Spontaneous-stimulated Raman co-localization single cell substructure atlas analysis system and method

Abstract

The invention relates to the technical field of spectral imaging detection, in particular to a spontaneous-stimulated Raman co-localization single-cell substructure atlas analysis system and a spontaneous-stimulated Raman co-localization single-cell substructure atlas analysis method. The system comprises a femtosecond pulse excitation module used for outputting two beams of broadband femtosecond pulse laser with different frequencies and continuously adjustable optical power; the Stokes light modulation module is used for modulating the Stokes light and carrying out space beam combination on the Stokes light and the pump light; the pump light modulation module is used for modulating pump light; the spontaneous Raman laser continuous adjusting module is used for outputting spontaneous Raman laser; the stimulated Raman exciting light co-positioning module is used for combining the combined laser and the spontaneous Raman laser again, focusing the combined laser and the spontaneous Raman laser on a sample, exciting a stimulated Raman scattering signal, converting the stimulated Raman scattering signal into a digital signal and carrying out data acquisition; and the spectrum detection module is used for collecting the spontaneous Raman scattering light which is reflected and returned along the original light path, and collecting a spectrum signal to form a Raman spectrum. The method has the advantage of improving the accuracy of single-cell heterogeneity structure and cancer cell type discrimination.

Description

Technical Field

[0001] The present invention relates to the technical field of spectral imaging detection, and in particular to a spontaneous-stimulated Raman co-localized single cell substructure spectrum analysis system and method. Background Art

[0002] In the progression of cellular carcinogenesis, factors such as genetic variation, metabolic alterations, and imbalances in cell proliferation and apoptosis all play a significant role. Different types of cancer also develop differently, often using multi-omics approaches for classification and tiered diagnosis and treatment. Raman spectroscopy, which detects molecular vibrational energy, is playing an increasingly important role in biomedical research, including multi-component analysis of cellular metabolism and diagnosis of cancer and its subtypes, due to its non-invasive and highly chemically specific properties. Numerous results have revealed differences between cancer cells and normal cells in terms of nucleic acid and protein content and types, as well as metabolites. Spontaneous Raman scattering microscopy allows for the full spectrum of complex molecules within a focal spot to be detected simultaneously. Currently, the focused Raman spot diameter is typically around one micron, while cell diameters are on the order of ten microns. Furthermore, the structural distribution of molecules within cells varies significantly, and spontaneous Raman scattering spectra are significantly affected by these differences in cellular structure. Current white-light microscopy, combined with spontaneous Raman scattering, struggles to image cellular structure without staining, and staining dyes can significantly interfere with Raman spectral acquisition. Therefore, current cell microscopic Raman identification techniques often rely on multiple blind measurements within cells and averaging to obtain Raman spectra that characterize cells. However, weak scattering efficiency limits Raman studies on single-cell heterogeneity, which is manifested in low efficiency in detecting trace single-cell components. Due to the influence of single-cell sample heterogeneity, this blind detection method reduces measurement efficiency and accuracy, is prone to cell photodamage, and the average spectral information masks the significant differences in cells in specific structural components, resulting in low detection accuracy.

[0003] Stimulated Raman scattering (SRS) is an avalanche-like collision process between photons and excited phonons, which stimulates the amplification of Raman signals and enables rapid spectral imaging analysis. The spectral detection range and spectral resolution of SRS are determined by the properties of the two excitation spectral bands. Femtosecond pulses with higher instantaneous power are ideal excitation light sources for SRS. However, the time-frequency characteristics of femtosecond pulses limit the detection band range of current spectrally focused stimulated Raman technology to around 300 wavenumbers, and the spectral resolution is difficult to meet the spectral resolution requirements of cell fingerprint detection below 10 wavenumbers. Achieving high spectral resolution and multi-band detection is the main research direction of SRS. The main method is to tune the central wavelength of the incident femtosecond laser to achieve Raman frequency shift detection in different bands, from the fingerprint region to the high wavenumber region.

[0004] 2800-3100cm -1 The high-wavenumber region of the spectrum exhibits intense CH molecular vibrations, and SRS typically targets this region to acquire information about components such as proteins and several lipids within single cells or tissues. However, due to the single peaks and high degree of crossover in this high-wavenumber region, the Raman peaks of major cellular components, such as nucleic acids, are obscured, leaving the fingerprint region rich in Raman molecular vibrations. SRS targeting the fingerprint region and combining it with high-wavenumber imaging is expected to separate single-cell nucleic acids from proteins and lipids, and to map the complete structural composition of a single cell. Dual-band SRS imaging can achieve an effect similar to brightfield staining and guide the cell Raman spectrometer to accurately acquire fingerprint spectral information about subcellular structures. Compared to blind Raman spectroscopy, this is expected to improve spectral acquisition efficiency and enhance the accuracy of studies on biological sample heterogeneity. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a spontaneous-stimulated Raman co-localized single cell substructure atlas analysis system and method.

[0006] The first object of the present invention is to provide a spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system, comprising:

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

[0008] a Stokes light modulation module, configured to modulate the Stokes light and spatially combine it with the pump light;

[0009] A pump light modulation module, used for modulating the pump light;

[0010] A spontaneous Raman laser continuous adjustment module for outputting spontaneous Raman laser;

[0011] Stimulated Raman excitation light co-localization module, including a spectrum scanning unit and a signal processing unit;

[0012] The spectrum scanning unit is used to combine the combined laser beam with the spontaneous Raman laser beam and focus the combined laser beam on the sample to stimulate stimulated Raman scattering signals; the signal processing unit is used to convert the stimulated Raman scattering signals into digital signals and perform data acquisition;

[0013] The spectral detection module is used to collect the spontaneous Raman scattered light that is reflected and returned along the original optical path, and to collect the spectral signal to form a Raman spectrum.

[0014] Preferably, the Stokes light modulation module comprises: a Stokes optical path glass rod, a time delay line, a first lens, an acousto-optic modulator, a beam splitting light block, a second lens, a third reflector, a short-pass dichroic mirror, a radio frequency driver, and a function generator;

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

[0016] The time delay line is composed of an electric displacement platform equipped with a pair of mutually perpendicular plane mirrors. The electric displacement platform can move back and forth along the arrow direction and change the relative optical path difference between the pump light and the Stokes light.

[0017] Preferably, a plurality of reflectors are provided in the Stokes light modulation module for changing the propagation direction of the light path to meet the spatial arrangement of the light path.

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

[0019] The first pump light path dichroic mirror and the second pump light path dichroic mirror are perpendicular to each other; the pump light stretches the femtosecond pulse width to a picosecond pulse after passing through the first glass rod of the pump light path, and is incident on the first pump light path dichroic mirror and the second pump light path dichroic mirror in sequence.

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

[0021] The central wavelength of the pump light is set to 897 nm. After passing through the first glass rod of the pump light path, the pump light widens the femtosecond pulse width to a picosecond pulse and is incident on the first pump light path dichroic mirror. After being reflected by the silver reflector, the pump light 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 loss due to the wavelength change. The laser light compensated by the second glass rod of the pump light path then passes through the second pump light path dichroic mirror to realize the modulation of the pump light.

[0022] Preferably, the spontaneous Raman laser continuous adjustment module includes: a spontaneous Raman laser, a half-wave plate, a polarization beam splitter prism, and a light block;

[0023] The half-wave plate is used to rotate the polarization angle of the initial laser line emitted by the spontaneous Raman laser; 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 light shielding baffle is used to block the polarized light reflected by the polarization beam splitter prism.

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

[0025] The combined laser beam passes through the 650nm long-pass dichroic mirror and then passes through the 532nm long-pass dichroic mirror together with the laser beam emitted by the spontaneous Raman laser continuous modulation module, and then is incident on the laser scanning microscope equipped with the two-dimensional galvanometer oscillator. The objective lens is directly opposite the sample stage where the sample is placed, and is used to focus the laser beam onto the sample to stimulate the stimulated Raman scattering signal. The condenser is used to collect the stimulated Raman scattered light. The short-wave pass filter is used to filter out excess Stokes light components in the stimulated Raman scattered light.

[0026] The two-dimensional galvanometer mirror is composed of a pair of mutually perpendicular silver-plated mirrors and is 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] The pump light passing through the short-wave pass filter enters the silicon photodiode and is demodulated into a digital signal by the lock-in amplifier; the digital signal is collected by the digital acquisition card and transmitted to the computer host, which performs final processing.

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

[0030] The 532nm excitation light interacts with the sample molecules at the focus of the microscope, and the Raman scattered light excited by it returns along the original optical path, is reflected by the 650nm long-pass dichroic mirror to the spectral detection module, and is retained after passing through the 532nm cut-off filter; the Raman scattered light is converged by the sleeve lens to the Raman spectrometer for Raman spectrum detection.

[0031] Preferably, the femtosecond pulse excitation module includes: a dual-path synchronous emission femtosecond laser, a pump light path power continuous adjustment group, and a Stokes light path power continuous adjustment group;

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

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

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

[0035] A second object of the present invention is to provide a method for analyzing the substructure of a single cell by spontaneous-stimulated Raman co-localization. The method uses a spontaneous-stimulated Raman co-localization substructure analysis system for single cell analysis, and specifically comprises the following steps:

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

[0037] S2. Modulating the high-wavenumber pump and Stokes light: Tuning the central wavelengths of the pump and Stokes light so that the excitation wavelength is in the high-wavenumber region; modulating the first glass rod in the pump and Stokes light paths, respectively, to stretch the femtosecond pulse width to a picosecond pulse;

[0038] S3. The modulated pump light and Stokes photosynthesis beam are combined and introduced into a laser scanning microscope to stimulate the SRS effect in single-cell samples. Hyperspectral acquisition and image analysis algorithms are then used to decompose the components and obtain an image of the protein and lipid composition distribution within the single cell.

[0039] S4. Modulating the pump light and Stokes light in the fingerprint region: Tuning the central wavelengths of the pump light and the Stokes light so that the excitation wavelength band is in the fingerprint region; modulating the pump light via the first and second glass rods in the pump light path, and modulating the Stokes light via the glass rods in the Stokes light path, thereby stretching the femtosecond pulse width to a picosecond pulse;

[0040] S5. The modulated pump light and Stokes light beam are combined and introduced into a laser scanning microscope to stimulate the SRS effect of the single-cell sample in the fingerprint region. By varying the time delay, the spectrum is focused on the peak of the nucleic acid molecule, allowing label-free imaging of the single-cell nucleic acid, resulting in a high-resolution image of the composition distribution of the nucleic acid within the single cell.

[0041] S6. Combine the high-resolution composition distribution images of nucleic acids within a single cell with the composition distribution images of proteins and lipids within a single cell, and construct a pseudo-color single-cell structure map;

[0042] S7. Turn off the dual synchronously emitting femtosecond lasers and turn on the spontaneous Raman laser. The laser beam is then aligned with the original femtosecond laser beam path via a 532nm long-pass dichroic mirror and incident on the sample to be measured. The image pixel information is converted into X-axis and Y-axis voltages for the two-dimensional galvanometer mirror to achieve co-localization control.

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

[0044] S9. Perform spectrum recognition and accuracy judgment on the spectral composite information through a spectral analysis method.

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

[0046] The present invention provides an imaging and spectral analysis system for the co-localization of spontaneous Raman and stimulated Raman. By using this analysis system, the accuracy of distinguishing the heterogeneous structure of single cells and the types of cancer cells can be improved.

[0047] Label-free SRS imaging achieves a staining-like effect, assisting spontaneous Raman in the targeted detection of single-cell substructures of interest, improving Raman detection efficiency and analysis accuracy. Compared to existing spontaneous Raman or stimulated Raman microscopy, the spontaneous-stimulated Raman co-localized single-cell substructure mapping method provided by this invention achieves a measurement accuracy of 93.2%. This method further enhances the ability to capture biological information and has the potential for in situ diagnosis of clinical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagram of the logical structure of the spontaneous-stimulated Raman co-localized single-cell substructure atlas analysis system provided according to an embodiment of the present invention.

[0049] Figure 2 Schematic diagram of the optical path of the spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system provided according to an embodiment of the present invention.

[0050] Figure 3 According to the embodiment of the present invention / the specific implementation mode of the present invention;

[0051] Figure 4 3 is a comparison chart of the co-localization test results provided by an embodiment of the present invention and the results of the traditional Raman spectroscopy blind test method.

[0052] Reference numerals:

[0053] 1. Femtosecond pulse excitation module;

[0054] 11. Dual-path synchronously emitting femtosecond laser; 12. Pump optical path half-wave plate; 13. Pump optical path polarization beam splitter prism; 14. Pump optical path light block; 15. Stokes optical path half-wave plate; 16. Stokes optical path polarization beam splitter prism; 17. Stokes optical path light block;

[0055] 2. Stokes optical modulation module;

[0056] 201, first reflector; 202, Stokes optical path glass rod; 203, time delay line; 204, second reflector; 205, first lens; 206, acousto-optic modulator; 207, beam splitter; 208, second lens; 209, third reflector; 210, 1000 nm short-pass dichroic mirror; 211, RF driver; 212, function generator;

[0057] 3. Pump light modulation module;

[0058] 31. First glass rod of the pump light path; 32. First dichroic mirror of the pump light path; 33. Second dichroic mirror of the pump light path; 34. Fourth reflector; 35. Second glass rod of the pump light path; 36. Fifth reflector;

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

[0060] 41. Spontaneous Raman laser; 42. Half-wave plate; 43. Polarization beam splitter prism; 44. Light shield;

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

[0062] 51. 650nm long-pass dichroic mirror; 52. 532nm long-pass dichroic mirror; 53. Sixth reflector; 54. Two-dimensional galvanometer galvanometer; 55. Objective lens; 56. Sample stage; 57. Condenser; 58. Seventh reflector; 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 reflector; 72. 532nm cutoff filter; 73. Tube lens; 74. Raman spectrometer. DETAILED DESCRIPTION

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

[0066] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with 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 of the present invention.

[0067] Figure 1-2 The spontaneous-stimulated Raman co-localization single-cell substructure mapping system is shown, including:

[0068] A femtosecond pulse excitation module 1 is configured to output two beams of broadband femtosecond pulse lasers with different frequencies and continuously adjustable optical power. The femtosecond pulse excitation module 1 comprises a dual-path synchronously emitting femtosecond laser 11, a pump optical path power continuous adjustment group, and a Stokes optical path power continuous adjustment group. The dual-path synchronously emitting femtosecond laser 11 is configured to output two beams of linearly polarized broadband femtosecond pulse lasers with different frequencies, with the high-frequency femtosecond pulse laser serving as the pump light and the low-frequency femtosecond pulse laser serving as the Stokes light.

[0069] The pump light path power continuous adjustment group and the Stokes light path power continuous adjustment group have the same structure; the pump light path power continuous adjustment group includes a pump light path half-wave plate 12, a pump light path polarization splitter prism 13, and a pump light path light block 14; the Stokes light path power continuous adjustment group includes a Stokes light path half-wave plate 15, a Stokes light path polarization splitter prism 16, and a Stokes light path light block 17; the pump light path half-wave plate 12 and the Stokes light path half-wave plate 15 respectively rotate the initial 532nm laser linear polarization direction by a specific angle, and rotate the linear polarization direction of the pump and Stokes light; the pump light path polarization splitter prism 13 and the Stokes light path polarization splitter prism 16 are used to transmit the light component in the horizontal polarization direction and reflect the light component in the vertical direction; the pump light path light block 14 and the Stokes light 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 split the linearly polarized light incident on its surface into two orthogonal directions, take the horizontally polarized light that passes through the polarization splitter prism, and continuously adjust the power of the horizontally polarized light by continuously rotating the half-wave plate.

[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 splitting light block 207, a second lens 208, a third reflector 209, a 1000 nm short-pass dichroic mirror 210, a radio frequency driver 211, and a function generator 212. After the Stokes light selective output component is reflected by the first reflector 201, it passes through the Stokes optical path glass rod 202 to widen the femtosecond pulse width to a picosecond pulse and is incident on the time delay line 203. The time delay line 203 consists of a pair of mutually perpendicular planes. The electromotive displacement platform of the surface reflector can be reciprocated and translated in 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 performs high-frequency periodic modulation on the Stokes light through the function generator 212 and the RF driver 211, emitting zero-order light and first-order light. The zero-order light is blocked by the beam splitter 207 (preventing the zero-order light from propagating and affecting 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, where the modulated Stokes light and the pump light are spatially combined.

[0072] In a specific embodiment, the Stokes light modulation module 2 is provided with a plurality of reflectors, which are mainly used to change the propagation direction of the light path 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-wavelength pass dichroic mirror. If the pump light and the Stokes light are exchanged, the dichroic mirror needs to correspond to a long-wavelength pass dichroic mirror to meet the actual requirements.

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

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

[0075] High-wavenumber region SRS imaging system: The initial broadband femtosecond pulse pump and Stokes light center wavelengths are set to 802 and 1045 nm, respectively. According to the Raman shift calculation formula The initial spectrum width of the femtosecond pulse tested by the fiber spectrometer is 2800-3100 cm-1. -1 ;

[0076] Fingerprint area SRS imaging system: Tune the pump light center wavelength to 897nm. Similarly, the fingerprint area 1500~1700cm -1 Raman frequency shift.

[0077] The hyperspectral SRS imaging system with co-localization of the fingerprint region and the high wavenumber region can achieve the following functions: protein and lipid imaging in single cells can be acquired through the high wavenumber region, and the fingerprint region spectrum can be focused on 1578 cm -1 Nucleic acid molecules are imaged, and through co-localization image combination, three different color imaging channels are combined to form a staining-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 polarization beam splitter prism 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, and the polarization beam splitter prism 43 polarizes the linear polarized light incident on its surface into two orthogonal directions, takes the horizontal polarized light transmitted through the polarization beam splitter prism, and blocks the vertical polarized light reflected by the polarization beam splitter prism 43 through the light shielding baffle 44; the power of the horizontal polarized light can be continuously adjusted by continuously rotating the half-wave plate 42.

[0079] Stimulated Raman excitation light co-localization module 5, including a spectrum 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 reflector 53, a two-dimensional galvanometer galvanometer mirror 54, an objective lens 55, a sample stage 56, a condenser 57, a seventh reflector 58, and a short-pass filter 59. After the combined laser light passes through the 650nm long-pass dichroic mirror 51, it is combined with the laser light emitted by the spontaneous Raman laser continuous adjustment module 4 through the 532nm long-pass dichroic mirror 52, and then reflected by the sixth reflector 53 to a laser scanning microscope equipped with a two-dimensional galvanometer galvanometer mirror 54. The two-dimensional galvanometer galvanometer mirror 54 is composed of a pair of mutually perpendicular silver-plated reflectors and is used for rapid laser scanning of samples. The objective lens 55 faces the sample stage 56 on which the sample is placed, and is used to converge the laser light onto the sample to stimulate stimulated Raman scattering signals. The condenser 57 is used to collect stimulated Raman scattered light. The short-wave pass filter 59 is used to filter out excess Stokes light components in the stimulated Raman scattered light.

[0081] The signal processing unit includes a silicon photodiode 61, a phase-locked amplifier 62, a digital acquisition card 63 and a computer host 64; the pump light passes through the short-wave pass filter 59 and enters the silicon photodiode 61. After the phase-locked amplifier 62 demodulates the digital signal, the digital signal is collected by the digital acquisition card 63 and transmitted to the computer host 64 for final processing.

[0082] The stimulated Raman excitation light co-localization module 5 includes two dichroic mirrors. The stimulated Raman excitation light is above 680nm, and the Raman frequency shift of the molecule is excited by the 532nm excitation light, which is within the single cell vibration range (400-4000cm -1 ), the Raman shift calculation formula indicates that the maximum wavelength of Raman scattered light does not exceed 650nm. The module's two dichroic mirrors—a 532nm long-wavelength dichroic mirror and a 650nm long-wavelength dichroic mirror—are used to separate the four frequency components: the stimulated Raman pump and Stokes light, the 532nm Raman excitation light, and the Raman scattered light between 532 and 650nm.

[0083] Spectral detection module 6, including a silver reflector 71, a 532nm cutoff filter 72, a tube lens 73, and a Raman spectrometer 74;

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

[0085] Spectral detection module 6 consists of a Raman cutoff filter, a tube lens, and a Raman spectrometer. The Raman cutoff filter, based on the excitation light wavelength, selects a 532nm cutoff filter to completely filter out the 532nm excitation light, allowing only the Raman scattered light to pass through. After passing through the tube lens, the Raman scattered light is incident on the slit of the Raman spectrometer, where it is split and the spectral signal is collected to form a Raman spectrum.

[0086] Example 1

[0087] This embodiment provides a method for analyzing the substructure of a single cell by spontaneous-stimulated Raman colocalization (see flowchart for details). Figure 3 ),use Figure 1-2 The spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system shown in the figure is analyzed. The specific steps are as follows:

[0088] S1. Turn on the dual-path synchronous emission femtosecond laser 11 to emit two femtosecond laser beams ω1 and ω2, which serve as the pump light and Stokes light of the SRS system respectively. The initial pulse widths of the two are τ 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 central wavelength that is tunable within a certain range. According to the Raman shift formula:

[0089]

[0090] The pump and Stokes light center wavelengths can be tuned to locate the SRS spectrum detection area.

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

[0092] S201. Tuning the pump and Stokes light center wavelengths, calculated by the Raman shift formula, the corresponding excitation wavelength is between 2800 and 3100 cm -1 , this region is called the Raman high wavenumber region.

[0093] S202.pump and Stokes light travel through lengths l p and l S The chirped medium (the first glass rod 31 in the pump optical path and the glass rod 202 in the Stokes optical path) has its pulse width broadened 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 that the light passes through, and the product of the two is the group delay (GDD) of the material to the pulse light;

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

[0097]

[0098] Where τ0 is the initial pulse width. According to the linear chirp parameter formula, the relationship between the chirped medium length required by the pump and Stokes for linear chirp matching can be obtained.

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

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

[0101] ΔΩ SRS =(S p / F)*(S S / F).

[0102] After the S3.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 stimulate the SRS effect of single-cell samples. Components are separated through hyperspectral acquisition and image analysis algorithms to obtain a component distribution image of proteins and lipids within single cells.

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

[0104] S401. Tune the center wavelengths of the pump and Stokes light, and calculate by the Raman shift formula that the wavelength band excited by the two is within the cell Raman fingerprint region.

[0105] S402.pump and Stokes light travel through a path of length l p′ and l S′ , chirped medium, the same as steps S202 to S204, calculate the pulse width, instantaneous bandwidth and spectral resolution of the two in the fingerprint area of ​​the system.

[0106] After the S5.pump and Stokes lights are spatiotemporally combined by a 1000nm short-pass dichroic mirror 210, they are jointly introduced into a laser scanning microscope to stimulate the SRS effect in the fingerprint region of the single-cell sample. By varying the time delay, the spectrum is focused on the spectral peak of the nucleic acid molecule, allowing label-free imaging of the single-cell nucleic acid to be performed, resulting in a high-definition component distribution image of the nucleic acid within the single cell.

[0107] S6. Combine high-resolution component distribution images of nucleic acids within single cells and component distribution images of proteins and lipids within single cells to construct a pseudo-color single-cell structure map.

[0108] S7. Turn off the dual-path synchronously emitting femtosecond laser 11 and turn on the spontaneous Raman laser 41. The laser is incident on the sample to be tested through the 532nm long-pass dichroic mirror 52 to ensure that the laser light path is spatially consistent with the original femtosecond laser light path. The laser light is then incident on the sample to be tested. Co-localization control is achieved by converting the image pixel information into the X and Y axis voltages in the two-dimensional galvanometer galvanometer mirror 54.

[0109] S8. By changing the voltage of the two-dimensional galvanometer oscillator 54 and moving the laser focus to the single-cell substructure area of ​​interest, and sampling the spectrum of this point through the Raman spectrometer 74, a co-localization image and spectral composite information are obtained.

[0110] S9. The spectral composite information obtained by sampling is subjected to spectral recognition and accuracy judgment through spectral analysis method.

[0111] Figure 4 The results of the analysis method of the present invention and the traditional Raman spectroscopy blind test method are compared; for tests on DU145 and SiHa cell lines, the traditional Raman spectroscopy blind test method, through the PCA-LDA algorithm, has a discrimination accuracy of 84.1% after ten-fold cross validation, while the colocalization test method gives a measurement accuracy of 93.2%.

[0112] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in the present disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in the present disclosure can be achieved. This is not limited herein.

[0113] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may be made based on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. Spontaneous-stimulated Raman co-localization single-cell substructure mapping analysis system, characterized by: include: Femtosecond pulse excitation module, used to output two beams of broadband femtosecond pulse lasers with different frequencies and continuously adjustable optical power, using the high-frequency linearly polarized broadband femtosecond pulse laser as pump light and the low-frequency linearly polarized broadband femtosecond pulse laser as Stokes light; a Stokes light modulation module, configured to modulate the Stokes light and spatially combine it with the pump light; A pump light modulation module, used for modulating the pump light; A spontaneous Raman laser continuous adjustment module for outputting spontaneous Raman laser; Stimulated Raman excitation light co-localization module, including a spectrum scanning unit and a signal processing unit; The spectrum scanning unit is used to combine the combined laser beam with the spontaneous Raman laser beam and focus the combined laser beam on the sample to stimulate stimulated Raman scattering signals; the signal processing unit is used to convert the stimulated Raman scattering signals into digital signals and perform data acquisition; The spectral detection module is used to collect the spontaneous Raman scattered light that is reflected and returned along the original optical path, and to collect the spectral signal to form a Raman spectrum.

2. The spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system according to claim 1, characterized in that: The Stokes light modulation module includes: a Stokes optical path glass rod, a time delay line, a first lens, an acousto-optic modulator, a beam splitting light block, a second lens, a third reflector, a short-pass dichroic mirror, a radio frequency driver, and a function generator; After the selected output component of the Stokes light passes through the Stokes optical path glass rod, the femtosecond pulse width is widened to a picosecond pulse, and the pulse is incident on the time delay line; the acousto-optic modulator performs high-frequency periodic modulation on the Stokes light through the function generator and the radio frequency driver, and emits zero-order light and first-order light; the zero-order light is blocked by the beam splitter, and the first-order light is collimated by the second lens and enters the short-pass dichroic mirror, and the modulated Stokes light and the modulated pump light are spatially combined by the short-pass dichroic mirror; The time delay line is composed of an electric displacement platform equipped with a pair of mutually perpendicular plane mirrors. The electric displacement platform can move back and forth along the arrow direction and change the relative optical path difference between the pump light and the Stokes light.

3. The spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system according to claim 2, characterized in that: A plurality of reflectors are provided in the Stokes light modulation module for changing the propagation direction of the light path to meet the spatial arrangement of the light path.

4. The spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system according to claim 3, characterized in that: The pump light modulation module includes: a first glass rod of the pump light path, a first dichroic mirror of the pump light path, and a second dichroic mirror of the pump light path; The first pump light path dichroic mirror and the second pump light path dichroic mirror are perpendicular to each other; the pump light stretches the femtosecond pulse width to a picosecond pulse after passing through the first glass rod of the pump light path, and is incident on the first pump light path dichroic mirror and the second pump light path dichroic mirror in sequence.

5. The spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system according to claim 4, characterized in that: The pump light modulation module further includes: a second glass rod in the pump light path and a silver reflector; The central wavelength of the pump light is set to 897 nm. After passing through the first glass rod of the pump light path, the pump light widens the femtosecond pulse width to a picosecond pulse and is incident on the first pump light path dichroic mirror. After being reflected by the silver reflector, the pump light 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 loss due to the wavelength change. The laser light compensated by the second glass rod of the pump light path then passes through the second pump light path dichroic mirror to realize the modulation of the pump light.

6. The spontaneous-stimulated Raman co-localized 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 polarization beam splitter prism, and a light block; The half-wave plate is used to rotate the polarization angle of the initial laser line emitted by the spontaneous Raman laser; 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 light shielding baffle is used to block the polarized light reflected by the polarization beam splitter prism.

7. The spontaneous-stimulated Raman co-localized single-cell substructure mapping analysis system according to claim 6, characterized in that: The spectrum scanning unit includes: a 650nm long-pass dichroic mirror, a 532nm long-pass dichroic mirror, a two-dimensional galvanometer oscillator, an objective lens, a sample stage, a condenser, and a short-pass filter; The combined laser beam passes through the 650nm long-pass dichroic mirror and then passes through the 532nm long-pass dichroic mirror together with the laser beam emitted by the spontaneous Raman laser continuous modulation module, and then is incident on the laser scanning microscope equipped with the two-dimensional galvanometer oscillator. The objective lens is directly opposite the sample stage where the sample is placed, and is used to focus the laser beam onto the sample to stimulate the stimulated Raman scattering signal. The condenser is used to collect the stimulated Raman scattered light. The short-wave pass filter is used to filter out excess Stokes light components in the stimulated Raman scattered light. The two-dimensional galvanometer mirror is composed of a pair of mutually perpendicular silver-plated mirrors and is 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; The pump light passing through the short-wave pass filter enters the silicon photodiode and is demodulated into a digital signal by the lock-in amplifier; the digital signal is collected by the digital acquisition card and transmitted to the computer host, which performs final processing.

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

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

10. A method for analyzing a spontaneous-stimulated Raman co-localized single-cell substructure map, comprising: using the spontaneous-stimulated Raman co-localized single-cell substructure map analysis system according to any one of claims 1 to 9, wherein: The specific steps include: S1. Turn on the dual-path synchronous femtosecond laser to emit two femtosecond laser beams ω1 and ω2, which serve as pump light and Stokes light, respectively. S2. Modulating the high-wavenumber pump and Stokes light: Tuning the central wavelengths of the pump and Stokes light so that the excitation wavelength is in the high-wavenumber region; modulating the first glass rod in the pump and Stokes light paths, respectively, to stretch the femtosecond pulse width to a picosecond pulse; S3. The modulated pump light and Stokes photosynthesis beam are combined and introduced into a laser scanning microscope to stimulate the SRS effect in single-cell samples. Hyperspectral acquisition and image analysis algorithms are then used to decompose the components and obtain an image of the protein and lipid composition distribution within the single cell. S4. Modulating the pump light and Stokes light in the fingerprint region: Tuning the central wavelengths of the pump light and the Stokes light so that the excitation wavelength band is in the fingerprint region; modulating the pump light via the first and second glass rods in the pump light path, and modulating the Stokes light via the glass rods in the Stokes light path, thereby stretching the femtosecond pulse width to a picosecond pulse; S5. The modulated pump light and Stokes light beam are combined and introduced into a laser scanning microscope to stimulate the SRS effect of the single-cell sample in the fingerprint region. By varying the time delay, the spectrum is focused on the peak of the nucleic acid molecule, allowing label-free imaging of the single-cell nucleic acid, resulting in a high-resolution image of the composition distribution of the nucleic acid within the single cell. S6. Combine the high-resolution composition distribution images of nucleic acids within a single cell with the composition distribution images of proteins and lipids within a single cell, and construct a pseudo-color single-cell structure map; S7. Turn off the dual synchronously emitting femtosecond lasers and turn on the spontaneous Raman laser. The laser beam is then aligned with the original femtosecond laser beam path via a 532nm long-pass dichroic mirror and incident on the sample to be measured. The image pixel information is converted into X-axis and Y-axis voltages for the two-dimensional galvanometer mirror to achieve co-localization control. S8. By changing the voltage of the two-dimensional galvanometer mirror, moving the laser focus to the single-cell substructure region to be measured, and sampling the spectrum of the single-cell substructure region to be measured using a Raman spectrometer, a co-localization image and spectral composite information are obtained; S9. Perform spectrum recognition and accuracy judgment on the spectral composite information through a spectral analysis method.

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