Spontaneous-stimulated raman co-localization single cell substructure mapping system and method

Abstract

The present application relates to the technical field of spectral imaging detection, and particularly relates to a spontaneous-stimulated Raman co-localization single cell substructure mapping analysis system and method. The system comprises: a femtosecond pulse excitation module for outputting two beams of broadband femtosecond pulse lasers with different frequencies and continuously adjustable optical power; a Stokes light modulation module for modulating Stokes light and spatially combining with pump light; a pump light modulation module for modulating pump light; a spontaneous Raman laser continuous adjustment module for outputting spontaneous Raman laser; a stimulated Raman excitation light co-localization module for re-combining the combined laser and the spontaneous Raman laser and focusing on the sample, exciting stimulated Raman scattering signals, converting the signals into digital signals, and collecting data; and a spectral detection module for collecting spontaneous Raman scattering light reflected and returned along the original light path and collecting spectral signals to form Raman spectra. The advantage lies in improving the single cell heterogeneity structure and the accuracy of cancer cell type discrimination.

Description

Technical Field

[0001] This invention relates to the field of spectral imaging detection technology, and in particular to a spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system and method. Background Technology

[0002] In the development of cellular carcinogenesis, factors such as genetic variations, metabolic changes, and imbalances in cell proliferation and apoptosis all play crucial roles. Different types of cancer develop differently, often requiring multi-omics approaches for classification and grading. Raman spectroscopy, which detects molecular vibrational energy, plays an increasingly important role in biomedical research, including multi-component analysis of cell metabolism and diagnosis of cancer and its subtypes, due to its non-invasive nature and strong chemical specificity. Many results have revealed differences between cancer cells and normal cells in terms of nucleic acid and protein content and types, and metabolites. Spontaneous Raman scattering microscopy can detect complex molecules within a single focal spot across the entire spectrum. Currently, the diameter of the Raman focusing spot is generally around one micrometer, while the cell diameter is on the order of ten micrometers. Furthermore, the structural differences in molecular distribution within cells are significant, and spontaneous Raman scattering spectroscopy is significantly affected by these structural differences. Currently, white light microscopy, used in conjunction with spontaneous Raman scattering, struggles to provide images of cell structure without staining, and the dyes used for staining severely interfere with Raman spectral acquisition. Therefore, current cell micro-Raman identification techniques often employ a method of multiple blind measurements within the cell and averaging the results to obtain Raman spectra characterizing the cell. However, the weak scattering efficiency limits Raman spectroscopy for studying single-cell heterogeneity, resulting in low detection efficiency for trace single-cell components. Due to the heterogeneity of single-cell samples, this blind testing method reduces measurement efficiency and accuracy, is prone to cell photodamage, and the average spectral information masks the significant differences in specific structural components of cells, leading to low detection accuracy.

[0003] Stimulated Raman scattering (SRS), based on third-order nonlinear polarization excitation, is an avalanche-like collision process between photons and stimulated phonons, which amplifies the Raman signal, enabling rapid spectral imaging analysis. The spectral range and resolution of SRS are determined by the properties of the two excitation spectral bands. Femtosecond pulses with higher instantaneous power are ideal excitation sources for SRS; however, the time-frequency characteristics of femtosecond pulses limit the detection range of current spectral focusing stimulated Raman techniques to around 300 wavenumbers, making it difficult to meet the spectral resolution requirements of cell fingerprinting below 10 wavenumbers. Achieving high spectral resolution and multi-band detection is the main research direction of SRS, primarily achieved by tuning the center wavelength of the incident femtosecond laser to detect Raman frequency shifts from the fingerprint region to the high-wavenumber region.

[0004] 2800-3100cm -1 The high wavenumber region of Raman spectroscopy exhibits intense CH molecule vibrations. SRS typically locates this region to obtain information on proteins and several lipids within single cells or tissues. However, due to the single and highly overlapping peaks in the high wavenumber region, the Raman peaks of major cellular components such as nucleic acids are masked. The fingerprint region contains abundant Raman molecular vibrations. SRS localization in the fingerprint region, combined with high wavenumber imaging, holds promise for separating nucleic acids from proteins and lipids in single cells and mapping complete component structure images of single cells. Dual-band SRS imaging can achieve effects similar to bright-field staining and guide cell Raman spectrometers to accurately acquire fingerprint spectral information of subcellular structures. Compared to blind Raman spectroscopy, this method is expected to improve spectral acquisition efficiency and enhance the accuracy of studies on the heterogeneity of biological samples. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a spontaneous-stimulated Raman co-localization single-cell substructure mapping system and method.

[0006] The primary objective of this invention is to provide a spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system, comprising:

[0007] The femtosecond pulse excitation module is used to output two broadband femtosecond pulse lasers with different frequencies and continuously adjustable optical power. The high-frequency linearly polarized broadband femtosecond pulse laser is used as the pump light, and the low-frequency linearly polarized broadband femtosecond pulse laser is used as the Stokes light.

[0008] A Stokes light modulation module is used to modulate the Stokes light and spatially combine it with the pump light.

[0009] A pump light modulation module is used to modulate the pump light;

[0010] A spontaneous Raman laser continuous adjustment module is used to output spontaneous Raman laser;

[0011] The stimulated Raman excitation light colocalization module includes a spectral scanning unit and a signal processing unit;

[0012] The spectral scanning unit is used to combine the combined laser beam with the spontaneous Raman laser beam again and focus it on the sample to excite stimulated Raman scattering signal; the signal processing unit is used to convert the stimulated Raman scattering signal into a digital signal and perform data acquisition.

[0013] The spectral detection module is used to collect spontaneous Raman scattered light that has been reflected and returned along the original optical path, and to acquire spectral signals and form Raman spectra.

[0014] Preferably, the Stokes optical modulation module includes: a Stokes optical path glass rod, a time delay line, a first lens, an acousto-optic modulator, a beam splitter, a second lens, a third mirror, a short-pass dichroic mirror, an RF driver, and a function generator;

[0015] The selected output component of the Stokes light, after passing through the glass rod of the Stokes light path, widens the femtosecond pulse width to a picosecond pulse and is incident on the time delay line; the acousto-optic modulator, through the function generator and the RF driver, performs high-frequency periodic modulation on the Stokes light, emitting zero-order and first-order light; the zero-order light is blocked by the beam splitter, and the first-order light, after being collimated by the second lens, enters the short-pass dichroic mirror, through which the modulated Stokes light and the modulated pump light are spatially combined;

[0016] The time delay line consists of an electrically driven displacement platform equipped with a pair of mutually perpendicular planar mirrors. The electrically driven displacement platform can reciprocate in the direction of the arrow and change the relative optical path difference between the pump light and the Stokes light.

[0017] Preferably, the Stokes light modulation module is equipped with multiple reflectors to change the direction of light propagation and meet the requirements of the optical path spatial arrangement.

[0018] Preferably, the pump light modulation module includes: a first glass rod for the pump light path, a first pump light path dichroic mirror, and a second pump light path dichroic mirror;

[0019] The first pump light path dichroic mirror and the second pump light path dichroic mirror are perpendicular to each other; the pump light passes through the first glass rod of the pump light path, which widens the femtosecond pulse width to a picosecond pulse, and then sequentially enters the first pump light path dichroic mirror and the second pump light path dichroic mirror.

[0020] Preferably, the pump light modulation module further includes: a second glass rod for the pump light path and a silver reflector;

[0021] The pump light's center wavelength is set to 897nm. After passing through the first glass rod of the pump light path, the femtosecond pulse width is widened to a picosecond pulse and incident on the first pump light path dichroic mirror. After being reflected by the silver mirror, it is incident on the second glass rod of the pump light path. The second glass rod of the pump light path compensates for the dispersion lost due to the wavelength change. The laser light compensated by the second glass rod of the pump light path is then modulated by passing through the second pump light path dichroic mirror.

[0022] Preferably, the spontaneous Raman laser continuous adjustment module includes: a spontaneous Raman laser, a half-wave plate, a polarizing beam splitter, and a beam stop;

[0023] The half-wave plate is used to rotate the polarization angle of the initial laser line emitted by the spontaneous Raman laser; the polarizing beam splitter is used to selectively transmit the light component with a fixed polarization direction while reflecting the light component with other directions; the light-shielding baffle is used to block the polarized light reflected by the polarizing beam splitter.

[0024] Preferably, the spectral scanning unit includes: a 650nm long-pass dichroic mirror, a 532nm long-pass dichroic mirror, a two-dimensional galvanometer galvanometer mirror, an objective lens, a sample stage, a condenser lens, and a short-pass filter;

[0025] The combined laser beam passes through the 650nm long-pass dichroic mirror, and then, together with the laser emitted from the spontaneous Raman laser continuous adjustment module, passes through the 532nm long-pass dichroic mirror for beam combining before entering the laser scanning microscope equipped with the two-dimensional galvanometer galvanometer. The objective lens faces the sample stage on which the sample is placed, and is used to focus the laser beam onto the sample to excite stimulated Raman scattering signals. The condenser lens is used to collect the stimulated Raman scattered light. The short-pass filter is used to filter out excess Stokes light components in the stimulated Raman scattered light.

[0026] The two-dimensional galvanometer galvanometer mirror consists of a pair of mutually perpendicular silver-plated mirrors, used for rapid laser scanning of samples;

[0027] The signal processing unit includes: a silicon photodiode, a lock-in amplifier, a digital acquisition card, and a computer host;

[0028] Pump light passing through the short-pass filter enters the silicon photodiode, and is demodulated into a digital signal by the lock-in amplifier. The digital signal is acquired by the digital acquisition card and transmitted to the computer host for final processing.

[0029] Preferably, the spectral detection module includes: a 532nm cutoff filter, a sleeve lens, and a Raman spectrometer;

[0030] The 532nm excitation light interacts with the sample molecules at the focal point of the microscope. The excited Raman scattered light returns along the original optical path and is reflected by the 650nm long-pass dichroic mirror to the spectral detection module. After passing through the 532nm cutoff filter, the Raman scattered light is retained. The Raman scattered light is focused by the sleeve lens to the Raman spectrometer for Raman spectral detection.

[0031] Preferably, the femtosecond pulse excitation module includes: a dual-channel synchronously emitting femtosecond laser, a pump optical path power continuously adjustable group, and a Stokes optical path power continuously adjustable group;

[0032] The dual-channel synchronous femtosecond laser is used to output pump light and Stokes light;

[0033] The pump optical path power continuous adjustment group includes a pump optical path half-wave plate, a pump optical path polarizing beam splitter, and a pump optical path light block; the pump optical path half-wave plate is used to rotate the polarization angle of the Stokes light; the pump optical path polarizing beam splitter is used to selectively transmit light components with a fixed polarization direction while reflecting light components with other directions; the pump optical path light block is used to block the polarized light reflected by the pump optical path polarizing beam splitter.

[0034] The Stokes optical path power continuous adjustment group includes a Stokes optical path half-wave plate, a Stokes optical path polarizing beam splitter, and a Stokes optical path light block; the Stokes optical path half-wave plate is used to rotate the polarization angle of the Stokes light, the Stokes optical path polarizing beam splitter is used to selectively transmit light components with a fixed polarization direction and reflect light components with other directions; the Stokes optical path light block is used to block the polarized light reflected by the Stokes optical path polarizing beam splitter.

[0035] The second objective of this invention is to provide a method for spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis, which employs a spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system and specifically includes the following steps:

[0036] S1. Turn on the dual-channel synchronous femtosecond laser to emit two femtosecond laser beams ω1 and ω2, which are used as pump light and Stokes light, respectively;

[0037] S2. Modulation of pump light and Stokes light in high wavenumber region: The center wavelengths of the pump light and the Stokes light are tuned to make the excitation band in the high wavenumber region; the femtosecond pulse width is broadened to a picosecond pulse by modulation by the first glass rod of the pump light path and the glass rod of the Stokes light path, respectively.

[0038] S3. The modulated pump light and Stokes light are combined and then introduced into the laser scanning microscope to excite the SRS effect of the single-cell sample. The components are then dissociated through hyperspectral acquisition and image analysis algorithms to obtain the component distribution image of proteins and lipids in the single cell.

[0039] S4. Modulation of pump light and Stokes light in fingerprint region: The center wavelengths of the pump light and the Stokes light are tuned so that the excitation band is in the fingerprint region; the pump light is modulated by the first glass rod and the second glass rod of the pump light path, and the Stokes light is modulated by the glass rod of the Stokes light path to broaden the femtosecond pulse width to a picosecond pulse.

[0040] S5. The modulated pump light and Stokes light are combined and introduced into the laser scanning microscope to excite the SRS effect of the single-cell sample in the fingerprint area. By changing the time delay, the spectrum is focused on the nucleic acid molecular peak, and label-free imaging of the single-cell nucleic acid is performed to obtain a high-resolution image of the component distribution of nucleic acid in the single cell.

[0041] S6. Combine high-resolution images of nucleic acid composition distribution within a single cell with images of protein and lipid composition distribution within a single cell to construct a pseudo-color single-cell structure map;

[0042] S7. Turn off the dual-path synchronous femtosecond laser, turn on the spontaneous Raman laser, and use a 532nm long-pass dichroic mirror to make the laser and the original femtosecond laser optical path spatially consistent, and incident on the sample to be tested, converting the image pixel information into the X-axis voltage and Y-axis voltage of the two-dimensional galvanometer galvanometer mirror to achieve co-positioning control;

[0043] S8. By changing the voltage of the two-dimensional galvanometer galvanometer mirror and moving the laser focus point to the single-cell substructure region to be tested, and sampling the spectrum of the single-cell substructure region to be tested using a Raman spectrometer, colocalization images and spectral composite information are obtained;

[0044] S9. The spectral composite information is used for spectral identification and accuracy determination through spectral analysis methods.

[0045] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0046] This invention provides an imaging and spectral analysis system for spontaneous and stimulated Raman co-localization. By using this analysis system, the accuracy of identifying single-cell heterogeneous structures and cancer cell types can be improved.

[0047] By employing label-free SRS imaging to achieve a staining-like effect, this invention assists spontaneous Raman spectroscopy in targeted detection of single-cell substructures of interest, thereby improving Raman detection efficiency and analytical accuracy. Compared to existing spontaneous or stimulated Raman microscopy, the spontaneous-stimulated Raman co-localization single-cell substructure mapping method provided by this invention achieves a measurement accuracy of 93.2%. This method further enhances the acquisition of biological information and possesses the potential for in-situ diagnosis of clinical samples. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the logical structure of a spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system provided in an embodiment of the present invention.

[0049] Figure 2 This is a schematic diagram of the optical path of a spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system provided in an embodiment of the present invention.

[0050] Figure 3 This is according to the embodiments of the present invention / specific implementation of the present invention;

[0051] Figure 4 This is a comparison chart of the co-location test results provided by the embodiments of the present invention and the results of the traditional blind Raman spectroscopy method.

[0052] Figure label:

[0053] 1. Femtosecond pulse excitation module;

[0054] 11. Dual-channel synchronous femtosecond laser; 12. Pump path half-wave plate; 13. Pump path polarizing beam splitter prism; 14. Pump path beam stop; 15. Stokes path half-wave plate; 16. Stokes path polarizing beam splitter prism; 17. Stokes path beam stop;

[0055] 2. Stokes optical modulation module;

[0056] 201. First reflecting mirror; 202. Stokes optical path glass rod; 203. Time delay line; 204. Second reflecting mirror; 205. First lens; 206. Acousto-optic modulator; 207. Beam splitter; 208. Second lens; 209. Third reflecting mirror; 210. 1000nm short-pass dichroic mirror; 211. Radio frequency driver; 212. Function generator;

[0057] 3. Pump light modulation module;

[0058] 31. First glass rod of the pump optical path; 32. Dichroic mirror of the first pump optical path; 33. Dichroic mirror of the second pump optical path; 34. Fourth reflecting mirror; 35. Second glass rod of the pump optical path; 36. Fifth reflecting mirror;

[0059] 4. Spontaneous Raman laser continuous adjustment module;

[0060] 41. Spontaneous Raman laser; 42. Half-wave plate; 43. Polarizing beam splitter; 44. Light-shielding baffle;

[0061] 5. Stimulated Raman excitation light co-localization module;

[0062] 51. 650nm long-pass dichroic mirror; 52. 532nm long-pass dichroic mirror; 53. Sixth reflecting mirror; 54. Two-dimensional galvanometer galvanometer mirror; 55. Objective lens; 56. Sample stage; 57. Condenser lens; 58. Seventh reflecting mirror; 59. Short-pass filter; 61. Silicon photodiode; 62. Lock-in amplifier; 63. Digital acquisition card; 64. Computer host;

[0063] 6. Spectral detection module;

[0064] 71. Silver mirror; 72. 532nm cutoff filter; 73. Tube lens; 74. Raman spectrometer. Detailed Implementation

[0065] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the 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 merely illustrative of the invention and do not constitute a limitation thereof.

[0067] Figure 1-2 This demonstrates a spontaneous-stimulated Raman co-localization single-cell substructure mapping system, including:

[0068] The femtosecond pulse excitation module 1 is used to output two broadband femtosecond pulse lasers with different frequencies and continuously adjustable optical power. The femtosecond pulse excitation module 1 includes a dual-channel synchronously emitting femtosecond laser 11, a pump optical path power continuously adjustable group, and a Stokes optical path power continuously adjustable group. The dual-channel synchronously emitting femtosecond laser 11 is used to output two linearly polarized broadband femtosecond pulse lasers with different frequencies, using the higher frequency femtosecond pulse laser as the pump light and the lower frequency femtosecond pulse laser as the Stokes light.

[0069] The pump optical path power continuous adjustment group and the Stokes optical path power continuous adjustment group have the same structure. The pump optical path power continuous adjustment group includes a pump optical path half-wave plate 12, a pump optical path polarizing beam splitter 13, and a pump optical path light block 14. The Stokes optical path power continuous adjustment group includes a Stokes optical path half-wave plate 15, a Stokes optical path polarizing beam splitter 16, and a Stokes optical path light block 17. The pump optical path half-wave plate 12 and the Stokes optical path half-wave plate 15 rotate the initial 532nm laser linear polarization direction by a specific angle, thus rotating the linear polarization direction of the pump and Stokes light. The pump optical path polarizing beam splitter 13 and the Stokes optical path polarizing beam splitter 16 are used to transmit the light component in the horizontal polarization direction and reflect the light component in the vertical direction. The pump optical path light block 14 and the Stokes optical path light block 17 are used to block the light component in the vertical direction to prevent it from affecting the optical system.

[0070] The principle of the femtosecond pulse excitation module is to polarize and split the linearly polarized light incident on its surface in two orthogonal directions, and then take the horizontally polarized light that has passed through the polarizing beam splitter. By continuously rotating the half-wave plate, the power of the horizontally polarized light can be continuously adjusted.

[0071] The Stokes light modulation module 2 includes a first reflector 201, a Stokes optical path glass rod 202, a time delay line 203, a second reflector 204, a first lens 205, an acousto-optic modulator 206, a beam splitter 207, a second lens 208, a third reflector 209, a 1000nm short-pass dichroic mirror 210, an RF driver 211, and a function generator 212. The Stokes light selection output component, after being reflected by the first reflector 201, passes through the Stokes optical path glass rod 202, widening the femtosecond pulse width to a picosecond pulse, and is then incident on the time delay line 203. The time delay line 203 consists of a pair of mutually perpendicular flat... The reflector is composed of an electrically displaced platform that can reciprocate along the direction of the arrow and change the relative optical path difference between the pump light and the Stokes light. The acousto-optic modulator 206 modulates the Stokes light at a high frequency periodically through the function generator 212 and the radio frequency driver 211, and emits zero-order light and first-order light. The zero-order light is blocked by the beam splitter 207 (preventing the propagation of the zero-order light to affect the optical path). The first-order light is collimated by the second lens 208 and reflected by the third reflector 209, and then enters the 1000nm short-pass dichroic mirror 210. The 1000nm short-pass dichroic mirror 210 spatially combines the modulated Stokes light and the pump light.

[0072] In a specific embodiment, the Stokes light modulation module 2 is equipped with multiple reflectors, mainly used to change the direction of light propagation to meet the spatial arrangement of the light path. Different numbers of plane reflectors can be selected according to actual needs to meet the spatial arrangement requirements of different systems. In addition, the dichroic mirror used is a short-pass dichroic mirror. If the pump light and the Stokes light are exchanged, the dichroic mirror needs to be a corresponding long-pass dichroic mirror to meet the actual requirements.

[0073] The pump light modulation module 3 includes a first glass rod 31 for the pump light path, a first dichroic mirror 32 for the first pump light path, a second dichroic mirror 33 for the second pump light path, a fourth reflector 34, a second glass rod 35 for the pump light path, and a fifth reflector 36. The first dichroic mirror 32 and the fourth reflector 34 are parallel, the second dichroic mirror 33 and the fifth reflector 36 are parallel, and the angles at which the first dichroic mirror 32 and the second dichroic mirror 33 are set are perpendicular to each other. After passing through the first glass rod 31, the pump light widens the femtosecond pulse width to a picosecond pulse and is incident on the first dichroic mirror 32.

[0074] In a specific embodiment, in the high wavenumber SRS imaging system, if the center wavelength of the pump light is set to 802nm, it will pass directly through two 850nm short-pass dichroic mirrors (first pump light path dichroic mirror 32 and second pump light path dichroic mirror 33) and be combined with the Stokes light after the 1000nm short-pass dichroic mirror 210. In the fingerprint SRS imaging system, if the center wavelength of the pump light is set to 897nm, it will be reflected by the first pump light path dichroic mirror 32 and the fourth reflecting mirror 34, and then the dispersion lost due to the change of wavelength will be compensated by the second glass rod 35 of the pump light path. Finally, it will be reflected by the fifth reflecting mirror 36 and the second pump light path dichroic mirror 33, so that the pump light and the Stokes light are combined after the 1000nm short-pass dichroic mirror 210.

[0075] High wavenumber SRS imaging system: The initial broadband femtosecond pulse pump and Stokes light center wavelengths were set to 802 nm and 1045 nm, respectively, based on the Raman shift calculation formula. The initial spectral width of the femtosecond pulse measured by the fiber optic spectrometer, and the excitation Raman shift region in the range of 2800–3100 cm⁻¹ -1 ;

[0076] Fingerprint SRS imaging system: The center wavelength of the pump light is tuned to 897nm. Similarly, the fingerprint area at 1500-1700cm² will be excited. -1 Raman shift.

[0077] The hyperspectral SRS imaging system, which uses co-localization of the fingerprint region and the high-wavenumber region, enables the following functions: single-cell protein and lipid imaging is performed through the high-wavenumber region, while the fingerprint region's spectrum is focused at 1578 cm⁻¹. -1 Nucleic acid molecules are imaged, and through co-localization image combination, three different color imaging channels are combined to form a stain-like label-free single-cell substructure imaging map.

[0078] The spontaneous Raman laser continuous adjustment module 4 includes a spontaneous Raman laser 41, a half-wave plate 42, a polarizing beam splitter 43, and a light-shielding baffle 44. The half-wave plate 42 rotates the initial 532nm laser linear polarization direction emitted by the spontaneous Raman laser 41 by a specific angle, while the polarizing beam splitter 43 polarizes the linearly polarized light incident on its surface in two orthogonal directions. The horizontally polarized light that has passed through the polarizing beam splitter 43 is selected, and the vertically polarized light reflected by the polarizing beam splitter 43 is blocked by the light-shielding baffle 44. The power of the horizontally polarized light can be continuously adjusted by continuously rotating the half-wave plate 42.

[0079] Stimulated Raman excitation light colocalization module 5 includes a spectral scanning unit and a signal processing unit;

[0080] The spectral scanning unit includes a 650nm long-pass dichroic mirror 51, a 532nm long-pass dichroic mirror 52, a sixth reflecting mirror 53, a two-dimensional galvanometer galvanometer mirror 54, an objective lens 55, a sample stage 56, a condenser lens 57, a seventh reflecting mirror 58, and a short-pass filter 59. The combined laser beam passes through the 650nm long-pass dichroic mirror 51 and then, together with the laser emitted from the spontaneous Raman laser continuous adjustment module 4, passes through the 532nm long-pass dichroic mirror 52 for beam combining. The beam is then reflected by the sixth reflecting mirror 53 to the laser scanning microscope equipped with the two-dimensional galvanometer mirror 54. The two-dimensional galvanometer mirror 54 consists of a pair of mutually perpendicular silver-plated mirrors used for rapid laser scanning of the sample. The objective lens 55 faces the sample stage 56, which focuses the laser beam onto the sample to excite stimulated Raman scattering signals. The condenser lens 57 collects the stimulated Raman scattered light. The short-pass filter 59 filters out excess Stokes light components from the stimulated Raman scattered light.

[0081] The signal processing unit includes a silicon photodiode 61, a lock-in amplifier 62, a digital acquisition card 63, and a computer host 64. Pump light passing through the short-pass filter 59 enters the silicon photodiode 61, and after being demodulated by the lock-in amplifier 62, the digital signal is acquired by the digital acquisition card 63 and transmitted to the computer host 64 for final processing.

[0082] The stimulated Raman excitation light colocalization module 5 includes two dichroic mirrors. Both stimulated Raman excitation lights are above 680 nm, while the Raman frequency shift of molecules is excited by a 532 nm excitation light, falling within the single-cell vibrational range (400-4000 cm⁻¹). -1 The Raman shift calculation formula is used to determine that the maximum wavelength of the Raman scattered light does not exceed 650 nm. The module has two dichroic mirrors: one a 532 nm long-pass dichroic mirror and the other a 650 nm long-pass dichroic mirror. These are used to separate the four frequency components: stimulated Raman pump light, Stokes light, 532 nm Raman excitation light, and Raman scattered light between 532 and 650 nm.

[0083] The spectral detection module 6 includes a silver reflector 71, a 532nm cutoff filter 72, a sleeve lens 73, and a Raman spectrometer 74.

[0084] The spontaneous Raman laser interacts with the sample molecules at the focal point of the laser scanning microscope. The excited Raman scattered light returns along the original optical path and is reflected by the 650nm long-pass dichroic mirror 51 to the spectral detection module 6. After passing through the silver reflector 71 and the 532nm cutoff filter 72, the Rayleigh light in the Raman scattered light is completely filtered out, leaving only the Raman scattered light. The light is then focused by the sleeve lens 73 to the Raman spectrometer 74, and the Raman spectrum is detected by the spectrometer.

[0085] The spectral detection module 6 consists of a Raman cutoff filter, a sleeve lens, and a Raman spectrometer. Based on the excitation wavelength, a 532nm cutoff filter is selected to completely filter out the 532nm excitation light, allowing only the Raman scattered light to pass through. The Raman scattered light passes through the sleeve lens and enters the slit of the Raman spectrometer, where it is dispersed and the spectral signal is collected to form a Raman spectrum.

[0086] Example 1

[0087] This embodiment provides a method for spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis (flowchart shown). Figure 3 ),use Figure 1-2 The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system shown was used for analysis, and the specific steps are as follows:

[0088] S1. The dual-channel synchronous emission femtosecond laser 11 is activated, emitting two femtosecond laser beams ω1 and ω2, which serve as the pump light and Stokes light of the SRS system, respectively. Their initial pulse widths are τ and ω2, respectively. 0p and τ 0S The wavelengths of the two are λ. p and λ S Of the two beams, at least one beam must have a center wavelength that is tunable within a certain range, according to the Raman shift formula:

[0089]

[0090] The SRS spectral detection area can be located by tuning the center wavelength of the pump and Stokes light.

[0091] S2. Modulation of pump light and Stokes light in the high wavenumber region;

[0092] S201. The center wavelengths of the tuned pump and Stokes light, calculated using the Raman shift formula, correspond to an excitation band in the range of 2800–3100 cm⁻¹. -1 This region is called the high wavenumber region of Raman.

[0093] S202.pump and Stokes light beams have lengths of l respectively. p and l S The pulse width broadening of the chirped media (first glass rod 31 in the pump optical path and glass rod 202 in the Stokes optical path) can be given by the chirped dispersion formula:

[0094]

[0095] Where k″ is the group velocity dispersion (GVD) of the material, l is the length of the dispersive material through which the light passes, and the product of the two is the group delay (GDD) of the material for the pulsed light.

[0096] The linear chirp parameter β is expressed as a function of l in terms of τ0 and τ:

[0097]

[0098] In the formula, τ0 is the initial pulse width; according to the linear chirp parameter formula, the relationship between the length of the pump and the chirp medium required for linear chirp matching can be obtained.

[0099] S203. Measure the initial spectral width S of the pump and Stokes light using a spectrometer. p and S S The broadening factor F is the ratio of the dispersed pulse width to the initial pulse width. The instantaneous bandwidth is reduced to 1 / F of the initial bandwidth, meaning that the instantaneous bandwidths of the two pulses after broadening are S and S, respectively. p / F and S S / F.

[0100] S204. Under linear chirped matching conditions, the spectral resolution ΔΩ of the spectral system is represented by the instantaneous bandwidth convolution between pump and Stoeks. SRS :

[0101] ΔΩ SRS =(S p / F)*(S S / F).

[0102] After the S3 pump and Stokes light are spatiotemporally combined through a 1000nm short-pass dichroic mirror 210, they are jointly introduced into a laser scanning microscope to excite the SRS effect in a single-cell sample. The components are then dissociated using hyperspectral acquisition and image analysis algorithms to obtain images of the protein and lipid composition distribution within the single cell.

[0103] S4. Modulation of pump light and Stokes light in the fingerprint area;

[0104] S401. The center wavelengths of the tuned pump and Stokes light are calculated using the Raman shift formula to determine the Raman fingerprint region of the cells excited by both.

[0105] S402.pump and Stokes light have lengths of l respectively. p′ and l S′ For the chirped medium, in the same way as steps S202 to S204, calculate the pulse width, instantaneous bandwidth and spectral resolution of the system fingerprint region.

[0106] After the S5 pump and Stokes light are spatiotemporally combined by a 1000nm short-pass dichroic mirror 210, they are jointly introduced into a laser scanning microscope to excite the SRS effect in the fingerprint region of a single-cell sample. By changing the time delay, the spectrum is focused on the nucleic acid molecular peak, and label-free imaging of single-cell nucleic acids is performed to obtain a high-resolution image of the component distribution of nucleic acids within a single cell.

[0107] S6. Combine high-resolution images of nucleic acid composition distribution within single cells with images of protein and lipid composition distribution within single cells to construct a pseudo-color single-cell structure map.

[0108] S7. Turn off the dual-path synchronous femtosecond laser 11 and turn on the spontaneous Raman laser 41. Through the 532nm long-pass dichroic mirror 52, the laser and the original femtosecond laser optical path are kept spatially consistent. The laser is incident on the sample to be tested. By converting the image pixel information into the X and Y axis voltages in the two-dimensional galvanometer galvanometer mirror 54, co-positioning control is achieved.

[0109] S8. By changing the voltage of the two-dimensional galvanometer galvanometer mirror 54 and moving the laser focus point to the substructure region of interest in a single cell, the spectrum of that point is sampled by the Raman spectrometer 74 to obtain colocalization image and spectral composite information.

[0110] S9. The sampled spectral composite information is used for spectral identification and accuracy determination through spectral analysis methods.

[0111] Figure 4 The results of the analytical method of the present invention are compared with those of the traditional blind Raman spectroscopy method. For the DU145 and SiHa cell lines, the traditional blind Raman spectroscopy method, with PCA-LDA algorithm, has a discrimination accuracy of 84.1% with 10x cross-validation, while the colocalization test method gives a measurement accuracy of 93.2%.

[0112] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0113] The specific embodiments described above do not constitute a limitation on the scope of protection of this 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 principles of this invention should be included within the scope of protection of this invention.

Claims

1. A spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system, characterized in that, include: The femtosecond pulse excitation module is used to output two broadband femtosecond pulse lasers with different frequencies and continuously adjustable optical power. The high-frequency linearly polarized broadband femtosecond pulse laser is used as the pump light, and the low-frequency linearly polarized broadband femtosecond pulse laser is used as the Stokes light. A Stokes light modulation module is used to modulate the Stokes light and spatially combine it with the pump light. A pump light modulation module is used to modulate the pump light; A spontaneous Raman laser continuous adjustment module is used to output spontaneous Raman laser; The stimulated Raman excitation light colocalization module includes a spectral scanning unit and a signal processing unit; The spectral scanning unit is used to combine the combined laser beam with the spontaneous Raman laser beam again and focus it on the sample to excite stimulated Raman scattering signal; the signal processing unit is used to convert the stimulated Raman scattering signal into a digital signal and perform data acquisition. The spectral detection module is used to collect spontaneous Raman scattered light that has been reflected and returned along the original optical path, and to acquire spectral signals and form Raman spectra.

2. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 1, characterized in that, The Stokes optical modulation module includes: a Stokes optical path glass rod, a time delay line, a first lens, an acousto-optic modulator, a beam splitter, a second lens, a third reflector, a short-pass dichroic mirror, an RF driver, and a function generator; The selected output component of the Stokes light, after passing through the glass rod of the Stokes light path, widens the femtosecond pulse width to a picosecond pulse and is incident on the time delay line; the acousto-optic modulator, through the function generator and the RF driver, performs high-frequency periodic modulation on the Stokes light, emitting zero-order and first-order light; the zero-order light is blocked by the beam splitter, and the first-order light, after being collimated by the second lens, enters the short-pass dichroic mirror, through which the modulated Stokes light and the modulated pump light are spatially combined; The time delay line consists of an electrically driven displacement platform equipped with a pair of mutually perpendicular planar mirrors. The electrically driven displacement platform can reciprocate in the direction of the arrow and change the relative optical path difference between the pump light and the Stokes light.

3. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 2, characterized in that, The Stokes light modulation module is equipped with multiple mirrors to change the direction of light propagation and meet the spatial arrangement of the light path.

4. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 3, characterized in that, The pump light modulation module includes: a first glass rod for the pump light path, a first pump light path dichroic mirror, and a second pump light path dichroic mirror; The first pump light path dichroic mirror and the second pump light path dichroic mirror are perpendicular to each other; the pump light passes through the first glass rod of the pump light path, which widens the femtosecond pulse width to a picosecond pulse, and then sequentially enters the first pump light path dichroic mirror and the second pump light path dichroic mirror.

5. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 4, characterized in that, The pump light modulation module also includes: a second glass rod for the pump light path and a silver reflector; The pump light's center wavelength is set to 897nm. After passing through the first glass rod of the pump light path, the femtosecond pulse width is widened to a picosecond pulse and incident on the first pump light path dichroic mirror. After being reflected by the silver mirror, it is incident on the second glass rod of the pump light path. The second glass rod of the pump light path compensates for the dispersion lost due to the wavelength change. The laser light compensated by the second glass rod of the pump light path is then modulated by passing through the second pump light path dichroic mirror.

6. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 5, characterized in that, The spontaneous Raman laser continuous adjustment module includes: a spontaneous Raman laser, a half-wave plate, a polarizing beam splitter, and a beam stop; The half-wave plate is used to rotate the polarization angle of the initial laser line emitted by the spontaneous Raman laser; the polarizing beam splitter is used to selectively transmit the light component with a fixed polarization direction while reflecting the light component with other directions; the light-shielding baffle is used to block the polarized light reflected by the polarizing beam splitter.

7. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 6, characterized in that, The spectral scanning unit includes: a 650nm long-pass dichroic mirror, a 532nm long-pass dichroic mirror, a two-dimensional galvanometer galvanometer mirror, an objective lens, a sample stage, a condenser lens, and a short-pass filter. The combined laser beam passes through the 650nm long-pass dichroic mirror, and then, together with the laser emitted from the spontaneous Raman laser continuous adjustment module, passes through the 532nm long-pass dichroic mirror for beam combining before entering the laser scanning microscope equipped with the two-dimensional galvanometer galvanometer. The objective lens faces the sample stage on which the sample is placed, and is used to focus the laser beam onto the sample to excite stimulated Raman scattering signals. The condenser lens is used to collect the stimulated Raman scattered light. The short-pass filter is used to filter out excess Stokes light components in the stimulated Raman scattered light. The two-dimensional galvanometer galvanometer mirror consists of a pair of mutually perpendicular silver-plated mirrors, used for rapid laser scanning of samples; The signal processing unit includes: a silicon photodiode, a lock-in amplifier, a digital acquisition card, and a computer host; Pump light passing through the short-pass filter enters the silicon photodiode, and is demodulated into a digital signal by the lock-in amplifier. The digital signal is acquired by the digital acquisition card and transmitted to the computer host for final processing.

8. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 7, characterized in that, The spectral detection module includes: a 532nm cutoff filter, a sleeve lens, and a Raman spectrometer; The spontaneous Raman laser interacts with the sample molecules at the focal point of the laser scanning microscope. The excited Raman scattered light returns along the original optical path and is reflected by the 650nm long-pass dichroic mirror to the spectral detection module. After passing through the 532nm cutoff filter, the Raman scattered light is retained. The Raman scattered light is focused by the sleeve lens to the Raman spectrometer for Raman spectral detection.

9. The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to claim 8, characterized in that, The femtosecond pulse excitation module includes: a dual-channel synchronous femtosecond laser, a pump optical path power continuously adjustable group, and a Stokes optical path power continuously adjustable group; The dual-channel synchronous femtosecond laser is used to output pump light and Stokes light; The pump optical path power continuous adjustment group includes a pump optical path half-wave plate, a pump optical path polarizing beam splitter, and a pump optical path light block; the pump optical path half-wave plate is used to rotate the polarization angle of the Stokes light; the pump optical path polarizing beam splitter is used to selectively transmit light components with a fixed polarization direction while reflecting light components with other directions; the pump optical path light block is used to block the polarized light reflected by the pump optical path polarizing beam splitter. The Stokes optical path power continuous adjustment group includes a Stokes optical path half-wave plate, a Stokes optical path polarizing beam splitter, and a Stokes optical path light block; the Stokes optical path half-wave plate is used to rotate the polarization angle of the Stokes light, the Stokes optical path polarizing beam splitter is used to selectively transmit light components with a fixed polarization direction and reflect light components with other directions; the Stokes optical path light block is used to block the polarized light reflected by the Stokes optical path polarizing beam splitter.

10. A method for spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis, comprising using the spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system according to any one of claims 1-9, characterized in that, Specifically, the steps include the following: S1. Turn on the dual-channel synchronous femtosecond laser to emit two femtosecond laser beams ω1 and ω2, which are used as pump light and Stokes light, respectively; S2. Modulation of pump light and Stokes light in high wavenumber region: The center wavelengths of the pump light and the Stokes light are tuned to make the excitation band in the high wavenumber region; the femtosecond pulse width is broadened to a picosecond pulse by modulation by the first glass rod of the pump light path and the glass rod of the Stokes light path, respectively. S3. The modulated pump light and Stokes light are combined and then introduced into the laser scanning microscope to excite the SRS effect of the single-cell sample. The components are then dissociated through hyperspectral acquisition and image analysis algorithms to obtain the component distribution image of proteins and lipids in the single cell. S4. Modulation of pump light and Stokes light in fingerprint region: The center wavelengths of the pump light and the Stokes light are tuned so that the excitation band is in the fingerprint region; the pump light is modulated by the first glass rod and the second glass rod of the pump light path, and the Stokes light is modulated by the glass rod of the Stokes light path to broaden the femtosecond pulse width to a picosecond pulse. S5. The modulated pump light and Stokes light are combined and introduced into the laser scanning microscope to excite the SRS effect of the single-cell sample in the fingerprint area. By changing the time delay, the spectrum is focused on the nucleic acid molecular peak, and label-free imaging of the single-cell nucleic acid is performed to obtain a high-resolution image of the component distribution of nucleic acid in the single cell. S6. Combine high-resolution images of nucleic acid composition distribution in single cells with images of protein and lipid composition distribution in single cells to construct a pseudo-color single-cell structure map; S7. Turn off the dual-path synchronous femtosecond laser, turn on the spontaneous Raman laser, and use a 532nm long-pass dichroic mirror to make the laser and the original femtosecond laser optical path spatially consistent, and incident on the sample to be tested, converting the image pixel information into the X-axis voltage and Y-axis voltage of the two-dimensional galvanometer galvanometer mirror to achieve co-positioning control; S8. By changing the voltage of the two-dimensional galvanometer galvanometer mirror and moving the laser focus point to the single-cell substructure region to be tested, and sampling the spectrum of the single-cell substructure region to be tested using a Raman spectrometer, colocalization images and spectral composite information are obtained; S9. The spectral composite information is used for spectral identification and accuracy determination through spectral analysis methods.

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