CN113433108B - Stomach peeping biopsy histopathology imaging method based on stimulated Raman scattering - Google Patents

Abstract

The invention belongs to the technical field of nonlinear optical imaging, in particular to a gastric endoscopic biopsy histopathological imaging method based on stimulated Raman scattering. The method of the invention utilizes the fast, processing-free and label-free properties of stimulated Raman scattering microscopic imaging, and can acquire the histopathological image information of gastric biopsy in a short time. The present invention is the first clinical application of stimulated Raman scattering microscopy in gastroscopic endoscopic biopsy. Compared with existing traditional histopathological techniques, its advantages are reflected in: fast imaging speed, high imaging quality, and no need for pretreatment , non-invasively retain the original tissue, and can image each plane within a certain depth.

Description

Histopathological Imaging Method of Gastric Endoscopic Biopsy Based on Stimulated Raman Scattering

technical field

The invention belongs to the technical field of nonlinear optical imaging, in particular to a gastric endoscopic biopsy histopathological imaging method based on stimulated Raman scattering.

Background technique

Gastric cancer is a major disease affecting human health. According to the latest annual report of the National Cancer Registry Center, the number of new cases of gastric cancer exceeds 680,000 each year, and about 500,000 people die, making it the second deadliest tumor in my country. The key to the prevention and control of gastric cancer is early diagnosis and early treatment. Timely detection of early cancer, delay and reversal of precancerous lesions can effectively reduce the incidence and mortality of gastric cancer. The occurrence of gastric cancer goes through multiple steps such as chronic inflammation-precancerous lesions-early cancer-invasive cancer. The development of gastric cancer usually requires decades of development and accumulation of events. Even if an early cancer develops, it usually takes about 44 months to progress to an advanced stage. Therefore, taking gastric precancerous lesions and early cancers as the key nodes in the prevention and control of gastric cancer, and giving necessary intervention and treatment in time will effectively reduce the incidence and mortality of gastric cancer and reduce the harm of gastric cancer. However, due to the lack of obvious morphological characteristics of early gastric cancer and precancerous lesions, endoscopic findings alone cannot be clearly defined, and histopathological diagnosis must be relied on. The latter needs to go through multiple steps such as dehydration, embedding, slicing, staining, and film reading, which has an obvious time lag. Therefore, it is very important to quickly obtain histopathological images during the operation without time and labor intensive.

Raman spectroscopy technology can quickly, accurately and non-invasively detect molecular information of lesions. It has the advantages of comparing pathological results in effect and surpassing traditional pathology in detection time. Raman spectroscopy is a non-destructive analysis technique based on the principle of inelastic scattering effect of incident light and substances, which can describe the molecules of samples to be tested. Characteristic vibrations of chemical bonds/groups under incident light. Therefore, the Raman spectrum contains the fingerprint information of the molecule. Its important advantage is that it can directly identify the type of substance based on the Raman fingerprint characteristics of sample molecules, especially for fingerprint identification of disease molecules in the field of medical diseases. Compared with other detection methods, such as fluorescence detection, Raman detection not only has the obvious advantages of molecular fingerprint identification. Compared with other detection methods, such as fluorescence detection, Raman detection not only has the characteristics of molecular fingerprint recognition, but also has the characteristics of no need for sample treatment, water insensitivity (especially useful for medical biological detection containing water, which cannot be achieved by infrared spectroscopy) and micro-area detection. It has advantages such as trace detection, which is unmatched by other detection technologies at present. The second advantage of Raman spectroscopy is "fast". Raman spectrum collection can be completed within a few seconds, and the cost of detection is relatively low. In actual detection, only the sample needs to be placed at the Raman checkpoint, and a quick judgment can be made after the detection is completed.

However, traditional spontaneous Raman spectroscopy cannot achieve spatial imaging of samples, and biological samples often have strong spatial heterogeneity, for example, the distribution of lesions is not uniform, and the severity is different. Stimulated Raman scattering combines Raman scattering and Einstein's stimulated radiation principle, which can achieve a gain of 103~105 of the Raman effect and achieve high-speed imaging at video speed. Stimulated Raman spectroscopy inherits the molecular fingerprint characteristics of spontaneous Raman spectroscopy, has good molecular specificity, and can represent different molecules in a form similar to pathological images according to their characteristic vibration spectra. Therefore, the main advantage is that it can realize spatial imaging of the sample, and if it can locate and screen suspicious clinical areas to be tested, combined with rapid imaging technology to accurately obtain Raman spectra at different positions of the sample, it will be more conducive to improving the accuracy of diagnosis and targeting.

Contents of the invention

The purpose of the present invention is to provide a gastric endoscopic biopsy histopathological imaging method with fast imaging speed and high imaging quality, so as to solve the problems in the prior art such as complicated sample pretreatment, labor-intensive processing, and obvious time lag. .

The histopathological imaging method of gastric endoscopic biopsy provided by the present invention is based on stimulated Raman scattering technology, and the specific steps are:

S1. For the biochemical components existing in gastric tissue, according to the characteristics of concern in histopathology, select appropriate biomolecules as the substance to be detected, and use the stimulated Raman scattering microscopic imaging system to detect the corresponding standard of the substance to be tested sample to obtain the optimal state of specific parameters in the stimulated Raman imaging system; the parameters include: pump light wavelength and Stokes light wavelength, the relative ratio between pump light and Stokes light delay;

S2. Set the experimental parameters according to the results of S1, select the area of the substance to be detected in the gastric biopsy tissue, and then perform rapid microscopic imaging for each area: specifically, after the scanning of each frame of image, according to the original area The designed position moves the sample stage, and then scans the next frame of image, and so on, until the entire selected area is scanned completely, and the block image of the entire field of view is obtained, and finally it is stitched into a large one by stitching algorithm size image;

When it is necessary to image multiple substances to be detected such as lipids, proteins, and collagen, after scanning each frame of image, the experimental parameters are automatically switched to adjust to scan another channel of the substance to be detected, and then proceed The sample stage is translated; at the same time, the multi-modal method is used to image other substances at the same time;

S3. Write a stitching algorithm for the small images scanned in multiple areas, and stitch them into a large image with a complete field of view; specifically, read the sequence of images into the algorithm program, and first arrange the small images into a complete large image according to the serial number The estimated position of the picture, and then splicing adjacent pictures, cropping or averaging the edge part of the adjacent picture to remove the repeated part, and then using the averaging algorithm to calculate the number of rows or columns of pixels on the edge of the adjacent picture value difference, regenerate the compensation matrix to compensate for the unevenness of the image value caused by the unevenness of the light spot in each small image, and finally obtain several tissue sample images of different channels in the complete field of view;

S4. Synthesize the pseudo-color images of different channel substances obtained in S3; specifically, the images of different channels are linearly combined, and they are mapped to images of different colors using look-up tables of different colors, and then superimposed using the superposition method. Synthesize a histopathological-like image with various chemical components to provide for subsequent pathological diagnosis.

In step S1, if there are more than one substance to be detected, and the Raman peak wavenumber distance of different substances covers the spectral range of the pulsed laser used, then the middle value of the Raman peak wavenumber is selected as the compromise. The required Raman wavenumber is used to select the wavelength of the pump light, so that all substances to be tested 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 combined with the duration of the laser's limit pulse.

The present invention verifies the ability of stimulated Raman to rapidly and non-destructively image biomacromolecular components in gastric tissue through the example analysis of lipid, protein and collagen, and conducts x-y full field of view imaging of the whole fresh tissue Complete scanning, combined with multi-modality to complete the two-dimensional scanning of the three components, obtained the relative distribution of lipids, proteins, and collagen in fresh tissues, and compared them with traditional histopathological images to verify their consistency. Verification is provided for the practicability of the present invention.

The stimulated Raman scattering microscopic imaging technology based on the present invention is a new type of label-free nondestructive imaging means, and a nonlinear coherent Raman scattering process, which not only has the specific resolution of molecular chemical bonds by spontaneous Raman spectroscopy Moreover, under the stimulated radiation of Stokes light, the weak Raman signal is increased by 4-8 orders of magnitude, making it capable of real-time imaging with high signal-to-noise ratio. At the same time, stimulated Raman scattering is a nonlinear optical process, which can only be produced when the light intensity density at the light focus is sufficiently high, and has super-resolution capabilities in multi-dimensional space.

The rapid histopathological imaging method of gastric biopsy tissue in endoscope proposed by the present invention is the first application of stimulated Raman scattering microscopic technique in gastroscopic endoscopic biopsy. The size of gastroscopic biopsy tissue targeted by the present invention is on the order of millimeters. On the basis of making full use of the distribution characteristics of biological macromolecules in the tissue and forming the basic tissue structure, the rapid imaging and x-y plane translation by means of stimulated Raman scattering microscopic imaging counting Advantages such as table scanning, combined with image stitching algorithms, finally give images similar to traditional histopathological features.

Compared with the traditional method, the present invention has a major breakthrough, which is embodied in that after the gastric tissue is collected from gastroscopic biopsy or surgical margin surgery and soaked in formalin, no pretreatment (including paraffin embedding or freezing) is required. Section staining after processing), it can be placed on a glass slide, and the histopathological image of its specific tissue components can be given under the condition of ensuring non-invasive tissue.

The invention has fast imaging speed, no need for pretreatment, non-invasively retains the original tissue, and can image each plane within a certain depth, the operation process is simple, the scanning time of a single frame image is about 1.1s, and the imaging quality is high.

The invention provides an example for expanding the application field of the stimulated Raman scattering microscopic imaging technology.

Description of drawings

Fig. 1 is a stimulated Raman scattering microscope system used in the embodiment of the present invention.

Fig. 2 is a diagram showing the selection and division of the x-y axis scanning area of the sample in Fig. 1 .

Fig. 3 is a flow chart of using picture stitching and pseudo-color mapping software in an embodiment of the present invention.

Fig. 4 is a single-frame image formed by the stimulated Raman scattering imaging system corresponding to the three substances to be tested.

Figure 5 is the complete area stimulated Raman single-channel image/multi-channel mosaic image corresponding to the three substances to be tested, and the corresponding image of traditional histopathological HE staining.

Figure 5 is the complete area stimulated Raman single-channel image/multi-channel mosaic image corresponding to the three substances to be tested, and the corresponding image of traditional histopathological HE staining.

Numbers in the figure: 1 is the femtosecond laser, 1-1 is the pump light output port, 1-2 is the Stokes light output port, 2-1 is the first combination of components for adjusting optical power, 2-2 is the The second component combination for adjusting optical power, 3-1 is SF57 dispersion glass, 3-2 is the second SF57 dispersion glass, 4 is electro-optic modulator, 5 is precision translation stage, 6 is dichroic mirror, 7 is Two-dimensional scanning galvanometer, 8 is a dichroic mirror, 9 is an objective lens, 10 is a sample translation stage, 11 is a condenser, 12 is a short-pass filter, 13 is a photodetector, 14 is a lock-in amplifier, and 15 is a photoelectric Doubling the security, 16 is the computer.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

Example 1

A stimulated Raman scattering microscopy imaging system was built, as shown in Figure 1. In the system, the laser 1 generates femtosecond pulsed laser, and the pump light output port 1-1 can be femtosecond laser with a tunable wavelength of 680nm-1300nm as the pump light, and the Stokes light output port 1-2 at the other end outputs Femtosecond laser with a fixed wavelength of 1040nm, as Stokes light. After the pump light and Stokes light are adjusted through the combination 2-1 and 2-2 of the half-wave plate and the polarization beam splitter prism, the linear chirp process is completed through the SF57 dispersion glass 3-1 and 3-2, so that the flying The second light is stretched to picosecond light, and the spectrum is arranged in time and space during the chirping process, providing the stimulated Raman scattering system with a spectral resolution of 15cm ^-1 full width at half maximum. Then, the Stokes light is digitally modulated by the electro-optic modulator 4 according to a certain frequency to achieve 0, 1 digital modulation of the light pulse, and the optical path of the Stokes light is changed by the precision displacement stage 5 to adjust the Stokes light and The relative time delay between the pump light, and then, after the Stokes light and the pump light combine with the dichroic mirror 6, they are reflected by the dichroic mirror 8 under the action of the two-dimensional scanning galvanometer 7 After reaching the objective lens 9, the vibrating process of the vibrating mirror will change the focus of the objective lens 9 on the tissue sample on the translation stage 10. With the vibration of the vibrating mirror, a certain focal plane will be scanned repeatedly. After the light is transmitted through the sample, it will pass through the focusing lens 11 The transmitted light is focused, and after the Stokes light is filtered out by the filter 12, the pump light after stimulated Raman scattering is detected by the photodetector 13, and then demodulated by the lock-in amplifier 14 to obtain the stimulated Raman loss signal. In the reflected light returning along the original path at the translation stage 10, the second harmonic wave with a shorter wavelength will be collected by the photomultiplier tube 15 after being transmitted through the dichroic mirror 8 and converted into an electrical signal, combined with a lock-in amplifier 14. The demodulated Raman signal is transmitted to the computer 16 for display. After one frame of image of one channel is scanned, the position of precision translation stage 5 is changed, and the above process is repeated to scan the image of another channel.

Stomach tissue generally mainly contains components such as lipid, protein, and collagen. In this embodiment, lipid, protein, and collagen are used as substances to be detected. The Raman peak of lipid is located at a Raman shift of ²⁸⁴⁵ cm The Raman peak is located at the Raman shift of 2930cm ^-1 , and the collagen is collected by the second harmonic generation, so the central Raman shift is taken as the median wave number of 2887cm ^-1 . When the wavelength of the Stokes light is fixed at 1040nm, The calculated wavelength of the pump light is 801nm, so the collagen signal wavelengths generated by the second harmonic are 520nm and 400.5nm. After scanning, images of each frame of the three substances to be tested are obtained (as shown in Figure 4).

In this example, the experimental parameters of the stimulated Raman scattering microscopy imaging system were calibrated using the components in the gastric biopsy tissue, and the feasibility of the imaging of gastric biopsy by stimulated Raman scattering microscopy was verified, which is the example in Example 2. Scan the complete pathology image to lay the foundation.

Example 2

In this embodiment, lipid, protein and collagen are selected as the substances to be detected. With reference to Example 1, the rapid histopathological imaging method of gastric biopsy tissue includes the following steps:

S1. Obtain the experimental parameters of the respective Raman peaks in the stimulated Raman scattering microscope system from the actual situation of imaging of lipids and proteins. The parameters include: the fixed Stokes wavelength is 1040nm, and the pump wavelength is Choose 801nm, and the wavenumber corresponding to the corresponding time delay is selected as 2845cm ^-1 and 2930cm ^-1 , and the specific time delay position is obtained according to the actual experimental conditions;

S2. First set up the two outputs of the laser, and then set the corresponding wavenumber of the relative time delay between the Stokes light and the pump light to 2845cm ^-1 , after selecting the focal plane, move the sample translation stage 10 to find the desired imaging point area, and record the location of each boundary;

S3, open the second harmonic channel, and set the trigger signal to automatically switch the lipid and protein channels; run the automatic movement program of the sample translation stage, as shown in Figure 2, in each area of view, the lipid, protein channels and After the collagen scanning of multimodal simultaneous imaging is completed, the translation stage automatically moves to the next area for scanning and imaging, and this process is repeated until all the pre-set scanning areas are scanned.

S4. Import the image sequence obtained in S3 into the image stitching software, and complete the generation of stimulated Raman images by using the software as shown in FIG. 3 , thereby generating stimulated Raman histopathological images similar to traditional histopathological images.

In this example, based on the distribution state of lipids, proteins and collagen with the property of generating second harmonics in gastric tissue corresponding to CH ₂ and CH ₃ chemical bonds, as well as the tissue structure presented by traditional histopathological HE staining, obtained Stimulated Raman scattering images are the main histopathological images, and their consistency with traditional histopathological images has been verified.

According to the above-mentioned embodiments, the gastric endoscopic biopsy histopathological imaging method based on stimulated Raman scattering can be summarized as two process steps: 1. Scanning the gastric tissue in the focal plane along the x-y axis, acquiring 2. Import the image sequence into the image stitching and pseudo-color mapping software in sequence to obtain stimulated Raman histopathological images.

Figure 5 shows the complete area stimulated Raman single-channel image/multi-channel mosaic image corresponding to the three substances to be tested, and the corresponding image of traditional histopathological HE staining. in:

Lipid: Stimulated Raman images of gastric tissue acquired at a Raman shift of 2845 cm ^-1 .

Protein: Stimulated Raman images of gastric tissue acquired at a Raman shift of 2930cm ^-1 .

Collagen: Collagen fiber image generated by the second harmonic wave of gastric tissue collected by photomultiplier tube.

SRS: An image synthesized by performing pseudo-color mapping on the above three images using the three channels of RGB.

SRH: An image synthesized by mapping the above three images separately using pathologically similar pseudo-colors.

HE: Standard histopathological images stained using H&E.

The embodiment selects a gastroscopic biopsy tissue as a sample, and specifically illustrates the experimental idea and characteristics of the present invention. The protection scope of the present invention is not limited to the above-mentioned embodiments. Therefore, any application of the stimulated Raman scattering endoscopic biopsy technique according to the present invention falls within the scope of protection of 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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