Stimulated Raman synchronous excitation line scanning spectral imaging system and method

Abstract

The invention relates to the technical field of spectrum detection and analysis, in particular to a stimulated Raman synchronous excitation line scanning spectrum imaging system and method. The system comprises a pulse modulation module, wherein a laser outputs modulation light and detection light which are synchronous but have different center frequencies; the modulated light is divided into two paths, one path generates a plurality of beams of deflected light through an acousto-optic deflector, and the other path interferes with the deflected light after being subjected to frequency deviation through an acousto-optic frequency shifter, and is transmitted to a dichroscope after being combined; after the optical path of the detection light is adjusted by the optical delay line and the detection light is modulated by the binary diffraction element, the detection light and the modulated light interfere and are combined at the dichroscope to realize time-space synchronization; the stimulated Raman signal excitation module is used for focusing three beams of synchronous light on a sample to form a column of equidistant focusing light spots and performing linear scanning; the stimulated Raman signal detection and acquisition module is used for collecting stimulated Raman scattering signals, filtering out omega1 modulation light, detecting omega2 detection light intensity, converting optical signals into electric signals and recording the electric signals, and completing line excitation and sampling.

Description

Technical Field

[0001] The present invention relates to the technical field of spectral detection and analysis, and particularly to a stimulated Raman synchronous excitation line-scanning spectral imaging system and method. Background Art

[0002] Raman spectroscopy is a molecular scattering spectrum, which can reflect the intrinsic vibration information of molecular covalent bonds. Through cell Raman spectroscopy, information on the molecular species and content of cells can be obtained. Single-cell Raman spectroscopy technology has the detection advantages of being label-free and non-destructive, and has important application value in the field of spectral analysis of living cell tissues. Conventional spontaneous Raman signals are weak and the detection speed is slow, which cannot meet the requirements of high-speed cell spectral imaging. In recent years, new stimulated Raman microscopy technologies have promoted the development of high-speed cell Raman spectroscopy imaging instruments. Stimulated Raman significantly improves the scattering efficiency of Raman spectra through third-order nonlinear effects. Two pulsed lasers (pump light Pump and Stokes light Stokes) are used. When the frequency difference between the two is consistent with the vibration frequency of the molecular covalent bond, Pump photons will be significantly converted into Stokes photons. Therefore, the stimulated Raman signal is manifested as the weakening of the Pump light or the enhancement of the Stokes light at a specific frequency difference.

[0003] In the current stimulated Raman imaging system, to eliminate the influence of strong excitation light background noise on Raman signals, high signal-to-noise ratio detection is achieved through high-frequency modulation and lock-in amplification techniques. Specifically, for one of the light paths, an acousto-optic modulator or an electro-optic modulator is used to modulate the intensity of the excitation light at a high frequency (above megahertz), and then the signal amplitude at this frequency in the other light path is detected by a lock-in amplifier. Therefore, the current stimulated Raman imaging system strongly relies on the lock-in amplifier and requires high-speed optoelectronic signal conversion at dozens of megahertz, which limits the use of array detectors. Thus, the current stimulated Raman system adopts a point-by-point excitation and point-by-point detection method, that is, the deflection angle of the excitation light is changed by a two-dimensional galvanometer, focused on different positions in the field of view through a microscope, stimulated Raman is excited point by point, the corresponding optical signals are detected point by point, and the Raman signal at this point is demodulated by a lock-in amplifier, and the field of view is scanned according to a specific path. Subsequently, a two-dimensional image is reconstructed based on the scanning path and the corresponding signals. If large-field imaging is required, after the galvanometer scanning is completed, a two-dimensional translation stage is used to switch the field of view, and finally large-field imaging is achieved through field-of-view stitching. This method is limited by the scanning speed of the galvanometer, especially the scanning speed of the fast axis in the two-dimensional galvanometer. The current single-field imaging speed can reach the level of ten frames per second. Although this point-by-point scanning stimulated Raman spectroscopic imaging technology can meet the imaging requirements of general sliced tissue and cell samples, it faces many challenges when performing millimeter-scale large-field and micron-scale high-resolution Raman spectroscopic microscopy imaging on in vivo biological tissue samples. To ensure the viability of the sample or the time resolution of the observation, tissue imaging usually needs to be completed within a few minutes. However, the current point-by-point scanning technology is limited by device parameters such as the scanning speed of the galvanometer and the repetition frequency of the femtosecond pulsed laser, resulting in mutual constraints among the imaging speed, field-of-view range, resolution, and integration time. In this case, it is often necessary to sacrifice one parameter to meet other requirements, making it difficult for the current technical solution to meet the application requirements of large-field and high-speed spectroscopic imaging of in vivo tissues. Summary of the Invention

[0004] To solve the above problems, the present invention provides a stimulated Raman synchronous excitation line-scanning spectroscopic imaging system and method.

[0005] The first object of the present invention is to provide a stimulated Raman synchronous excitation line-scanning spectroscopic imaging system, including: a pulse modulation module, a stimulated Raman signal excitation module, and a stimulated Raman signal detection and acquisition module; The pulse modulation module includes: a laser, a first optical path adjustment group, a second optical path adjustment group, and a dichroic mirror; the laser is used to output two beams of pulsed lasers that are synchronous but have different central frequencies, denoted as ω1 and ω2 respectively, one beam is the modulation light, and the other beam is the detection light; the first optical path adjustment group includes a beam splitter, an acousto-optic deflector, an acousto-optic frequency shifter, a binary diffraction element, and a dichroic mirror; the modulation light is split into two paths by the beam splitter, one path passes through the acousto-optic deflector to generate multiple deflected beams, and the other path is frequency-shifted by the acousto-optic frequency shifter and then interferes with the deflected beams, and after merging, it is transmitted to the dichroic mirror; the second optical path adjustment group includes an optical delay line and a binary diffraction element; the detection light is modulated by adjusting the optical path through the optical delay line and the binary diffraction element, and then interferes and merges with the modulation light at the dichroic mirror to achieve spatio-temporal synchronization; The stimulated Raman signal excitation module includes two groups of 4-f lens groups, a one-dimensional scanning galvanometer, an objective lens, and a two-dimensional translation stage; the three synchronous beams output by the pulse modulation module are projected onto the mirror plane of the one-dimensional scanning galvanometer after passing through a group of 4-f lens groups, and then the beams are coupled to the objective lens through another group of 4-f lens groups, and the objective lens focuses the beams on the sample plane on the two-dimensional translation stage to form a series of equally spaced focused spots; the one-dimensional scanning galvanometer is used to perform linear scanning on the sample plane, and the scanning direction is perpendicular to the beam arrangement direction; The stimulated Raman signal detection and acquisition module includes a condenser lens, a filter, a photodiode detector, and a computer; the molecules at each focused spot generate stimulated Raman scattering with the excitation light, and the scattered signals are collected by the condenser lens, and the collected signals are filtered by the filter to remove the modulation light with the original central frequency of ω1; the photodiode detector detects the intensity of the excitation light with the central frequency of ω2, and after converting the collected optical signal into an electrical signal, it is quickly recorded by the computer to complete the primary line excitation and sampling of the stimulated Raman signal of the sample.

[0006] Preferably, the frequency ω1 is a fixed value; the frequency ω2 is tunable, and by changing the value of ω2, the Raman frequency shift |ω2 - ω1| detected by the stimulated Raman synchronous excitation line scanning spectroscopic imaging system is changed; When the modulation light is the pump light, the detection light is the Stokes light; or when the modulation light is the Stokes light, the detection light is the pump light.

[0007] Preferably, the first optical path adjustment group includes a first beam splitter, an acousto-optic deflector, an acousto-optic frequency shifter, a first binary diffraction element, a second beam splitter, and a dichroic mirror; A laser beam with a central frequency of ω1 is incident on the first beam splitter and is divided into two beams with the same intensity; one beam is reflected by the first mirror and then enters the acousto-optic deflector, causing the incident laser to simultaneously generate different deflection angles, and the beam is divided into N beams; the other beam of excitation light passing through the first beam splitter enters the acousto-optic frequency shifter, and after being modulated by the first binary diffraction element for the excitation light, the laser generates the same N beams with an angle of θ as the light passing through the acousto-optic deflector i of the deflected light, i = 1, 2, ……, N; the two sets of deflected beams passing through the acousto-optic deflector and the first binary diffraction element interfere at the beam splitting plane of the second beam splitter, spatially coincide, and continue to be transmitted to the dichroic mirror; at this time, the intensity of each transmitted light after interference is as follows: ; In the formula: I represents the intensity of the beam after interference; a is the amplitude of the two laser beams; t is the time; ω si represents the ith modulation frequency in the modulation frequency comb signal of the acousto-optic deflector, and the deflection angle is θ i of the light, and the frequency of the light generates an offset of ω si ; ω L represents the single-frequency signal of the acousto-optic frequency shifter, and the light passing through the acousto-optic frequency shifter will generate a frequency offset of ω L ; ω si -ω L represents the frequency difference between the ith deflected beam and the frequency-shifted beam; after interference, the intensity of the beam with a deflection angle of θ i appears with a periodic modulation of the intensity corresponding to a frequency of ω si -ω L .

[0008] Preferably, the second optical path adjustment group includes a mirror, an optical delay line, and a second binary diffraction element; a beam of light with a central frequency of ω2 emitted by the laser is reflected by the mirror, and after the optical path is adjusted by the optical delay line, it is reflected by the mirror and modulated by the second binary diffraction element, and is divided into N beams with an angle of θ i ; i = 1, 2, ……, N; The beam output by the second optical path adjustment group and each intensity-modulated beam with a central frequency of ω1 output by the first optical path adjustment group pass through the dichroic mirror, respectively generating reflected light and transmitted light, and spatially coincide.

[0009] Preferably, the beam splitter is a 50:50 beam splitter; The objective lens is installed on the piezoelectric actuator, and the piezoelectric actuator is used to drive the objective lens to perform sub-micron fine movement along the optical axis direction to realize stimulated Raman spectroscopy imaging of different axial position planes.

[0010] Preferably, in the stimulated Raman signal detection and acquisition module, the specific method for the primary line excitation and sampling of the sample stimulated Raman signal is as follows: Molecules at each focused light spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by a condenser lens. After collection, the signals pass through a filter to filter out the modulation light with a central frequency of ω1. The sum of the detection light signals with a central frequency of ω2 is detected and collected using a photodiode detector. After converting the optical signal into an electrical signal, the electrical signal contains a series of electrical signals with all periodic intensity variations at a frequency of ω si -ω L ; then perform a Fourier transform on this electrical signal to extract all signal intensity values corresponding to the frequency of ω si -ω L . The stimulated Raman signals corresponding to the light spots at N positions in the column are in one-to-one correspondence with the signal intensity at the frequency of ω si -ω L , and image reconstruction is performed according to the position to obtain a column of image signals, where i = 1, 2,..., N; The calculation method of the electrical signal sampling time t is shown in the following formula: ; In the formula: k is a positive integer, ω sN and ω s1 are respectively the maximum and minimum frequencies in the frequency comb signal input to the acousto-optic deflector, and N is the number of generated focused light spots.

[0011] The second object of the present invention is to provide a stimulated Raman synchronous excitation line scanning spectroscopic imaging method, which adopts a stimulated Raman synchronous excitation line scanning spectroscopic imaging system, specifically including the following steps: S1. The laser generates two beams of synchronous but different-frequency pulsed lasers, namely modulation light and detection light; the modulation light is divided into two paths by a beam splitter. One path generates multiple deflected light beams through an acousto-optic deflector, and the other path is frequency-shifted by an acousto-optic frequency shifter and then interferes with the deflected light beams. After merging, they are transmitted to a dichroic mirror; after the detection light adjusts the optical path through an optical delay line, it interferes and merges with the modulation light at the dichroic mirror to achieve spatio-temporal synchronization; S2. The three synchronized light beams pass through the first 4-f lens group and are projected onto the mirror plane of a one-dimensional scanning galvanometer, and then pass through the second 4-f lens group to couple the light beams into an objective lens. The objective lens focuses the light beams on the sample plane of a two-dimensional translation stage to form a column of equally spaced focused light spots; S3. Molecules at each focused light spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by a condenser lens, and after passing through a filter to filter out the modulation light with a central frequency of ω1, the optical signal is received by a photodiode detector and converted into an electrical signal; the electrical signal is quickly recorded by an acquisition card on a computer to complete the primary line excitation and sampling of the sample stimulated Raman signal; S4. Perform Fourier transform on the collected electrical signals, or process the electrical signals using a multi-channel lock-in amplifier to extract the signal intensities at all frequencies ω si -ω L The stimulated Raman signals corresponding to the light spots at N positions in the column are in one-to-one correspondence with the signal intensities at ω si -ω L frequencies, and image reconstruction is performed according to the positions to obtain a column of image signals; i = 1, 2,..., N; S5. Use a one-dimensional scanning galvanometer to scan along the Y direction to gradually complete the stimulated Raman line-scanning spectroscopy imaging of the two-dimensional image; use a two-dimensional translation stage to move the sample along the XY direction to complete the field-of-view switching and stitching to achieve large-field imaging of the plane; drive the objective lens to move along the Z-axis direction through a piezoelectric displacement device to achieve the stimulated Raman spectroscopy imaging of different axial-position planes; S6. Gradually perform field-of-view scanning and stitching to finally achieve label-free three-dimensional stimulated Raman line-scanning imaging of the sample.

[0012] Preferably, step S1 specifically includes the following sub-steps: S101. Use a laser to generate two pulses of laser light that are synchronized but have different central frequencies, namely the modulation light and the detection light, with frequencies ω1 and ω2 respectively; where ω1 is a fixed value and ω2 is tunable; the modulation light is frequency-modulated, and the detection light is used for subsequent signal detection; S102. Modulation optical path adjustment: The laser beam with a central frequency of ω1 is incident on the first beam splitter and is divided into two beams with the same intensity; one beam enters the acousto-optic deflector; under the drive of an equally spaced frequency comb signal, the acousto-optic deflector causes the incident laser to generate different deflection angles θi, and the beam is divided into N beams; the other ω1 excitation beam passing through the first beam splitter enters the acousto-optic frequency shifter, and the acousto-optic frequency shifter shifts the frequency of the incident laser to ω1 + ω L ; the deflected beam passes through the first binary diffraction element for modulation to make the laser generate N deflected lights with the same angle θi as the light passing through the acousto-optic deflector; the two groups of deflected beams interfere at the second beam splitter, making the transmitted light and the reflected light coincide in space and continue to be transmitted to the dichroic mirror; S103. Detection optical path adjustment: One beam of light with a central frequency of ω2 emitted by the laser is reflected by the second mirror and then passes through an optical delay line for optical path adjustment. After modulation by the second binary diffraction element, the adjusted beam is divided into N beams with an angle of θi; the beam output by the second optical path adjustment group interferes with the beam output by the first optical path adjustment group at the dichroic mirror and is combined into one beam and continues to propagate; where i = 1, 2,..., N.

[0013] Preferably, the objective lens is mounted on a piezoelectric translator, and the piezoelectric translator is used to drive the objective lens to perform fine movement at the sub-micron level along the optical axis direction, so as to realize stimulated Raman spectroscopy imaging of different axial position planes; In step S3, the electrical signal is quickly recorded by an acquisition card on the computer.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects: In the imaging system and method of the present invention during microscopic stimulated Raman testing, an acousto-optic deflector driven by an equally spaced frequency comb signal causes the modulated light to have different deflection angles and frequency offsets. And through the interference of two beams with similar frequencies, periodic modulation of the specific frequency of the excitation light intensity of the beams with different deflection angles is achieved. Different deflection angles correspond to different modulation frequencies, and spatial synchronization of the intensity-modulated beam and another excitation light is achieved simultaneously; the role of the acousto-optic frequency shifter is to cause the frequency of the incident light to have a frequency offset of ω under a modulation signal with a single frequency of ω. L A series of uniformly spaced light spots are generated on the sample image plane, stimulating the stimulated Raman signals at each point in this column position on the sample, and demodulating the spectral information of each point by performing a Fourier transform on the overall signal obtained, thereby realizing line-scanning detection of stimulated Raman spectroscopy imaging. L Compared with the mode of the traditional stimulated Raman scattering spectroscopy imaging system that sequentially excites the signals at each point on the image plane and sequentially detects them, the present invention can increase the stimulated Raman spectroscopy imaging speed by one to two orders of magnitude. Only one scan by a one-dimensional scanning galvanometer is required to obtain the two-dimensional image information of the sample surface. It is expected that the stimulated Raman label-free imaging speed in a single field of view can be increased to several hundred frames per second to one thousand frames per second.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 FIG. is an optical path diagram of a stimulated Raman synchronous excitation line-scanning spectroscopy imaging system provided according to an embodiment of the present invention.

[0017] Figure 2 FIG. is an enlarged view of the interference and beam combination optical path of each beam in the second optical path adjustment group in a stimulated Raman synchronous excitation line-scanning spectroscopy imaging system provided according to an embodiment of the present invention.

[0018] Figure 3 FIG. is a schematic diagram of equally spaced focused light spots provided according to an embodiment of the present invention.

[0019] Reference numerals: 1. Laser; 2. First beam splitter; 3. First reflector; 4. Acousto-optic deflector; 401. Arbitrary waveform generator; 402. First power amplifier; 5. Second beam splitter; 6. Dichroic mirror; 7. Acousto-optic frequency shifter; 701. Signal generator; 702. Second power amplifier; 8. First binary diffraction element; 9. First 4-f lens group; 10. One-dimensional scanning galvanometer; 11. Second 4-f lens group; 12. Piezoelectric displacement transducer; 13. Objective lens; 14. Two-dimensional translation stage; 15. Condenser lens; 16. Filter; 17. Photodiode detector; 18. Computer; 19. Second reflector; 20. Optical delay line; 21. Third reflector; 22. Fourth reflector; 23. Second binary diffraction element; 24. Fifth reflector; 25. Excitation light focused spot. Detailed implementation mode

[0020] In the following, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are denoted by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, their detailed descriptions will not be repeated.

[0021] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation to the present invention.

[0022] The present invention provides a stimulated Raman synchronous excitation line-scanning spectroscopic imaging system (see Figures 1-3 ), including: a pulse modulation module, a stimulated Raman signal excitation module, and a stimulated Raman signal detection and acquisition module; The pulse modulation module is capable of generating a set of modulated light, the modulated light has different spatial deflection angles, and the excitation light corresponding to each angle has different frequencies, intensities and periodic modulations; at the same time, detection light spatially coincident with the modulated light is generated; the pulse modulation module includes: laser 1, first optical path adjustment group, second optical path adjustment group, dichroic mirror 6; A laser 1 for outputting two beams of pulsed lasers (with frequencies ω1 and ω2) that are synchronized but have different central frequencies; among them, one beam is the modulated light and the other beam is the detection light; when the modulated light is the pump light, the detection light is the Stokes light; or when the modulated light is the Stokes light, the detection light is the pump light; In the present invention, an example is given where the modulated light is the Stokes light and the detection light is the pump light; the Stokes light is used as the modulated light, and the frequency is denoted as ω1; the pump light is used as the detection light, and the frequency is denoted as ω2; where ω1 is a fixed value and ω2 can be tuned, and by changing the value of ω2, the Raman frequency shift |ω2 - ω1| detected by the system can be changed; The first optical path adjustment group includes a first beam splitter 2, an acousto-optic deflector 4, an acousto-optic frequency shifter 7, a first binary diffraction element 8, a second beam splitter 5, and a dichroic mirror 6; Specifically, both the first beam splitter 2 and the second beam splitter 5 are 50:50 beam splitters; A laser beam with a central frequency of ω1 is incident on the first beam splitter 2 and is divided into two beams with the same intensity; one beam of light is reflected by the first mirror 3 and then enters the acousto-optic deflector 4. Driven by an equally spaced frequency comb signal composed of multi-frequency sine signals, the acousto-optic deflector 4 causes the incident laser to simultaneously generate different deflection angles θi (i = 1, 2,..., N), and the beam is divided into N beams; the central frequency of the frequency comb signal is ω s1 ,ω s2 ,……ω sN ,These frequencies are equally spaced, and the frequency interval between any two adjacent frequencies is equal, that is, ω sn -ω s(n-1) is a fixed value, n = 2, 3... N; and the magnitudes of the frequencies in the frequency comb are in the order of hundreds of megahertz, which is much smaller than the excitation light frequencies ω1 and ω2 in the terahertz order. Each modulation frequency ω si causes the incident light to generate a deflection angle of θ i and causes the frequency of the deflected laser to correspondingly become ω1 + ω si ,i = 1, 2... N; The other path of ω1 excitation light passing through the first beam splitter 2 is reflected by the fifth mirror 24 and then enters the acousto-optic frequency shifter 7. The acousto-optic frequency shifter 7 shifts the frequency of the incident laser to ω1 + ω L under the drive of a single frequency signal with a frequency of ω L ,and then passes through the first binary diffraction element 8. By modulating parameters such as the phase of the excitation light, the laser generates the same N beams as the light passing through the acousto-optic deflector 4 with an angle of θ iThe deflected light of (i = 1, 2... N); the two sets of deflected light beams passing through the acousto-optic deflector 4 and the first binary diffraction element 8 are symmetric with respect to the beam splitting plane of the second beam splitter 5. The two sets of light beams interfere with each other on this beam splitting plane. Subsequently, the transmitted light of the acousto-optic deflector 4 passing through the second beam splitter 5 and the reflected light of the acousto-optic frequency shifter 7 are spatially coincident and continue to be transmitted to the dichroic mirror 6; at this time, the intensity of each transmitted light beam after interference is shown in the following formula (1): (1) In the formula: I represents the intensity of the light beam after interference; a is the amplitude of the two laser beams; t is time; ω si represents the i-th modulation frequency in the frequency comb signal, which is a component of the multi-frequency sine signal and is used to drive the acousto-optic deflector 4 to generate an angular deflection. The deflection angle is θ i of the light to produce an offset of ω si ; ω L represents the single-frequency signal of the acousto-optic frequency shifter 7, which shifts the frequency of the incident laser light. The light passing through the acousto-optic frequency shifter 7 will produce an offset of ω L ; ω si -ω L represents the frequency difference between the i-th deflected light beam and the frequency-shifted light beam. This frequency difference determines the periodic modulation frequency of the interference light intensity; after interference, the intensity of the light beam with the deflection angle θ i appears with a frequency of ω si -ω L for corresponding intensity periodic modulation; The second optical path adjustment group includes an optical delay line 20 and a second binary diffraction element 23; a beam of light with a central frequency of ω2 emitted by the laser 1 is reflected by the second mirror 19, and then the optical path is adjusted by the optical delay line 20 (the purpose is to synchronize and coincide in time with the pulse with a central frequency of ω1). It is successively reflected by the third mirror 21, the fourth mirror 22 and modulated by the second binary diffraction element 23, and is divided into N beams of light with an angle of θ i (i = 1, 2,... N); The light beams output by the second optical path adjustment group and the intensity-modulated light beams with a central frequency of ω1 output by the first optical path adjustment group pass through the dichroic mirror 6, respectively generating reflected light and transmitted light, and are spatially coincident.

[0023] The enlarged view of the interference and beam combination optical paths of each light beam passing through the second beam splitter 5 and the dichroic mirror 6 is as shown in Figure 2 ; In Figure 2 , the drive signal of the acousto-optic frequency shifter 7 comes from the single frequency (ω LThe output signal after the signal is amplified by the second power amplifier 702; the driving signal of the acousto-optic deflector 4 comes from an equally spaced frequency comb signal (ω si ) The output signal after being amplified by the first power amplifier 402. Among them, ω L The function is to adjust the amplitude modulation frequency ω of the ω1 beam after interference si -ω L , so that this frequency is reduced to within the detector response frequency range and at the same time away from the modulation frequency bandwidth of the acousto-optic deflector 4 (ω sN -ω s1 ), so the setting range of ω L is 0 < ω L < 2ω s1 -ω sN . Among them, ω s1 is the starting frequency of the equally spaced frequency comb, and ω sN is the termination frequency of the equally spaced frequency comb.

[0024] As Figure 2 shown, at the interference plane of the second beam splitter 5, the beam deflected by the acousto-optic deflector 4 with a deflection angle of θ i is symmetric with respect to the mirror surface of the second beam splitter 5 with the corresponding beam that generates the same θ i deflection angle after passing through the first binary diffraction element 8; the two beams of light interfere at the second beam splitter 5, and the transmitted light and the reflected light after interference coincide in space and are combined into one beam.

[0025] At the dichroic mirror 6, the beam with a central frequency of ω2 is deflected by the second binary diffraction element 23 to generate a deflection angle of θ i beam, which is symmetric with respect to the mirror surface of the dichroic mirror 6 with the corresponding beam in the ω1 beam, the two beams of light interfere at the dichroic mirror 6, the reflected light output by the second optical path adjustment group coincides in space with the transmitted light of the ω1 beam of the first optical path adjustment group, and is combined into one beam of light and continues to propagate. By adjusting the optical delay line 20, the spatio-temporal synchronization of the pulses is realized.

[0026] Stimulated Raman signal excitation module, used to focus the excitation light and excite the sample to generate a stimulated Raman scattering signal; the stimulated Raman signal excitation module includes two groups of 4-f lens groups, a one-dimensional scanning galvanometer 9, an objective lens 13, and a two-dimensional translation stage 14; The direction of the one-dimensional scanning galvanometer 10 is perpendicular to the beam arrangement direction; The objective lens 13 is installed on the piezoelectric displacement device 12, so that the objective lens 13 can perform sub-micron fine movement along the optical axis direction; by driving the objective lens 13 to move axially through the piezoelectric displacement device 12, the stimulated Raman spectroscopy imaging of different axial position planes can be realized; The pulse modulation module outputs three synchronized light beams. After passing through the first 4-f lens group 9 composed of two lenses, the light beams are projected onto the mirror plane of the one-dimensional scanning galvanometer 10. Subsequently, the light beams are coupled into the objective lens 13 through the second 4-f lens group 11 and focused on the sample plane on the two-dimensional translation stage 14 through the objective lens 13, forming a series of equally spaced focused light spots as shown in Figure 3 shown.

[0027] The stimulated Raman signal detection and acquisition module is used to collect, filter, convert and process the stimulated Raman scattering signals to complete the reconstruction and imaging of the image; the stimulated Raman signal detection and acquisition module includes a condenser lens 15, a filter 16, a photodiode detector 17 and a computer 18; Molecules at each focused light spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by the condenser lens 15. After collection, the signals pass through the filter 16 to filter out the modulation light with the original central frequency of ω1, and only the photodiode detector 17 is used to detect the intensity of the excitation light with the central frequency of ω2. After converting the collected optical signals into electrical signals, the signal is quickly recorded by the acquisition card on the computer 18 to complete the one-line excitation and sampling of the stimulated Raman signal of the sample. The calculation method of the signal sampling time t of this series is shown in the following formula: (2) where k is a positive integer, ω sN is the maximum frequency in the frequency comb signal input to the acousto-optic deflector 4, ω s1 is the minimum frequency, N is the number of frequency components in the frequency comb and is also the number of deflected light beams after deflection, and its number is equal to Figure 3 the number of generated focused light spots.

[0028] As Figure 3 shown, light beams with each deflection angle θ i generate corresponding light spots i (i = 1, 2... N) on the image plane after passing through the objective lens, that is, the excitation light focused light spots 25. These light spots respectively contain excitation light signals with frequencies of ω1 + ω si (i = 1, 2,..., N) and ω2. ω si is much smaller than ω1 and ω2. Therefore, when calculating the Raman frequency shift corresponding to each light spot, it can be ignored. The Raman frequency shift signals corresponding to each light spot can be approximated as |ω2 - ω1|. However, in each light spot, the intensity of the excitation light of ω1 has a periodic change with a frequency of ω si -ω L . After jointly exciting the stimulated Raman signal of the molecules in the sample with the excitation light with a frequency of ω2, it will cause the excitation light of ω2 to generate a frequency of ω si -ω LThe periodic intensity transformation signal, where this frequency signal corresponds one-to-one with the spatial position of the generated light spot. Thus, after collecting the sum of the excitation light signals at all positions ω2 using only a single photodiode detector 17, the electrical signal contains frequencies of ω si -ω L (i = 1, 2... N) of all electrical signals with periodic intensity variations. After obtaining the signal in the time period t as shown in formula (2), perform a Fourier transform on this electrical signal to extract all ω si -ω L (i = 1, 2... N) frequencies corresponding signal intensities, which is Figure 3 The stimulated Raman signal intensities corresponding to the light spots at each position in this column (i = 1, 2,..., N). Reconstruct according to the position to obtain the image signal of this column.

[0029] After obtaining the image signal of this column, use a one-dimensional scanning galvanometer 10 to scan along the Y direction as shown in Figure 3 That is, scan in the direction perpendicular to the distribution direction of the line light spot. Gradually use the Fourier transform to solve the signal, and then the stimulated Raman line-scanning spectral imaging of the two-dimensional image can be completed, and the imaging of this field of view can be completed; use a two-dimensional translation stage 14 to move the sample along the XY direction. After completing the field-of-view switching, repeat the line-stimulated Raman image scanning of the image, and then reconstruct and splice the image according to the field-of-view switching sequence to achieve planar large-field imaging. After completing the planar imaging, drive the objective lens 13 to move axially through the piezoelectric actuator 12 to achieve the stimulated Raman spectral imaging of different axial position planes; in this way, gradually perform field-of-view scanning and splicing, and finally the label-free three-dimensional stimulated Raman line-scanning imaging of the sample can be achieved. Subsequently, by adjusting the center frequency of ω2, the stimulated Raman spectral imaging of different Raman frequency shifts (|ω2 - ω1|) can be achieved.

[0030] Based on the above system (see Figures 1-3 ), a stimulated Raman synchronous excitation line-scanning spectral imaging method is provided, which specifically includes the following steps: S1. Generation and modulation of pulsed laser: Use a laser 1 to generate two pulsed lasers that are synchronous but have different frequencies, namely the pump light and the Stokes light; the Stokes light is split into two paths by a beam splitter. One path generates multiple deflected lights through an acousto-optic deflector 4, and the other path is frequency-shifted by an acousto-optic frequency shifter 7 and then interferes with the deflected lights, and after merging, it is transmitted to a dichroic mirror 6; after the optical path of the Stokes light is adjusted by an optical delay line 20, it interferes and merges with the Stokes light at the dichroic mirror 6 to achieve spatio-temporal synchronization; specifically includes the following sub-steps: S101. Use laser 1 to generate two beams of pulsed lasers that are synchronized but have different central frequencies, namely pump light and Stokes light, with frequencies ω1 and ω2 respectively; among them, ω1 is a fixed value, and ω2 can be tuned; the Stokes light (ω1) is used for frequency modulation, and the pump light (ω2) is used for subsequent signal detection; the pump light and the Stokes light are jointly used to excite the stimulated Raman signal; S102. Adjustment of the Stokes optical path: The laser beam with a central frequency of ω1 is incident on the first beam splitter 2 and is divided into two beams with the same intensity; one beam of light is reflected by the first mirror 3 and then enters the acousto-optic deflector 4; under the drive of an equally spaced frequency comb signal composed of multi-frequency sine signals, the acousto-optic deflector 4 causes the incident laser to generate different deflection angles θi (i = 1, 2,..., N), and the beam is divided into N beams; the other ω1 excitation light passing through the first beam splitter 2 is reflected by the fifth mirror 24 and then enters the acousto-optic frequency shifter 7; the acousto-optic frequency shifter 7 shifts the frequency of the incident laser to ω1 + ω L under the drive of a single-frequency (ω L ) signal; the deflected beam passes through the first binary diffraction element 8, and by modulating parameters such as the phase of the excitation light, the laser generates N deflected lights with the same angle of θi (i = 1, 2,..., N) as the light passing through the acousto-optic deflector 4; the two sets of deflected beams interfere at the second beam splitter 5, and the transmitted light and the reflected light coincide spatially and continue to be transmitted to the dichroic mirror 6.

[0031] S103. Adjustment of the pump optical path: One beam of light with a central frequency of ω2 emitted by laser 1 is reflected by the second mirror 19, and then passes through the optical delay line 20 for optical path adjustment to achieve time synchronization and spatial coincidence with the pulse with a central frequency of ω1; the adjusted beam is successively reflected by the third mirror 21, the fourth mirror 22, and modulated by the second binary diffraction element 23, and is divided into N beams with an angle of θi (i = 1, 2,..., N); the beam output by the second optical path adjustment group and the beam output by the first optical path adjustment group generate spatial coincidence at the dichroic mirror 6, and the reflected light and the transmitted light coincide spatially and are combined into one beam and continue to propagate.

[0032] S2. Excitation of the stimulated Raman signal: The three synchronized beams output by the pulse modulation module pass through the first 4-f lens group 9 and are projected onto the mirror plane of the one-dimensional scanning galvanometer 10, and then pass through the second 4-f lens group 11 to couple the beam to the objective lens 13. The objective lens 13 focuses the beam on the sample plane of the two-dimensional translation stage 14 to form a series of equally spaced focused spots; The direction of the one-dimensional scanning galvanometer 10 is perpendicular to the direction of the beam arrangement; the objective lens 13 is installed on the piezoelectric actuator 12, and the piezoelectric actuator 12 is used to drive the objective lens 13 to perform sub-micron fine movement along the optical axis direction to realize stimulated Raman spectroscopic imaging of different axial position planes.

[0033] S3. Detection and acquisition of stimulated Raman signals: Molecules at each focused light spot generate stimulated Raman scattering with the excitation light, and the scattered signals are collected by the condenser lens 15; After the collected signals pass through the filter 16 to filter out the modulated light with the central frequency of ω1 (only retaining the intensity of the excitation light with the central frequency of ω2), the optical signal is received by the photodiode detector 17 and converted into an electrical signal; The electrical signal is quickly recorded by the acquisition card on the computer 18 to complete the one-line excitation and sampling of the stimulated Raman signals of the sample.

[0034] S4. Perform Fourier transform on the collected electrical signals, or use a multi-channel lock-in amplifier to process the electrical signals to extract all frequencies ω si -ω L (i = 1, 2,..., N) of the signal intensity values; The stimulated Raman signals corresponding to the light spots at N positions in the column are in one-to-one correspondence with the signal intensities at the frequencies of ω si -ω L frequency, and are reconstructed according to the position to demodulate the stimulated Raman signals of the pixels at the corresponding positions of the column pixels in the image, that is, the image signals of the column. S5. Use the one-dimensional scanning galvanometer 10 to scan along the Y direction to gradually complete the stimulated Raman line-scanning spectral imaging of the two-dimensional image; Use the two-dimensional translation stage 14 to move the sample along the XY direction to complete the field-of-view switching and stitching to achieve planar large-field imaging; Drive the objective lens 13 to move along the axial direction (Z-axis) through the piezoelectric displacement device 12 to achieve the stimulated Raman spectral imaging of different axial position planes. S6. Gradually perform field-of-view scanning and stitching to finally achieve label-free three-dimensional stimulated Raman line-scanning imaging of the sample. In steps S5 and S6, only by using the one-dimensional galvanometer to push and scan and repeating the above excitation and demodulation process, fast spectral imaging of the two-dimensional field-of-view can be achieved; After obtaining the image of this field-of-view, then cooperate with the two-dimensional translation stage to switch the horizontal field-of-view and use the piezoelectric objective lens driver to switch the axial field-of-view, and three-dimensional large-field high-resolution label-free stimulated Raman spectral imaging can be achieved through the stitching of the corresponding fields-of-view.

[0035] In the present invention, the binary diffraction element can be replaced by a cylindrical lens, but the cylindrical lens cannot achieve discrete θi angle beam splitting; At the same time, when demodulating the signals, in addition to using Fourier transform, a multi-channel lock-in amplifier can also be used to input ω si -ω L (i = 1, 2... N) frequency reference signals to demodulate the information of each pixel.

[0036] The stimulated Raman synchronous excitation line-scanning spectral imaging system of the present invention can achieve rapid and high-resolution imaging of samples; the imaging method adjusts the central frequency of ω2 to change the Raman frequency shift (|ω2 - ω1|) detected by the system, and realizes stimulated Raman spectral imaging with different Raman frequency shifts. This method combines the fine modulation of pulsed lasers, the precise focusing and scanning of light beams, the efficient collection and processing of signals, and the reconstruction and stitching of images, and finally realizes label-free three-dimensional stimulated Raman spectral imaging of samples.

[0037] In the microscopic stimulated Raman test of the imaging system and method of the present invention, an acousto-optic frequency shifter driven by an equally spaced frequency comb signal causes the stimulated Raman excitation light to have different deflection angles and frequency offsets, and realizes the periodic modulation of the specific frequency of the excitation light intensity of the light beams with different deflection angles through the interference of two beams with similar frequencies. Different deflection angles correspond to different modulation frequencies, and at the same time, the spatial synchronization of the intensity-modulated light beam and another excitation light is realized, generating a series of uniformly spaced light spots on the sample image plane, simultaneously exciting the stimulated Raman signals at each point in this column position on the sample, and demodulating the spectral information of each point by performing a Fourier transform on the obtained overall signal, realizing the line-scanning detection of stimulated Raman spectral imaging. Compared with the traditional stimulated Raman scattering spectral imaging system, which excites the signals at each point on the image plane one by one and detects them one by one, this mode can increase the imaging speed of stimulated Raman spectroscopy by one to two orders of magnitude. Only one scan of a one-dimensional scanning galvanometer is required to obtain the two-dimensional image information on the sample surface. It is expected that the label-free imaging speed of a single field of view of stimulated Raman can be increased to several hundred frames per second to one thousand frames per second, which is not available and cannot be achieved by the previous traditional methods. In addition to being used for rapid imaging of biological tissues, the present invention can also be applied to high-speed molecular Raman spectral imaging application fields such as large-area material detection and defect analysis.

[0038] It should be understood that various forms of the processes shown above can be used, with steps reordered, added, or deleted. For example, the steps described in the disclosure of the present invention can be executed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in the present invention can be achieved, and no limitations are imposed herein.

[0039] The above specific embodiments do not constitute a limitation on the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A stimulated Raman synchronous excitation line-scanning spectroscopic imaging system, characterized in that: Comprising: A pulse modulation module, a stimulated Raman signal excitation module, and a stimulated Raman signal detection and acquisition module; The pulse modulation module includes: a laser, a first optical path adjustment group, a second optical path adjustment group, and a dichroic mirror; The laser is used to output two beams of pulsed lasers with the same frequency but different central frequencies, denoted as ω1 and ω2 respectively. One beam is the modulation light, and the other beam is the detection light. The first optical path adjustment group includes a beam splitter, an acousto-optic deflector, an acousto-optic frequency shifter, a binary diffraction element, and a dichroic mirror. The modulation light is split into two paths by the beam splitter. One path passes through the acousto-optic deflector to generate multiple deflected beams, and the other path is frequency-shifted by the acousto-optic frequency shifter and then interferes with the deflected beams. After merging, it is transmitted to the dichroic mirror. The second optical path adjustment group includes an optical delay line and a binary diffraction element. The detection light is adjusted in optical path by the optical delay line and modulated by the binary diffraction element, and then interferes and merges with the modulation light at the dichroic mirror to achieve spatio-temporal synchronization; The stimulated Raman signal excitation module includes two groups of 4-f lens groups, a one-dimensional scanning galvanometer, an objective lens, and a two-dimensional translation stage. The three beams of synchronized light output by the pulse modulation module are projected onto the mirror plane of the one-dimensional scanning galvanometer after passing through a group of 4-f lens groups, and then coupled to the objective lens through another group of 4-f lens groups. The objective lens focuses the light beam on the sample plane on the two-dimensional translation stage, forming a series of equally spaced focused spots. The one-dimensional scanning galvanometer is used for linear scanning on the sample plane, and the scanning direction is perpendicular to the light beam arrangement direction; The stimulated Raman signal detection and acquisition module includes a condenser lens, a filter, a photodiode detector, and a computer. The molecules at each focused spot generate stimulated Raman scattering with the excitation light. The scattered signal is collected by the condenser lens, and the collected signal passes through the filter to filter out the modulation light with the original central frequency of ω1. The photodiode detector detects the intensity of the excitation light with the central frequency of ω2, and after converting the collected optical signal into an electrical signal, the computer quickly records the signal to complete a single-line excitation and sampling of the stimulated Raman signal of the sample.

2. The stimulated Raman synchronous excitation line scanning spectral imaging system according to claim 1, wherein: The frequency ω1 is a fixed value; the frequency ω2 is tunable, and the Raman frequency shift |ω2 - ω1| detected by the stimulated Raman synchronous excitation line-scanning spectroscopic imaging system is changed by changing the value of ω2; When the modulation light is the pump light, the detection light is the Stokes light; or when the modulation light is the Stokes light, the detection light is the pump light.

3. The stimulated Raman synchronous excitation line scanning spectroscopic imaging system according to claim 2, wherein: The first optical path adjustment group includes a first beam splitter, an acousto-optic deflector, an acousto-optic frequency shifter, a first binary diffraction element, a second beam splitter, and a dichroic mirror; A laser beam with a central frequency of ω1 is incident on the first beam splitter and is divided into two beams with the same intensity; one beam is reflected by the first mirror and then enters the acousto-optic deflector, causing the incident laser to simultaneously generate different deflection angles, and the beam is divided into N beams; the other beam of excitation light passing through the first beam splitter enters the acousto-optic frequency shifter, and after being modulated by the first binary diffraction element for the excitation light, the laser generates N beams of deflection light with the same angle θ as the light passing through the acousto-optic deflector, i = 1, 2, ……, N; the two sets of deflected beams passing through the acousto-optic deflector and the first binary diffraction element interfere at the beam splitting plane of the second beam splitter, spatially coincide, and continue to be transmitted to the dichroic mirror; at this time, the intensity of each transmitted light after interference is as follows: i where i = 1, 2, ……, N; the two sets of deflected beams passing through the acousto-optic deflector and the first binary diffraction element interfere at the beam splitting plane of the second beam splitter, spatially coincide, and continue to be transmitted to the dichroic mirror; at this time, the intensity of each transmitted light after interference is as follows: ； Where: I represents the intensity of the light beam after interference; a is the amplitude of the two laser beams; t is time; ω si represents the i-th modulation frequency in the modulation frequency comb signal of the acou-optic deflector, and the deflection angle is θ i of the light generates an offset of ω si ; ω L represents the single-frequency signal of the acou-optic frequency shifter, and the light passing through the acou-optic frequency shifter will generate an offset of ω L ; ω si -ω L represents the frequency difference between the i-th deflected light beam and the frequency-shifted light beam; after interference, θ i the intensity of the light beam with the deflection angle appears at a frequency of ω si -ω L of the corresponding intensity is periodically modulated.

4. A stimulated Raman synchronous excitation line scanning spectroscopic imaging system according to claim 2, characterized in that: The second optical path adjustment group includes a reflector, an optical delay line, and a second binary diffraction element; a beam of light with a central frequency of ω2 emitted by the laser is reflected by the reflector, and after the optical path is adjusted by the optical delay line, it is reflected by the reflector and modulated by the second binary diffraction element, and then divided into N beams of light with an angle of θ i ; i = 1, 2,..., N; The beams output by the second optical path adjustment group and the intensity-modulated beams with the central frequency of ω1 output by the first optical path adjustment group respectively generate reflected light and transmitted light after passing through the dichroic mirror, and are spatially coincident.

5. A stimulated Raman synchronous excitation line scanning spectroscopic imaging system according to claim 1, characterized in that: The beam splitter is a 50:50 beam splitter; The objective lens is installed on a piezoelectric displacement device, and the objective lens is driven by the piezoelectric displacement device to perform sub-micron fine movement along the optical axis direction to achieve stimulated Raman spectroscopic imaging of different axial position planes.

6. The stimulated Raman synchronous excitation line-scanning spectroscopic imaging system according to claim 1, wherein: In the stimulated Raman signal detection and acquisition module, the specific method for single-line excitation and sampling of the stimulated Raman signal of the sample is as follows: Molecules at each focused light spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by a condenser lens. After collection, the signals pass through a filter to remove the modulated light with a central frequency of ω1. The sum of the detected light signals with a central frequency of ω2 is detected and collected using a photodiode detector. After converting the optical signal into an electrical signal, the electrical signal contains a series of electrical signals with all periodic intensity variations at a frequency of ω si -ω L ; Then, perform a Fourier transform on this electrical signal to extract all signal intensity values corresponding to the frequency of ω si -ω L . The stimulated Raman signals corresponding to the light spots at N positions in the column are in one-to-one correspondence with the signal intensities at the frequency of ω si -ω L , and image reconstruction is performed according to the position to obtain a column of image signals, where i = 1, 2,..., N; The calculation method of the electrical signal sampling time t is shown by the following formula: ； where: k is a positive integer, ω sN and ω s1 are respectively the maximum and minimum frequencies in the frequency comb signal input to the acou-optic deflector, and N is the number of generated focused light spots.

7. A stimulated Raman synchronous excitation line-scanning spectroscopic imaging method, which uses a stimulated Raman synchronous excitation line-scanning spectroscopic imaging system as described in claim 1, is characterized in that: Specifically, it includes the following steps: S1. The laser generates two pulses of laser light that are synchronous but have different frequencies, namely, the modulation light and the detection light. The modulation light is split into two paths by a beam splitter. One path passes through an acousto-optic deflector to generate multiple deflected beams, and the other path is frequency-shifted by an acousto-optic frequency shifter and then interferes with the deflected beams. After merging, they are transmitted to a dichroic mirror. The detection light is adjusted in optical path through an optical delay line and then interferes and merges with the modulation light at the dichroic mirror to achieve spatio-temporal synchronization. S2. The three synchronized light beams pass through the first 4-f lens group and are projected onto the mirror plane of a one-dimensional scanning galvanometer. Then, they pass through the second 4-f lens group to couple the light beams to an objective lens. The objective lens focuses the light beams onto the sample plane on a two-dimensional translation stage, forming a series of equally spaced focused spots. S3. The molecules at each focused spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by a condenser lens and then filtered by a filter to remove the modulation light with a central frequency of ω1. The light signals are then received by a photodiode detector and converted into electrical signals. The electrical signals are quickly recorded by an acquisition card on a computer, completing one-line excitation and sampling of the stimulated Raman signals of the sample. S4. Perform Fourier transform on the collected electrical signals, or use a multi-channel lock-in amplifier to process the electrical signals to extract the signal intensities at all frequencies ω si -ω L The stimulated Raman signals corresponding to the light spots at N positions in the column are in one-to-one correspondence with the signal intensities at ω si -ω L The frequencies, and image reconstruction is performed according to the positions to obtain a column of image signals; i = 1, 2,..., N; S5. Use a one-dimensional scanning galvanometer to scan along the Y direction to gradually complete the stimulated Raman line-scanning spectral imaging of a two-dimensional image. Use the two-dimensional translation stage to move the sample along the XY directions to complete field-of-view switching and stitching to achieve large-field-of-view imaging on a plane. Drive the objective lens to move along the Z-axis direction through a piezoelectric displacement actuator to achieve stimulated Raman spectral imaging of planes at different axial positions. S6. Gradually perform field-of-view scanning and stitching to finally achieve label-free three-dimensional stimulated Raman line-scanning imaging of the sample.

8. A stimulated Raman synchronous excitation line-scanning spectroscopic imaging method according to claim 7, characterized in that: The specific steps of step S1 include the following sub-steps: S101. Use a laser to generate two pulses of laser light that are synchronous but have different central frequencies, namely, the modulation light and the detection light, with frequencies of ω1 and ω2 respectively. Among them, ω1 is a fixed value and ω2 is tunable. The modulation light is frequency-modulated, and the detection light is used for subsequent signal detection. S102. Modulation optical path adjustment: A laser beam with a central frequency of ω1 is incident on the first beam splitter and is divided into two beams with the same intensity; one beam enters the acousto-optic deflector; under the drive of an equally spaced frequency comb signal, the incident laser generates different deflection angles θi after the acousto-optic deflector, and the beam is divided into N beams; the other ω1 excitation beam passing through the first beam splitter enters the acousto-optic frequency shifter, and the acousto-optic frequency shifter shifts the frequency of the incident laser to ω1 + ω L ; the deflected beam after the shift passes through the first binary diffraction element for modulation, so that the laser generates N deflected beams with the same angle θi as the light passing through the acousto-optic deflector; the two groups of deflected beams interfere at the second beam splitter, so that the transmitted light and the reflected light coincide spatially and continue to be transmitted to the dichroic mirror; S103. Detection optical path adjustment: A beam of light with a central frequency of ω2 emitted by the laser is reflected by a second mirror and then the optical path is adjusted through an optical delay line. The adjusted light beam is modulated by a second binary diffraction element and split into N beams with an angle of θi. The light beam output by the second optical path adjustment group interferes with the light beam output by the first optical path adjustment group at the dichroic mirror and merges into a single beam of light for continued propagation. Here, i = 1, 2,..., N.

9. A stimulated Raman synchronous excitation line-scanning spectroscopic imaging method according to claim 7, characterized in that: The objective lens is mounted on a piezoelectric displacement actuator, and the objective lens is driven by the piezoelectric displacement actuator to perform sub-micron-level fine movement along the optical axis direction to achieve stimulated Raman spectral imaging of planes at different axial positions. In step S3, the electrical signals are quickly recorded by an acquisition card on a computer.

Images