances are imaged simultaneously by using a multi-mode;
s3, compiling a splicing algorithm for the small images scanned in the plurality of areas, and splicing the small images into a large image with a complete view field; reading a picture sequence into an algorithm program, firstly arranging small pictures to the expected position of a complete large picture according to the sequence number, splicing adjacent pictures, cutting or averaging the edge parts of the adjacent pictures to remove repeated parts, then using an averaging algorithm to calculate the difference of pixel point values of certain rows or columns of the edges of the adjacent pictures, generating a compensation matrix to compensate the image value unevenness of each small picture caused by the light spot unevenness, and finally obtaining tissue sample pictures of a plurality of different channels of the complete visual field;
s4, synthesizing different channel substances obtained in the S3 into a pseudo-color image; the method specifically comprises the steps of linearly combining images of different channels, respectively mapping the images into images of different colors by using lookup tables of different colors, and then synthesizing the images into a tissue-like pathological image with various chemical components by using an overlapping method so as to provide the tissue-like pathological image for subsequent pathological diagnosis.
In step S1, if there is more than one substance to be detected and the raman peak position wave number distance of different substances falls within the spectral range of the used pulse laser, the middle value of the raman peak wave number is selected as the required raman wave number in a compromise manner, and the wavelength of the pump light is selected accordingly, so that all substances to be detected can measure the raman peak without changing the wavelength of the pump light. The spectral range covered by the pulsed laser is calculated from the fourier transform in combination with the duration of the laser limit pulses.
The invention verifies the nondestructive rapid imaging capability of stimulated Raman on biomacromolecule components in stomach tissues through the example analysis of three substances of lipid, protein and collagen, performs complete scanning of x-y plane full view field on the whole fresh tissues, completes two-dimensional scanning of three components in combination with multiple modes, obtains the relative distribution state of lipid, protein and collagen in the fresh tissues, compares the relative distribution state with the traditional histopathology image, verifies the consistency of the two-dimensional scanning, and provides verification for the practicability of the invention.
The stimulated Raman scattering microscopic imaging technology based on the invention is a novel label-free nondestructive imaging means, is a nonlinear coherent Raman scattering process, not only has the advantage of specific resolution of spontaneous Raman spectrum to molecular chemical bonds, but also improves weak Raman signals by 4-8 orders of magnitude under the action of stimulated radiation of Stokes light, so that the Raman signals have high signal-to-noise ratio real-time imaging capability. Meanwhile, the stimulated Raman scattering belongs to a nonlinear optical process, and can be generated only when the light intensity density at the optical focus is large enough, so that the stimulated Raman scattering has the super-resolution capability of a multi-dimensional space.
The invention provides a rapid histopathology imaging method of stomach biopsy tissue in an endoscope, which is the first application of a stimulated Raman scattering microscopy technology in endoscopic biopsy of a gastroscope. The invention aims at that the size of the gastroscope biopsy tissue is in millimeter magnitude, and finally provides an image similar to the traditional histopathology characteristics by virtue of the advantages of rapid imaging of stimulated Raman scattering microscopic imaging counting, scanning of an x-y plane translation table and the like and combining with an image splicing algorithm on the basis of fully utilizing the distribution characteristics of biological macromolecules in the tissue to form a basic tissue structure.
Compared with the traditional method, the invention has a great breakthrough, which is specifically embodied in that: after the stomach tissue is collected from gastroscopic biopsy or incisal margin operation and soaked in formalin, the stomach tissue can be placed on a glass slide without any pretreatment work (including paraffin embedding or section staining after freezing treatment), and a histopathology image of specific tissue components of the stomach tissue can be given under the condition of ensuring that the tissue is not invasive.
The invention has the advantages of high imaging speed, no need of preprocessing, no wound retention of original tissues, capability of imaging each plane in a certain depth, simple and convenient operation process, about 1.1s of scanning time of a single-frame image and high imaging quality.
The invention provides an example for the expansion of the stimulated Raman scattering microscopic imaging technology in the application field.
Drawings
FIG. 1 is a stimulated Raman scattering microscopy system employed in an embodiment of the present invention.
Fig. 2 is a diagram for selecting and dividing the x-y axis scanning area of the sample in fig. 1.
Fig. 3 is a flowchart illustrating the use of the image stitching and pseudo-color mapping software according to the embodiment of the present invention.
Fig. 4 is a single-frame image of the stimulated raman scattering imaging system corresponding to three substances to be measured.
Fig. 5 is a stimulated raman single-channel image/multi-channel mosaic image of the complete region corresponding to three substances to be tested, and an image corresponding to conventional histopathology HE staining.
Fig. 5 is a stimulated raman single-channel image/multi-channel mosaic image of the complete region corresponding to three substances to be tested, and a corresponding image of conventional histopathology HE staining.
Reference numbers in the figures: the device comprises a femtosecond laser 1, a pump light output port 1-1, a stokes light output port 1-2, a first light power adjusting component combination 2-1, a second light power adjusting component combination 2-2, SF57 dispersion glass 3-1, a second SF57 dispersion glass 3-2, an electro-optical modulator 4, a precision displacement table 5, a dichroic mirror 6, a two-dimensional scanning galvanometer 7, a dichroic mirror 8, an objective lens 9, a sample translation table 10, a condenser lens 11, a short-pass filter 12, a photoelectric detector 13, a phase-locked amplifier 14, a photomultiplier 15 and a computer 16.
Detailed Description
The invention is further illustrated with reference to the following specific embodiments and the accompanying drawings.
Example 1
A stimulated raman scattering microscopy imaging system was set up as shown in figure 1. In the system, a laser 1 generates femtosecond pulse laser, a pump light output port 1-1 can be femtosecond laser with tunable wavelength of 680nm-1300nm as pump light, and a Stokes light output port 1-2 at the other end outputs femtosecond laser with fixed wavelength of 1040nm as Stokes light. After the pump light and the Stokes light respectively pass through the half-wave plate and the polarization beam splitter prism combination 2-1 and 2-2 to adjust power, a linear chirp process is completed through SF57 dispersion glass 3-1 and 3-2, so that the femtosecond light is stretched into picosecond light, and in the chirp process, a spectrum is arranged according to time and space to provide a stimulated Raman scattering system with the full width at half maximum of 15cm -1 The spectral resolution of (a). Then, the Stokes light is digitally modulated by an electro-optical modulator 4 according to a certain frequency by 0 and 1 of light pulses, and the optical path of the Stokes light is changed through a precise displacement table 5 to adjust the Stokes lightThe relative time delay between the Turks light and the pump light, then, after the Stokes light and the pump light are combined with the dichroscope 6, under the action of the two-dimensional scanning galvanometer 7, after the Stokes light and the pump light are reflected to the objective lens 9 through the dichroscope 8, the focus of the objective lens 9 focused on the tissue sample on the translation stage 10 is changed in the vibrating process of the galvanometer, a certain focal plane can be repeatedly scanned along with the vibration of the galvanometer, after the light transmits through the sample, the transmitted light is focused through the focusing lens 11, after the Stokes light is filtered through the optical filter 12, the pump light after the stimulated Raman scattering effect is detected by the photoelectric detector 13, and then the stimulated Raman loss signal is obtained through demodulation of the lock-in amplifier 14. In the reflected light returning along the original path at the translation stage 10, the second harmonic with shorter wavelength is transmitted by the dichroic mirror 8, then is collected by the photomultiplier 15 and converted into an electric signal, and is transmitted to the computer 16 to be displayed in combination with the raman signal demodulated by the phase-locked amplifier 14, after one frame of image of one channel is scanned, the position of the precision displacement stage 5 is changed, and the process is repeated to scan the image of the other channel.
The stomach tissue generally contains mainly lipid, protein, collagen, etc., and in this embodiment, lipid, protein, collagen are used as the substance to be detected, and the raman peak of lipid is located at the raman shift of 2845cm -1 The Raman peak of the protein is located at a Raman shift of 2930cm -1 Here, collagen is collected by means of second harmonic generation, thus taking the wavenumber median 2887cm -1 For central raman shift, the wavelength of the pump light was calculated to be 801nm when the wavelength of the stokes light was fixed to 1040nm, and thus the collagen signal wavelengths generated by the second harmonic were 520nm and 400.5nm. After scanning, images of each frame of the three substances to be detected are obtained (as shown in fig. 4).
In the embodiment, the experimental parameters of the stimulated Raman scattering microscopic imaging system are calibrated by using the components in the stomach biopsy tissue, the imaging feasibility of the stimulated Raman scattering microscopic technology on the stomach biopsy is verified, and a foundation is laid for scanning a complete pathological image in the embodiment 2.
Example 2
In this example, lipids, proteins and collagen were selected as the analytes to be detected. In conjunction with example 1, the method for rapid histopathological imaging of gastric biopsy comprises the following steps:
s1, obtaining experimental parameters of respective Raman peaks in a stimulated Raman scattering microscope system from imaging practical conditions of lipid and protein, wherein the parameters comprise: the fixed Stokes wavelength is 1040nm, the pump wavelength is 801nm, and the corresponding wave number for the time delay is 2845cm -1 And 2930cm -1 Obtaining a specific time delay position according to actual experimental conditions;
s2, firstly setting two paths of output of the laser, and setting the wave number corresponding to the relative time delay of the Stokes light and the pump light to 2845cm -1 After a focal plane is selected, moving the sample translation stage 10 to search for an area to be imaged, and recording the position of each boundary;
s3, opening a second harmonic channel, and setting a trigger signal to automatically switch lipid and protein channels; and (3) running a sample translation stage automatic moving program, as shown in fig. 2, after the collagen scanning of lipid, protein channels and multi-mode simultaneous imaging in each area visual field is completed, automatically moving the translation stage to the next area for scanning imaging, and repeating the process until all preset scanning areas are scanned.
And S4, importing the picture sequence acquired in the S3 into picture splicing software, and finishing the generation of the stimulated Raman image by using a software use process shown in the figure 3, so as to generate the stimulated Raman histopathology image similar to the traditional histopathology image.
This embodiment is based on CH 2 ，CH 3 The distribution state of the lipid, protein and collagen with the property of generating second harmonic in stomach tissue corresponding to chemical bonds and the tissue structure presented by the traditional histopathology HE staining obtain a histopathology image mainly based on a stimulated Raman scattering image, and verify the consistency of the histopathology image and the traditional histopathology image.
According to the above described embodiments, the method of stimulated raman scattering based histopathological imaging of an endoscopic biopsy can be summarized in two process steps: 1. scanning the stomach tissue at the focal plane along the x-y axis to obtain a two-dimensional image having a series of selected regions; 2. and (5) sequentially guiding the picture sequences into picture splicing and pseudo-color mapping software to obtain the stimulated Raman histopathology image.
Fig. 5 shows a stimulated raman single-channel image/multi-channel mosaic image of the complete region corresponding to three substances to be detected, and an image corresponding to conventional histopathology HE staining. Wherein:
and (3) Lipid: at 2845cm -1 A stimulated raman image acquired of the stomach tissue at the raman shift.
Protein: at 2930cm -1 A stimulated raman image acquired of the stomach tissue at the raman shift.
Collagen: images of collagen fibers generated by the second harmonic of stomach tissue collected using a photomultiplier tube.
SRS: and performing pseudo-color mapping synthesis on the three images by using three channels of RGB.
SRH: and (3) an image obtained by mapping and synthesizing the three images by using similar pathological pseudo colors.
HE: standard histopathology images of H & E staining were used.
In the embodiment, a certain gastroscope biopsy tissue is selected as a sample, and the experimental thought and characteristics of the invention are specifically explained. The protective scope of the invention is not limited to the embodiments described above. Therefore, it is within the scope of the present invention to apply the stimulated raman scattering endoscopic biopsy technique according to the present invention.
Claims (2)
1. A histopathology imaging method of an intragastric endoscopic biopsy based on stimulated Raman scattering is characterized by comprising the following specific steps:
s1, selecting proper biomolecules as a substance to be detected according to characteristics concerned in histopathology aiming at biochemical components in stomach tissues, and detecting a standard sample corresponding to the substance to be detected by using a stimulated Raman scattering microscopic imaging system to obtain the optimal state of specific parameters in the stimulated Raman imaging system; the parameters include: pump and stokes wavelengths, relative time delays between pump and stokes;
s2, setting experiment parameters according to the result of the S1, carrying out region selection on a substance to be detected in the stomach biopsy tissue, and then carrying out rapid microscopic imaging on each region: after scanning of each frame of image is finished, moving the sample stage according to the designed position of the original region, then scanning the next frame of image, repeating the steps until the whole selected region is completely scanned to obtain a blocked image of the field of view of the whole region, and finally splicing the blocked image into a large-size image through a splicing algorithm;
when multiple substances to be detected such as lipid, protein and collagen need to be imaged, after each frame of image is scanned, the experimental parameters are automatically switched to be adjusted to scan another substance channel to be detected, and then the sample platform is translated; meanwhile, a multi-modal mode is used for imaging the substance;
s3, compiling a splicing algorithm for the small images scanned in the plurality of areas, and splicing the small images into a large image with a complete view field; reading a picture sequence into an algorithm program, firstly arranging small pictures to the expected position of a complete large picture according to the sequence number, splicing adjacent pictures, cutting or averagely processing the edge part of the adjacent pictures to remove repeated parts, then using an averaging algorithm to calculate the difference of pixel point values of certain rows or columns of the edge of the adjacent pictures, generating a compensation matrix to compensate the image value unevenness of each small picture caused by the uneven light spots, and finally obtaining a plurality of tissue sample pictures of different channels of a complete view field;
s4, synthesizing different channel substances obtained in the S3 into a pseudo-color image; the method specifically comprises the steps of linearly combining images of different channels, respectively mapping the images into images of different colors by using lookup tables of different colors, and then synthesizing the images into a tissue-like pathological image with various chemical components by using an overlapping method so as to provide the tissue-like pathological image for subsequent pathological diagnosis.
2. The method for histopathological imaging of endoscopic biopsy according to claim 1, wherein in step S1, when there is more than one substance to be detected and the raman peak wave number distance of different substances is within the spectrum range of the used pulse laser, the middle value of the raman peak wave number is selected as the required raman wave number in the compromise, and the wavelength of the pump light is selected accordingly, so that all substances to be detected can measure the raman peak without changing the wavelength of the pump light; the spectral range covered by a pulsed laser is calculated from the fourier transform in combination with the duration of the laser limit pulses.
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