Axial automatic positioning method and system for single-cell spontaneous-stimulated Raman spectrum detection

Abstract

The invention relates to the technical field of cell detection, in particular to a single-cell spontaneous-stimulated Raman spectrum detection axial automatic positioning method and system. Comprising the steps of building a positioning system; after the cells are roughly focused and positioned through white light, a three-dimensional space tomography stack of the cells is obtained through stimulated Raman spectrum imaging; the axial displacement distance of the precise displacement platform is determined through an equal spacing method or a PID control algorithm, and the axial displacement distance H of the precise displacement platform is adjusted; setting the position of the precise displacement platform as H + L after correction; switching an electric diaphragm, setting spontaneous Raman spectrum acquisition parameters, and performing spontaneous Raman spectrum acquisition of the cells to complete fine Raman spectrum acquisition of the cells; a known cell Raman spectrum is collected, and a cell type discrimination model is established; and discriminant analysis is carried out on an unknown type of cell sample to realize accurate positioning. The device and the method have the advantages that spatial distribution of molecules such as lipid in cells is obtained in a label-free and non-staining manner, and accurate sampling of the cell Raman spectrum is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cell detection, and particularly to a method and system for axial automatic positioning of single-cell spontaneous-stimulated Raman spectroscopy detection. Background Art

[0002] Cells are the basic units that make up life and are mainly composed of molecules such as proteins, lipids, and nucleic acids. There are differences in molecular composition and content among different cells and different developmental stages of the same cell. For example, there are usually metabolic differences during the process of cell carcinogenesis. By detecting the molecular components inside cells, the identification of different types of cells can be achieved. Raman spectroscopy is a molecular scattering spectrum that can reflect the intrinsic vibration information of molecular covalent bonds. Thus, through cell Raman spectroscopy, the molecular types and content information of cells can be obtained. The single-cell Raman spectroscopy technology has the technical advantages of label-free, non-destructive, and in-situ detection, and has important application values in the fields of life sciences such as cell differentiation and carcinogenesis research and precision medicine. However, cells have complex structures and compositional differences, and there are significant molecular differences both horizontally and axially inside the cells. This kind of difference will make the detection signals of cells have significant uncertainties, resulting in a serious decline in subsequent analysis accuracy and repeatability. Therefore, currently, to achieve precise analysis of single cells based on Raman spectroscopy detection, it is necessary to achieve precise positioning of the spatial position of cell spectrum acquisition.

[0003] Currently, for cell Raman spectroscopy detection, spontaneous Raman is usually combined with a white light microscope. The analysis sample is placed under the microscope, and the Raman excitation spot is focused on the cell by the microscope. After exciting the cell Raman signal, it is collected by the objective lens, and the scattered signal is transmitted to the spectrometer to obtain the cell Raman spectrum for subsequent analysis. To further obtain the spatial image information of cells, conventional Raman spectroscopy technology is integrated with emerging imaging technologies for detection. Commonly used technologies include fluorescence spectroscopy analysis technology, optical coherence tomography technology, and stimulated Raman technology. Fluorescent labeling and staining methods are currently mature technical means for providing cell image information. The methods of fluorescent labeling and staining modify cells with exogenous markers such as dyes or fluorescent groups to increase the contrast of intracellular molecules and obtain the spatial position information of cells. Optical coherence tomography (OCT) is an emerging optical imaging technology that obtains information such as the refractive index of biological tissues through the optical coherence analysis of photons returned by the medium and the reference light, and can achieve non-contact, three-dimensional tomographic scanning of living tissues. Stimulated Raman is based on two pulsed lasers with different wavelengths. Through their third-order nonlinear effects with matter molecules, the Raman scattering signal is amplified, and by combining the cell structure and molecular spectral information, the internal image information of cells is obtained.

[0004] The spot size of the Raman excitation light focused by the microscope objective is usually smaller than the cell size. The traditional microscope obtains a planar structure image of a certain layer in the cell. The cell sample is placed at the position of the focused spot through a galvanometer or a translation stage for horizontal (lateral) positioning. In the horizontal direction, the cell contour has a relatively clear boundary with the external environment, which can assist in cell positioning. However, in the direction of the microscope optical axis, the microscope focal length is manually adjusted by the operator to image different layers of the cell and perform axial (longitudinal) positioning. The inside of the cell is an almost transparent sample, and the cell Raman detection cannot stain the sample. The internal structure of the cell has low contrast, and the difference between layers can hardly be distinguished. Therefore, an ordinary white light microscope cannot effectively distinguish the morphological differences of cells in the axial direction, that is, on different layers, and has low axial resolution. It can only rely on the experience of the experimenter to judge, resulting in low axial positioning accuracy of the cell Raman excitation spot, and the consistency of the excitation spectrum cannot be guaranteed, introducing sampling errors in subsequent analysis and leading to a decrease in the overall technical analysis accuracy. Therefore, other imaging techniques are needed to provide cell axial position information.

[0005] The dyes or fluorescent groups and other exogenous markers used in the fluorescence labeling and staining methods will seriously interfere with the life activities of cells, affect the metabolic activities of small molecules inside the cells, and even make it impossible to observe live cells. Combining fluorescence imaging with Raman technology will destroy the technical advantage of Raman spectroscopy label-free detection. Optical coherence tomography technology is a label-free detection technology, but the specificity of the technology comes from the refractive index differences of different media and cannot reflect the intrinsic properties of molecules. Its imaging resolution, especially the axial resolution, is on the order of 10 microns, and the single cell size is generally also about 10 microns. Therefore, it is difficult to meet the needs of fine analysis of the internal structure of single cells.

[0006] Stimulated Raman enhances the Raman scattering efficiency based on the optical nonlinear effect, can achieve Raman spectroscopic imaging at video rate, and has sub-micron imaging resolution. Its spectral signal is consistent with the spontaneous Raman spectrum. However, limited by the spectral characteristics of femtosecond lasers, its spectral resolution is low and cannot meet the requirements of cell fingerprint spectrum analysis. It is commonly used for high wavenumber band spectral imaging with lower cell spectral resolution requirements. Our research group previously proposed a method of obtaining cell images based on stimulated Raman spectroscopic imaging for lateral positioning detection of cell substructures to improve the accuracy of analysis. However, this method still needs to rely on the experimenter to manually adjust the microscope focal length and judge the axial position of the detection based on the experimenter's experience. Moreover, the axial focusing position of stimulated Raman is different from that of the spontaneous Raman mode, and the experimenter still needs to judge the axial focusing position of spontaneous Raman based on experience, resulting in a decrease in axial detection accuracy, uncertainty, and repeatability of the analysis results. Summary of the Invention

[0007] In order to solve the above problems, the present invention provides a method and system for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection.

[0008] The present invention aims to provide a method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection, which specifically comprises the following steps: S1. Build an axial automatic positioning system for single-cell spontaneous-stimulated Raman spectroscopy detection, including a Raman excitation light source unit, a signal excitation and collection unit, a spatial position scanning unit, and a signal processing and system control unit; place the cell sample on the stage of the signal excitation and collection unit, turn on the white light source and camera, and roughly adjust the focus in the white light mode to make the cells appear in the field of view; S2. Open the stimulated Raman excitation light source motorized diaphragm of the Raman excitation light source unit, close the spontaneous Raman light source motorized diaphragm of the Raman excitation light source unit, and set the stimulated Raman detection band to the Raman frequency shift of lipid molecules; S3. Set the motion range and single step range of the precision displacement platform of the axial position scanning mechanism, and drive the microscope objective of the signal excitation and collection unit to perform sub-micron stepping along the axial direction by the precision displacement platform. Each time the precision displacement platform steps once, stimulated Raman spectroscopy imaging of the cell is performed to obtain a stimulated Raman image; after the movement is completed, a three-dimensional stimulated Raman tomography stack of the cell is obtained; S4. Determine the axial displacement distance of the precision displacement platform by an equal spacing method or a PID control algorithm, and adjust the axial displacement distance H of the precision displacement platform; perform stimulated Raman spectroscopy imaging on the cells, and locate the spontaneous Raman detection position as the light spot where the lipid molecules are focused in the stimulated Raman image; S5. Calculate the spontaneous Raman and stimulated Raman axial focus position correction L using the spontaneous Raman and stimulated Raman axial focus position compensation method, and set the position of the precision displacement platform to H+L; S6. Close the stimulated Raman excitation light source electric diaphragm of the Raman excitation light source unit, turn off the white light source, open the spontaneous Raman light source electric diaphragm of the Raman excitation light source unit, set the spontaneous Raman spectrum acquisition parameters, perform cell spontaneous Raman spectrum acquisition, and complete cell fine Raman spectrum acquisition; S7. Collect Raman spectra of known types of cell samples according to steps S1 to S6, establish a Raman spectrum database of different types of cells, and obtain different types of cell discrimination models; then train and optimize each cell discrimination model; S8. Collect the Raman spectrum of the unknown type of the cell sample to be tested according to steps S1 to S6, substitute it into the cell discrimination model for discrimination and obtain the discrimination result, so as to achieve accurate analysis of the cells.

[0009] Preferably, the Raman excitation light source unit includes a stimulated Raman excitation light source module and a spontaneous Raman excitation light source module. The stimulated Raman excitation light source module and the spontaneous Raman excitation light source module correspond to two spectral detection modes of the system. The stimulated Raman excitation light source module is used to emit two femtosecond pulsed laser signals with different central wavelengths. After modulation, they are transmitted to the signal excitation and collection unit to excite stimulated Raman signals. The spontaneous Raman excitation light source module is used to emit narrow-band laser with a fixed wavelength, which is transmitted to the signal excitation and collection unit to excite Raman signals of cell samples. Both the stimulated Raman excitation light source module and the spontaneous Raman excitation light source module include an electric aperture. The switching between the spontaneous Raman light source and the stimulated Raman excitation light source is realized by the opening and closing of the electric aperture. The signal excitation and collection unit is used to focus the stimulated Raman excitation light on the sample to excite energy changes, and then modulate the excitation light to obtain stimulated Raman signals. The spatial position scanning unit includes a transverse position scanning mechanism and an axial position scanning mechanism. Three-dimensional spatial tomography imaging of cells is realized through the cooperation of the transverse position scanning mechanism and the axial position scanning mechanism. The system control unit includes a computer, which is used to coordinate the collaborative work of each unit.

[0010] Preferably, the stimulated Raman excitation light source module includes a femtosecond laser, a first optical path adjustment group, a second optical path adjustment group, a beam combining mirror group, and a stimulated Raman excitation light source electric aperture. The femtosecond laser emits two femtosecond pulsed laser signals with different central wavelengths. After passing through the first optical path adjustment group and the second optical path adjustment group respectively, they are spatially combined by the beam combining mirror group, and after combination, they are transmitted to the signal excitation and collection unit to excite stimulated Raman signals. The spontaneous Raman excitation light source module includes a continuous wave laser and a spontaneous Raman light source electric aperture. The continuous wave laser is used to emit narrow-band laser with a fixed wavelength, which is transmitted to the signal excitation and collection unit to excite Raman signals of cell samples. By controlling the opening and closing of the spontaneous Raman light source electric aperture or / and the stimulated Raman excitation light source electric aperture, it is respectively realized whether the spontaneous Raman light source or the stimulated Raman excitation light source can be transmitted to the signal excitation and collection unit to excite Raman signals.

[0011] Preferably, the first optical path adjustment group sequentially includes a half-wave plate, a polarization beam splitter prism, and a dispersion medium along the optical path direction. The second optical path adjustment group sequentially includes a half-wave plate, a polarization beam splitter prism, a reflector, a delay line, a reflector, a modulator, and a dispersion medium along the optical path direction. The beam combining mirror group is used to spatially combine the femtosecond pulsed laser signals passing through the first optical path adjustment group and the second optical path adjustment group. In the first optical path adjustment group and the second optical path adjustment group: The half-wave plate is used to rotate the polarization angle of light; the polarization beam splitter prism is used to selectively transmit the light component with a fixed polarization direction and reflect the light components in other directions; the combination of the half-wave plate and the polarization beam splitter prism is used to adjust the output power of the femtosecond laser; the dispersion medium is used to adjust the chirp degree of the two femtosecond pulse laser signals. In the second optical path adjustment group, the delay line changes the characteristic wavelength of the stimulated Raman detection mode by translation.

[0012] Preferably, the modulator is sequentially connected to the power amplifier and the signal generator. The spatial light modulator is used to receive the high-frequency modulation signal output by the signal generator and amplified by the power amplifier and perform amplitude high-frequency modulation; the modulator is a spatial light modulator, an electro-optic modulator or an acousto-optic modulator.

[0013] Preferably, the signal excitation and collection unit includes a microscope objective lens, a stage, a condenser lens, a filter, a photodetector and a lock-in amplifier. After the stimulated Raman excitation light is focused on the sample on the stage by the microscope objective lens to generate the stimulated Raman scattering phenomenon, the excitation energy changes. Subsequently, the excitation light passes through the sample and is reflected by the beam splitter, and one path of the excitation light modulated by the modulator is filtered by the filter, and the other path of the excitation light is collected and detected by the photodetector, and then the signal is transmitted to the lock-in amplifier to extract the stimulated Raman signal. The plane of the lateral position scanning mechanism is perpendicular to the optical axis of the microscope objective lens, and the axis of the axial position scanning mechanism is parallel to the optical axis of the microscope objective lens; the lateral position scanning mechanism includes a two-dimensional galvanometer group and a 4-f lens group composed of two lenses, which are used to perform stimulated Raman spectroscopic scanning imaging on cells; the axial position scanning mechanism includes a precision displacement platform, and the precision displacement platform drives the microscope objective lens to perform axial movement with sub-micron accuracy along the optical axis direction.

[0014] Preferably, step S4 determines the axial displacement distance of the precision displacement platform by an equal-spacing method and adjusts the axial displacement distance H of the precision displacement platform; specifically, it includes the following sub-steps: S401. The method of equal-interval step scanning: Calculate the variance A of the pixel intensity of each point in each obtained stimulated Raman image; assuming that the pixel intensity is denoted as Ixy, the specific calculation formula for the pixel intensity variance A is as follows: ; In the formula, m represents the total number of pixels in the x dimension, n represents the total number of pixels in the y dimension, represents the average intensity of all pixels in the image, I xy represents the pixel intensity; The calculation formula is as follows: ; In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. I xy represents the pixel intensity; Set the focusing coefficient F of the stimulated Raman image, which is expressed as follows: ; S402. Select the axial displacement H of the precision displacement platform corresponding to the maximum focusing coefficient, and set the axial displacement distance of the precision displacement platform to H; S403. Perform stimulated Raman spectroscopy imaging on the cells, and position the spontaneous Raman detection position at the spot where the lipid molecules are focused in the stimulated Raman image.

[0015] Preferably, step S4 determines the axial displacement distance of the precision displacement platform through the PID control algorithm and adjusts the axial displacement distance H of the precision displacement platform; specifically, it includes the following sub-steps: S401. Solve the focusing coefficient of the current stimulated Raman image: Let F(t) represent the focusing coefficient of the current stimulated Raman image, and F(t - 1) represent the focusing coefficient of the image collected at the previous moving position when the precision displacement platform moves to the current position; before the first movement of the precision displacement platform, t = 1; set F(0) to 0, and at this time the position of the precision displacement platform is h; the calculation method of the focusing coefficient F(t) of the current stimulated Raman image is as follows: Calculate the variance A of the pixel intensities at each point in the current stimulated Raman image; assume that the pixel intensity is denoted as Ixy, then the specific calculation formula for the pixel intensity variance A is as follows: ; In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. represents the average intensity of all pixels in the image. I xy represents the pixel intensity; The calculation formula is as follows: ; In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. I xy represents the pixel intensity; Then the focusing coefficient F(t) of the current stimulated Raman image is expressed as follows: ; The current focusing coefficient difference E(t) = F(t) - F(t - 1); E(t - 1) represents the focusing coefficient difference corresponding to the previous movement of the precision displacement platform. S402. Estimate the next moving distance Z of the precision displacement platform, and the calculation formula is as follows: ; Among them, k p , k i , k d It is a constant set according to the actual situation; Set the moving position of the precision displacement platform to H=h+Z; set h=H, F(t-1)=F(t); E(t-1)=E(t); repeat the above process, and stop when E(t) or Z is less than the set threshold; at this time, H is the axial positioning position of the precision displacement platform; S403. Select the H value obtained by iterating the PID control process, and set the axial displacement distance of the precision displacement platform to H; S404. Perform stimulated Raman spectroscopy imaging on the cells, and locate the spontaneous Raman detection position as the light spot where the lipid molecules are focused in the stimulated Raman image.

[0016] Preferably, in step S402, the initial focusing coefficient difference E(0) is 0; the value of the next moving distance Z of the precision displacement platform includes positive and negative values, and the positive and negative values ​​respectively indicate that the movement direction of the precision displacement platform is upward and downward.

[0017] Preferably, the precision displacement platform is a piezoelectric displacement device; the spontaneous Raman and stimulated Raman axial focus position compensation method in step S5 specifically comprises the following steps: S501. Adjust the position of the piezoelectric displacer to maximize the spontaneous Raman signal intensity of the collected lipid molecules, and record the displacement distance H of the piezoelectric displacer at this time. 1 ; S502. Calculate the spontaneous Raman and stimulated Raman axial focus position correction L, the expression is as follows: L = H 1 -H; In the formula, H 1 is the displacement distance of the piezoelectric displacer when the spontaneous Raman signal intensity of the lipid molecules reaches the maximum, and H is the axial displacement of the piezoelectric displacer corresponding to the maximum value of the pixel intensity variance; The step S8 uses principal component analysis or linear discriminant analysis to discriminate the unknown type of the cell sample to be tested.

[0018] Compared with the prior art, the present invention can achieve the following beneficial effects: With the positioning system provided by the present invention, a label-free and non-staining method can be adopted to obtain the three-dimensional spatial distribution of lipid molecules inside cells. Using the axial position of the lipid molecular layer in each cell sample as a reference, the three-dimensional spatial position calibration of cell Raman acquisition can be achieved, and accurate Raman spectrum sampling can be performed. Compared with traditional methods, the positioning method provided by the present invention selects the lipid molecular layer inside the cell as the detection reference for the axial position of Raman spectra of different cells, reduces the sampling error of the axial position during the detection of Raman spectra of different cells, greatly improves the consistency and repeatability of the signals in each measurement during the cell Raman spectrum discrimination process, and thus greatly improves the accuracy of data-driven cell discrimination methods such as principal component analysis-linear discriminant analysis. At the same time, the method of the present invention only requires the participation of a small number of experimental personnel, and the acquisition process can be automatically carried out by a computer control system, greatly improving the acquisition efficiency. Description of the Drawings

[0019] Figure 1 is a schematic optical path diagram of a single-cell spontaneous-stimulated Raman spectroscopy detection axial automatic positioning system according to an embodiment of the present invention.

[0020] Figure 2 is the detection result of single-cell spontaneous-stimulated Raman spectroscopy detection axial automatic positioning according to an embodiment of the present invention; in the figure, A is the stimulated Raman axial tomography data, B is the axial distribution of lipid molecules in the cell, C is the stimulated Raman transverse spectral imaging of the cell after axial positioning, and D is the spontaneous Raman spectrum collected after three-dimensional spatial positioning.

[0021] Figure 3 is the result of cell Raman discrimination analysis of two cultured cell lines, DU145 and SIHA, according to an embodiment of the present invention; in the figure, A is the cell classification scatter plot obtained without axial position detection, and B is the classification scatter plot established by collecting Raman spectrum data using the method of the present invention.

[0022] Figure 4 is the PID positioning flow chart according to an embodiment of the present invention.

[0023] Reference Signs: 1: Femtosecond laser; 2: First half-wave plate; 3: First polarization beam splitter prism; 4: First dispersion medium; 5: Spectrometer; 6: Stimulated Raman excitation light source electric aperture; 7: Continuous wave laser; 8: Spontaneous Raman light source electric aperture; 9: Second dichroic mirror; 10: Third dichroic mirror; 11: Two-dimensional galvanometer group; 12: First lens; 13: Second lens; 14: First beam splitter; 15: Camera; 16: Piezoelectric displacement actuator; 17: Microscope objective; 18: Stage; 19: Condenser; 20: Second beam splitter; 21: White light source; 22: Filter; 23: Photoelectric detector; 24: Computer; 25: Phase-locked amplifier; 26: Signal generator; 27: Power amplifier; 28: Delay line; 29: First mirror; 30: Second polarization beam splitter prism; 31: Second half-wave plate; 32: Second mirror; 33: Spatial light modulator; 34: Second dispersion medium; 35: Third mirror; 36: First dichroic mirror. Detailed implementation manner

[0024] In the following, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are denoted by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, their detailed descriptions will not be repeated.

[0025] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation to the present invention.

[0026] See Figure 1 , the present invention provides a single-cell spontaneous-stimulated Raman spectroscopy detection axial automatic positioning system, including: a Raman excitation light source unit, a spatial position scanning unit, a signal excitation and collection unit, and a signal processing and system control unit; The Raman excitation light source unit includes a stimulated Raman excitation light source module and a spontaneous Raman excitation light source module, corresponding to two spectral detection modes of the system; during operation, first, the stimulated Raman excitation light source module is used to obtain a three-dimensional image of the cell, and the excitation position is automatically positioned to the region where molecules such as cell lipids aggregate, and then it is switched to the spontaneous Raman excitation light source module to achieve automatic and precise spontaneous Raman detection.

[0027] The stimulated Raman excitation light source module includes a femtosecond laser 1, a first optical path adjustment group, a second optical path adjustment group, a beam combining mirror group, and a stimulated Raman excitation light source electric aperture 6; the core of the stimulated Raman excitation light source module is a femtosecond laser 1; The femtosecond laser 1 emits two femtosecond pulse laser signals with different central wavelengths, which respectively enter the first optical path adjustment group and the second optical path adjustment group; The first optical path adjustment group sequentially includes a first half-wave plate 2, a first polarization beam splitter prism 3, and a first dispersion medium 4 along the optical path direction; the second optical path adjustment group sequentially includes a second half-wave plate 31, a second polarization beam splitter prism 30, a first mirror 29, a delay line 28, a second mirror 32, a spatial light modulator 33, and a second dispersion medium 34 along the optical path direction; the half-wave plate is used to rotate the polarization angle of light, and the polarization beam splitter prism is used to selectively transmit the light component with a fixed polarization direction and reflect the light components in other directions; the combination of the half-wave plate and the polarization beam splitter prism is used to adjust the output power of the femtosecond laser 1; the dispersion medium is used to adjust the chirp degree of the two femtosecond pulsed laser signals; the delay line 28 is composed of an electric displacement platform carrying a pair of mutually perpendicular plane mirrors, and the electric displacement platform can translate back and forth along the arrow direction and change the characteristic wavelength of the stimulated Raman detection mode; A signal generator 26, a power amplifier 27, and a spatial light modulator 33 are sequentially connected. The spatial light modulator 33 is used to receive the high-frequency modulation signal output by the signal generator 26 and amplified by the power amplifier 27 and perform amplitude high-frequency modulation; The spatial light modulator 33 can also be replaced by an electro-optic modulator or an acousto-optic modulator to achieve the modulation function; The beam combining mirror group includes a third mirror 35 and a first dichroic mirror 36, and is used for spatially combining the femtosecond pulsed laser signals passing through the first optical path adjustment group and the second optical path adjustment group; Brief description of the principle of the stimulated Raman excitation light source module: By adjusting the wavelength difference between femtosecond pulses and changing the position of the mirror pair on the delay line 28, the characteristic wavelength of the stimulated Raman detection mode can be changed, and the lipid molecules inside the cell will be focused into a specific layer at the middle position of the cell. In the present invention, it is innovatively proposed to use the axial position of the lipid molecule layer as a reference to calibrate the spatial axial position during the detection of different cell samples, thereby improving the consistency of detection data. Therefore, by setting the output wavelength of the femtosecond laser 1 and adjusting the delay line 28, the wavelength of the stimulated Raman spectroscopy imaging is the characteristic peak of lipids (including but not limited to 2850 cm -1 、2930 cm -1 etc.). The output power of the femtosecond laser 1 is adjusted by the combination of the half-wave plate and the polarization beam splitter prism, and the chirp degree of the two femtosecond pulsed laser signals is adjusted by the dispersion medium to improve the spectral resolution of the stimulated Raman detection. Any one of the two femtosecond pulses is amplitude high-frequency modulated by the spatial light modulator 33, and the modulation frequency is determined by the signal generator 26. Subsequently, the two femtosecond pulsed lasers are spatially combined by the first dichroic mirror 36 and transmitted to the signal excitation and collection unit to excite the stimulated Raman signal.

[0028] The spontaneous Raman excitation light source module includes a continuous-wave laser 7 and a motorized diaphragm 8 for the spontaneous Raman light source; the continuous-wave laser 7 is used to emit narrow-band laser with a fixed wavelength, which is transmitted to the signal excitation and collection unit to excite the Raman signal of the cell sample; the Raman signal of the cell sample is transmitted to the spectrometer 5 for Raman spectroscopy analysis; a filter is arranged at the entrance slit of the spectrometer 5, and the filter is used to filter out the laser excitation signal; for the convenience of system integration and volume reduction, the light incident on the spectrometer 5 can first pass through a mirror for optical path folding.

[0029] By controlling the switch of the motorized diaphragm 8 for the spontaneous Raman light source and / or the motorized diaphragm 6 for the stimulated Raman excitation light source, it is respectively realized whether the spontaneous Raman light source and / or the stimulated Raman excitation light source can be transported to the signal excitation and collection unit for Raman signal excitation.

[0030] In a specific embodiment, a first dichroic mirror 36, a motorized diaphragm 6 for the stimulated Raman excitation light source, a second dichroic mirror 9, and a third dichroic mirror 10 are sequentially arranged along the optical path propagation direction; the second dichroic mirror 9 and the third dichroic mirror 10 are used to reflect light of a certain wavelength or wavelength band and transmit light of another wavelength or wavelength band, which is convenient for separating light of different wavelengths or wavelength bands.

[0031] The signal excitation and collection unit includes a microscope objective 17, a stage 18, a condenser 19, a filter 22, a photodetector 23, and a lock-in amplifier 25. After the stimulated Raman excitation light is focused on the sample on the stage 18 by the microscope objective 17 to generate the stimulated Raman scattering phenomenon, the excitation energy changes. Subsequently, the changed excitation light passes through the sample and is reflected by the second beam splitter 20, and a path of excitation light modulated by the spatial light modulator 33 is filtered out by the filter 22. The other path of excitation light is collected and detected by the photodetector 23, and the signal is then transmitted to the lock-in amplifier 25 to extract the stimulated Raman signal.

[0032] The spatial position scanning unit includes a lateral position scanning mechanism and an axial position scanning mechanism, which cooperate to achieve three-dimensional spatial tomography imaging of cells; the plane of the lateral position scanning mechanism is perpendicular to the optical axis of the microscope objective 17, and the axis of the axial position scanning mechanism is parallel to the optical axis of the microscope objective 17; the lateral position scanning mechanism includes a two-dimensional galvanometer group 11 and a 4-f lens group composed of two lenses (the first lens 12 and the second lens 13), which is used for stimulated Raman spectroscopy scanning imaging of cells. The axial position scanning mechanism includes a precision displacement platform, and the precision displacement platform drives the microscope objective 17 to perform axial movement with sub-micron accuracy along the optical axis direction to achieve precise positioning; the precision displacement platform is a piezoelectric displacement actuator 16. Brief description of the principle of the spatial position scanning unit: The lateral position scanning mechanism (i.e., the galvanometer lateral scanning mechanism) and the axial position scanning mechanism (i.e., the microscopic objective axial scanning mechanism) are multiplexed. The microscopic objective axial scanning mechanism consists of a piezoelectric displacement device and a conventional optical objective. The piezoelectric displacement device drives the objective lens to perform axial movement with sub-micron accuracy to determine a specific axial position. The detection wavelength of the stimulated Raman mode is set as the characteristic wavelength of the lipid molecules of the target cells. Subsequently, the cells are scanned by a scanning galvanometer for stimulated Raman spectroscopic imaging to obtain a Raman spectroscopic image reflecting the spatial distribution of lipid molecules at this axial position. The variance value of the intensity of each pixel point in the image is used as the basis for judging the focusing degree of the cell image. The PID control algorithm or the method of equal-interval step scanning is used to automatically search for the axial position of the image with the largest variance value of the pixel point intensity, find the determined position of the lipid molecular layer in the cell, use the lipid molecular layer as the normalization positioning reference for different cell detections, determine the sampling spatial axial position, and then determine the spontaneous Raman lateral acquisition position according to the cell substructure information reflected by the stimulated Raman spectroscopic image at this axial position. Subsequently, the spontaneous Raman spectrum at this position is collected based on the spontaneous Raman spectrum unit.

[0033] The system control unit includes a computer 24, and the computer 24 is used to coordinate the collaborative work of each unit; as Figure 1 described, the computer 24 can be respectively connected to the signal generator 26, the photodetector 23, and the piezoelectric displacement device 16.

[0034] See Figures 1 - 3 , based on the above positioning system, a method for automatically positioning the axis of single-cell spontaneous-stimulated Raman spectroscopy detection is provided, which specifically includes the following steps: S1. Place the cell sample on the stage 18, turn on the white light source 21 and the camera 15, and roughly focus in the white light mode so that cells appear in the field of view.

[0035] S2. Turn on the electric aperture 6 of the stimulated Raman excitation light source, turn off the electric aperture 8 of the spontaneous Raman light source, and set the stimulated Raman detection band as the Raman shift of lipid molecules, such as 2850 cm -1 , 2930 cm -1 etc.

[0036] S3. Set the movement range and the single-step range of the piezoelectric displacement device 16. The piezoelectric displacement device 16 drives the microscopic objective lens 17 to perform extremely fine steps at the sub-micron level along the axis. Each time the piezoelectric displacement device 16 takes a step, a stimulated Raman spectroscopic imaging (XY plane imaging) is performed on the cells to obtain a stimulated Raman image; after the movement is completed, a three-dimensional spatial stimulated Raman tomography imaging stack of the cells is obtained, as Figure 2 shown in A in.

[0037] S4. Determine the axial displacement distance of the piezoelectric actuator 16 by the equal-spacing method or the PID control algorithm, adjust the axial displacement H of the piezoelectric actuator 16, perform stimulated Raman spectroscopy imaging on the cells, and position the spontaneous Raman detection position at the spot where the lipid molecules are focused in the stimulated Raman image; in this step, automatically search all axial positions of the stimulated Raman image by methods such as the PID control algorithm or equal-interval step scanning, and find the maximum value of the focusing coefficient among the focusing coefficient values of the stimulated Raman images corresponding to all axial positions; in a specific embodiment, the two methods are as follows: When the equal-spacing method is adopted, it specifically includes the following sub-steps: S401. Solve the focusing coefficient by the interval step scanning method: Calculate the variance A of the pixel intensity at each point in each obtained stimulated Raman image; assuming that the pixel intensity is denoted as Ixy, the specific calculation formula for the pixel intensity variance A is as follows: ; In the formula, m represents the total number of pixels in the x dimension, n represents the total number of pixels in the y dimension, represents the average intensity of all pixels in the image, I xy represents the pixel intensity; The calculation formula is as follows: ; In the formula, m represents the total number of pixels in the x dimension, n represents the total number of pixels in the y dimension, I xy represents the pixel intensity; Then the focusing coefficient F of the stimulated Raman image is expressed as follows: ; The axial distribution of the lipid molecular layer is as Figure 2 shown as B in; S402. Select the axial displacement of the piezoelectric actuator 16 corresponding to the maximum value of the focusing coefficient, and set the axial displacement distance of the piezoelectric actuator 16 to H; S403. Perform stimulated Raman spectroscopy imaging on the cells, and position the spontaneous Raman detection position at the spot where the lipid molecules are focused in the stimulated Raman image, as Figure 2 shown as C in.

[0038] When the PID control algorithm is adopted, it specifically includes the following sub-steps: S401. Solve the focusing coefficient of the current stimulated Raman image: Let F(t) represent the focusing coefficient of the current stimulated Raman image, and F(t - 1) represent the focusing coefficient of the image acquired at the previous moving position when the piezoelectric displacement actuator 16 moves to the current position; before the first movement of the piezoelectric displacement actuator 16, t = 1; set F(0) to 0, and at this time the position of the piezoelectric displacement actuator 16 is h; the calculation method of the focusing coefficient F(t) of the current stimulated Raman image is as follows: Calculate and obtain the variance A of the pixel intensities of each point in the current stimulated Raman image; assuming the pixel intensity is denoted as Ixy, the specific calculation formula for the pixel intensity variance A is as follows: ; In the formula, m represents the total number of pixels in the x dimension, n represents the total number of pixels in the y dimension, represents the average intensity of all pixels in the image, I xy represents the pixel intensity; The calculation formula is as follows: ; In the formula, m represents the total number of pixels in the x dimension, n represents the total number of pixels in the y dimension, I xy represents the pixel intensity; Then the focusing coefficient F(t) of the current stimulated Raman image is expressed as follows: ; Then the current focusing coefficient difference E(t) = F(t) - F(t - 1); E(t - 1) represents the focusing coefficient difference corresponding to the previous movement of the piezoelectric displacement actuator 16. Initially, the focusing coefficient difference E(0) is 0; S402. Estimate the next movement distance Z of the piezoelectric displacement actuator 16, and the calculation formula is as follows: ; where k p 、k i 、k d are constants set according to the actual situation; the value of Z has positive and negative values, respectively indicating the up and down movement directions of the piezoelectric displacement actuator 16; Set the moving position of the piezoelectric displacement actuator 16 to H = h + Z; let h = H, F(t - 1) = F(t); E(t - 1) = E(t); repeat the above process. When E(t) or Z is less than the set threshold, stop; at this time, H is the axial positioning position of the piezoelectric displacement actuator 16; the PID positioning flowchart is shown in Figure 4 ; S403. Select the H value obtained by iterating the PID control process, and set the axial displacement distance of the piezoelectric displacement actuator 16 to H; S404. Perform stimulated Raman spectroscopy imaging on the cells, and locate the spontaneous Raman detection position as the light spot where the lipid molecules are focused in the stimulated Raman image.

[0039] S5. Calculate the spontaneous Raman and stimulated Raman axial focus position correction amount L using the spontaneous Raman and stimulated Raman axial focus position compensation method, and set the position of the piezoelectric displacer 16 to H + L; The method for compensating the axial focusing position of spontaneous Raman and stimulated Raman specifically comprises the following steps: S501. Adjust the position of the piezoelectric displacer 16 so that the spontaneous Raman signal intensity of the collected lipid molecules reaches the maximum, and record the displacement distance H of the piezoelectric displacer 16 at this time. 1 ; S502. Calculate the spontaneous Raman and stimulated Raman axial focus position correction L, the expression is as follows: L = H 1 -H; In the formula, H 1 is the displacement distance of the piezoelectric displacer when the spontaneous Raman signal intensity of the lipid molecules reaches the maximum, and H is the axial displacement of the piezoelectric displacer corresponding to the maximum value of the pixel intensity variance.

[0040] S6. Close the stimulated Raman excitation light source electric diaphragm of the Raman excitation light source unit, turn off the white light source, open the spontaneous Raman light source electric diaphragm of the Raman excitation light source unit, set the spontaneous Raman spectrum acquisition parameters, perform cell spontaneous Raman spectrum acquisition, and complete cell fine Raman spectrum acquisition; The spontaneous Raman spectrum acquisition parameters include wavelength band, etc. S7. Collect Raman spectra of known types of cell samples according to steps S1 to S6, establish a Raman spectrum database of different types of cells, and obtain different types of cell discrimination models; then train and optimize each cell discrimination model; Use machine learning and other methods to train each cell discrimination model; S8. Collect the Raman spectrum of the unknown type of the cell sample to be tested according to steps S1 to S6, substitute it into the cell discrimination model for discrimination and obtain the discrimination result, so as to achieve accurate analysis of the cell; In this step, the unknown type of the cell sample to be tested is discriminated based on methods such as principal component analysis and linear discriminant analysis.

[0041] The advantages are: after collecting data to establish a Raman spectral database (standard sample library) of different types of cells, the same method is used to collect the Raman spectra of the cell samples to be tested, and the unknown cell samples are discriminated based on methods such as principal component analysis and linear discriminant analysis. Compared with traditional microscopic Raman technology, the discrimination accuracy can be greatly improved.

[0042] To ensure the discrimination accuracy, for the Raman spectra of the training set samples and subsequent test set samples, the experimental conditions for Raman spectrum acquisition need to be kept consistent. Therefore, the axial positioning of the excitation position of cells is crucial. However, there is currently no axial positioning method for cell Raman testing, which cannot guarantee the consistency of testing and leads to a reduction in analysis accuracy. At the same time, when collecting the Raman spectra of cells, the optimal axial position is the center position of the cells. The molecular composition at the cell center is rich, and the characteristic differences are obvious. Away from the cell axis position, there is a large difference in molecular concentration compared with the center. Therefore, the uncertainty of the axial position will result in significant differences in the Raman spectra of even the same type of cells. So, the present invention uses the internal lipid molecular layer of cells as the calibration benchmark for testing, and adopts the spontaneous-stimulated Raman dual-mode spectroscopy detection axial automatic positioning and precise discrimination method to normalize and standardize the sampling conditions of different cell Raman spectra, thereby improving the analysis accuracy. Cell Raman discrimination analysis was performed on two cultured cell lines, DU145 and SIHA, as shown in Figure 3 shown in Figure 3 Figure A in [reference] shows the classification results obtained by performing principal component-linear discriminant analysis on the Raman spectrum data of cells collected without axial positioning guidance. The accuracy rate of ten-fold cross-validation is 84%; Figure 3 Figure B in [reference] shows the discrimination results obtained after normalizing the axial position using the method of the present invention. The discrimination accuracy rate of the two types of cells is increased to 98% by ten-fold cross-validation.

[0043] It should be understood that various forms of the processes shown above can be used, steps can be reordered, added, or deleted. For example, the steps described in the disclosure of the present invention can be executed in parallel, sequentially, or in a different order, as long as the desired results of the technical solution disclosed in the present invention can be achieved. No limitation is imposed herein.

[0044] The above specific embodiments do not constitute a limitation to the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection, characterized in that: The specific steps include: S1. Build an axial automatic positioning system for single-cell spontaneous-stimulated Raman spectroscopy detection, including a Raman excitation light source unit, a signal excitation and collection unit, a spatial position scanning unit, and a signal processing and system control unit; Place the cell sample on the stage of the signal excitation and collection unit, turn on the white light source and camera, and roughly adjust the focus in white light mode so that cells appear in the field of view; S2. Open the stimulated Raman excitation light source motorized diaphragm of the Raman excitation light source unit, close the spontaneous Raman light source motorized diaphragm of the Raman excitation light source unit, and set the stimulated Raman detection band to the Raman frequency shift of lipid molecules; S3. Set the motion range and single step range of the precision displacement platform of the axial position scanning mechanism, and drive the microscope objective of the signal excitation and collection unit to perform sub-micron stepping along the axial direction by the precision displacement platform. Each time the precision displacement platform steps once, stimulated Raman spectroscopy imaging of the cell is performed to obtain a stimulated Raman image; after the movement is completed, a three-dimensional stimulated Raman tomography stack of the cell is obtained; S4. Determine the axial displacement distance of the precision displacement platform by an equal spacing method or a PID control algorithm, and adjust the axial displacement distance H of the precision displacement platform; perform stimulated Raman spectroscopy imaging on the cells, and locate the spontaneous Raman detection position as the light spot where the lipid molecules are focused in the stimulated Raman image; S5. Calculate the spontaneous Raman and stimulated Raman axial focus position correction L using the spontaneous Raman and stimulated Raman axial focus position compensation method, and set the position of the precision displacement platform to H+L; S6. Close the stimulated Raman excitation light source electric diaphragm of the Raman excitation light source unit, turn off the white light source, open the spontaneous Raman light source electric diaphragm of the Raman excitation light source unit, set the spontaneous Raman spectrum acquisition parameters, perform cell spontaneous Raman spectrum acquisition, and complete cell fine Raman spectrum acquisition; S7. Collect Raman spectra of known types of cell samples according to steps S1 to S6, establish a Raman spectrum database of different types of cells, and obtain different types of cell discrimination models; then train and optimize each cell discrimination model; S8. Collect the Raman spectrum of the unknown type of the cell sample to be tested according to steps S1 to S6, substitute it into the cell discrimination model for discrimination and obtain the discrimination result, so as to achieve accurate analysis of the cells.

2. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 1, characterized in that: The Raman excitation light source unit includes a stimulated Raman excitation light source module and a spontaneous Raman excitation light source module, and the stimulated Raman excitation light source module and the spontaneous Raman excitation light source module correspond to two spectral detection modes of the system; the stimulated Raman excitation light source module is used to emit two femtosecond pulse laser signals with different central wavelengths, which are transmitted to the signal excitation and collection unit after modulation to excite the stimulated Raman signal; the spontaneous Raman excitation light source module is used to emit a narrow-band laser with a fixed wavelength, which is transmitted to the signal excitation and collection unit to excite the Raman signal of the cell sample; the stimulated Raman excitation light source module and the spontaneous Raman excitation light source module both include an electric diaphragm, and the switching of the electric diaphragm is used to realize the switching of the spontaneous Raman light source and the stimulated Raman excitation light source; The signal excitation and collection unit is used to focus the stimulated Raman excitation light on the sample to excite energy changes, and then modulate the excitation light to obtain a stimulated Raman signal; The spatial position scanning unit comprises a transverse position scanning mechanism and an axial position scanning mechanism; the three-dimensional spatial tomographic imaging of cells is achieved through the cooperation of the transverse position scanning mechanism and the axial position scanning mechanism; The system control unit includes a computer, and the computer is used to coordinate the various units to work together.

3. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 2, characterized in that: The stimulated Raman excitation light source module comprises a femtosecond laser, a first optical path adjustment group, a second optical path adjustment group, a beam combining mirror group and a stimulated Raman excitation light source electric aperture; the femtosecond laser emits two femtosecond pulse laser signals with different central wavelengths, which are spatially combined by the beam combining mirror group after passing through the first optical path adjustment group and the second optical path adjustment group, and then transmitted to the signal excitation and collection unit to stimulate stimulated Raman signals; The spontaneous Raman excitation light source module includes a continuous wave laser and a spontaneous Raman light source electric aperture; the continuous wave laser is used to emit a narrow-band laser with a fixed wavelength, which is transmitted to the signal excitation and collection unit to excite the Raman signal of the cell sample; By controlling the switch of the spontaneous Raman light source electric aperture and / or the stimulated Raman excitation light source electric aperture, it is respectively realized whether the spontaneous Raman light source or the stimulated Raman excitation light source can be transmitted to the signal excitation and collection unit for Raman signal excitation.

4. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 3, characterized in that: The first optical path adjustment group includes a half-wave plate, a polarization beam splitter prism, and a dispersion medium in sequence along the optical path direction; The second optical path adjustment group includes a half-wave plate, a polarization beam splitter, a reflector, a delay line, a reflector, a modulator, and a dispersion medium in sequence along the optical path direction; The beam combining mirror group is used to spatially combine the femtosecond pulse laser signals passing through the first optical path adjustment group and the second optical path adjustment group; In the first optical path adjustment group and the second optical path adjustment group: the half-wave plate is used to rotate the polarization angle of light; the polarization beam splitter prism is used to selectively transmit the light component with a fixed polarization direction and reflect the light components in other directions; the combination of the half-wave plate and the polarization beam splitter prism is used to adjust the output power of the femtosecond laser; The dispersive medium is used to adjust the chirp degree of the two femtosecond pulse laser signals; In the second optical path adjustment group, the delay line changes the characteristic wavelength of the stimulated Raman detection mode by translation.

5. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 4, characterized in that: The modulator is connected to the power amplifier and the signal generator in sequence. The spatial light modulator is used to receive the high-frequency modulation signal output by the signal generator and amplified by the power amplifier and perform amplitude high-frequency modulation. The modulator is a spatial light modulator, an electro-optic modulator or an acousto-optic modulator.

6. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 1, characterized in that: The signal excitation and collection unit includes a microscope objective, a stage, a condenser, a filter, a photodetector and a lock-in amplifier; After the stimulated Raman excitation light is focused by the microscope objective onto the sample on the stage to generate stimulated Raman scattering, the excitation energy changes, and then the excitation light passes through the sample and is reflected by the spectroscope, and the filter removes one channel of the excitation light modulated by the modulator, and the other channel of the excitation light is collected and detected by the photodetector, and then the signal is transmitted to the phase-locked amplifier to extract the stimulated Raman signal; The plane of the transverse position scanning mechanism is perpendicular to the optical axis of the microscope objective lens, and the axis of the axial position scanning mechanism is parallel to the optical axis of the microscope objective lens; the transverse position scanning mechanism includes a two-dimensional galvanometer group and a 4-f lens group composed of two lenses, which are used to perform stimulated Raman spectroscopy scanning imaging of cells; the axial position scanning mechanism includes a precision displacement platform, which drives the microscope objective lens to perform axial movement with sub-micron precision along the optical axis direction.

7. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 1, characterized in that: The step S4 determines the axial displacement distance of the precision displacement platform by an equal spacing method, and adjusts the axial displacement distance H of the precision displacement platform; specifically includes the following sub-steps: S401. The method of equally spaced step scanning: Calculate the pixel intensity variance A of each point in each stimulated Raman image acquired; assuming that the pixel intensity is recorded as Ixy, the specific calculation formula of the pixel intensity variance A is as follows: ； In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. represents the average intensity of all pixels in the image, I xy represents pixel intensity; The calculation formula is as follows: ； In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. I xy represents pixel intensity; Set the focusing factor F of the stimulated Raman image, which is expressed as follows: ； S402. Selecting the axial displacement H of the precision displacement platform corresponding to the maximum value of the focusing coefficient, and setting the axial displacement distance of the precision displacement platform to H; S403. Perform stimulated Raman spectroscopy imaging on the cells, and locate the spontaneous Raman detection position as the light spot where the lipid molecules are focused in the stimulated Raman image.

8. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 1, characterized in that: The step S4 determines the axial displacement distance of the precision displacement platform by using a PID control algorithm, and adjusts the axial displacement distance H of the precision displacement platform; specifically, it includes the following sub-steps: S401. Solve the current stimulated Raman image focusing coefficient: F(t) represents the current stimulated Raman image focusing coefficient, and F(t-1) represents the focusing coefficient of the image collected at the previous moving position when the precision displacement platform moves to the current position; before the first movement of the precision displacement platform, t=1; set F(0) to 0, and the position of the precision displacement platform is h at this time; the current stimulated Raman image focusing coefficient F(t) is calculated as follows: Calculate and obtain the pixel intensity variance A of each point in the current stimulated Raman image; assuming that the pixel intensity is recorded as Ixy, the specific calculation formula of the pixel intensity variance A is as follows: ； In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. represents the average intensity of all pixels in the image, I xy represents pixel intensity; The calculation formula is as follows: ； In the formula, m represents the total number of pixels in the x dimension, and n represents the total number of pixels in the y dimension. I xy represents pixel intensity; Then the current stimulated Raman image focusing coefficient F(t) is expressed as follows: ； The current focusing coefficient difference E(t) = F(t) - F(t-1); E(t-1) represents the focusing coefficient difference corresponding to the last movement of the precision displacement platform; S402. Estimate the next moving distance Z of the precision displacement platform, and the calculation formula is as follows: ； Among them, k p , k i , k d It is a constant set according to the actual situation; Set the moving position of the precision displacement platform to H=h+Z; set h=H, F(t-1)=F(t); E(t-1)=E(t); repeat the above process, and stop when E(t) or Z is less than the set threshold; at this time, H is the axial positioning position of the precision displacement platform; S403. Select the H value obtained by iterating the PID control process, and set the axial displacement distance of the precision displacement platform to H; S404. Perform stimulated Raman spectroscopy imaging on the cells, and locate the spontaneous Raman detection position as the light spot where the lipid molecules are focused in the stimulated Raman image.

9. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 8, characterized in that: In the step S402, the initial focusing coefficient difference E(0) is 0; the value of the next moving distance Z of the precision displacement platform includes positive and negative values, and the positive and negative values ​​respectively indicate that the movement direction of the precision displacement platform is upward and downward.

10. The method for automatic axial positioning of single-cell spontaneous-stimulated Raman spectroscopy detection according to claim 1, characterized in that: The precision displacement platform is a piezoelectric displacer; the spontaneous Raman and stimulated Raman axial focus position compensation method in step S5 specifically includes the following steps: S501. Adjust the position of the piezoelectric displacer so that the intensity of the spontaneous Raman signal of the collected lipid molecules reaches the maximum, and record the displacement distance H1 of the piezoelectric displacer at this time; S502. Calculate the spontaneous Raman and stimulated Raman axial focus position correction L, the expression is as follows: L = H1-H; Where H1 is the displacement distance of the piezoelectric displacer when the spontaneous Raman signal intensity of the lipid molecule reaches the maximum, and H is the axial displacement of the piezoelectric displacer corresponding to the maximum value of the pixel intensity variance; The step S8 uses principal component analysis or linear discriminant analysis to discriminate the unknown type of the cell sample to be tested.

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