Stimulated Raman synchronous excitation line scanning spectral imaging system and method

Abstract

The present invention relates to the field of spectral detection and analysis technology, and in particular to a stimulated Raman synchronous excitation line scanning spectral imaging system and method. The system includes a pulse modulation module, in which a laser outputs modulated light and detection light that are synchronized but have different center frequencies; the modulated light is divided into two paths, one path generates multiple deflected light beams through an acousto-optic deflector, and the other path interferes with the deflected light after frequency shifting through an acousto-optic frequency shifter, and is then transmitted to a dichroic mirror after merging; the detection light is adjusted by an optical delay line and modulated by a binary diffraction element, and then interferes with the modulated light at the dichroic mirror and merges to achieve spatiotemporal synchronization; a stimulated Raman signal excitation module is used to focus three beams of synchronous light on a sample to form a row of equally spaced focused light spots and perform linear scanning; a stimulated Raman signal detection and acquisition module is used to collect stimulated Raman scattering signals, filter out the ω1 modulated light, detect the ω2 detection light intensity, convert the optical signal into an electrical signal, and record it, completing line excitation and sampling.

Description

Technical Field

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

[0002] Raman spectroscopy is a molecular scattering spectrum that reveals the intrinsic vibrations of molecular covalent bonds. Cell Raman spectroscopy can be used to determine the molecular species and content of cells. Single-cell Raman spectroscopy offers the advantages of label-free and non-destructive detection, making it valuable for spectral analysis of living tissues. Conventional spontaneous Raman signals are weak and slow, failing to meet the demands of high-speed cell spectral imaging. In recent years, novel stimulated Raman microscopy techniques have advanced the development of high-speed cell Raman spectroscopy instrumentation. Stimulated Raman significantly enhances Raman scattering efficiency through third-order nonlinear effects. It utilizes two pulsed laser beams (pump light and Stokes light). When the frequency difference between the two pulses aligns with the vibration frequency of a molecular covalent bond, pump photons are significantly converted to Stokes photons. Consequently, the stimulated Raman signal manifests as a decrease in the pump light or an increase in the Stokes light at a specific frequency difference.

[0003] To eliminate the influence of strong background noise from the excitation light on the Raman signal, current stimulated Raman imaging systems utilize high-frequency modulation and lock-in amplification to achieve high signal-to-noise ratio detection. Specifically, an acousto-optic modulator (AOM) or electro-optic modulator (EOM) is used to modulate the excitation light intensity at a high frequency (above MHz) in one beam channel. A lock-in amplifier is then used to detect the signal amplitude at that frequency in the other beam channel. Consequently, current stimulated Raman imaging systems rely heavily on the lock-in amplifier and require high-speed photoelectric signal conversion at tens of MHz, which limits the use of array detectors. Consequently, current stimulated Raman imaging systems employ a point-by-point excitation and detection approach. This involves using a two-dimensional galvanometer to deflect the excitation light, focusing it at different locations in the field of view through a microscope. Stimulated Raman is then detected point by point, and the corresponding light signal is demodulated using a lock-in amplifier to obtain the Raman signal at that point. The field of view is then scanned along a specific path, and a two-dimensional image is reconstructed based on the scan path and the corresponding signal. If large-field imaging is required, a two-dimensional translation stage is used to switch fields of view after the galvanometer scan is complete. Finally, field-of-view stitching is used to achieve large-field imaging. 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 imaging speed of a single field of view can reach ten frames per second. Although this point-by-point scanning stimulated Raman spectroscopy imaging technology can meet the imaging needs of general sliced tissue and cell samples, it faces many challenges when performing millimeter-level large-field-of-view and micron-level high-resolution Raman spectroscopy microscopy imaging of living biological tissue samples. In order to ensure the activity 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 galvanometer scanning speed and the repetition frequency of the femtosecond pulse laser, resulting in mutual constraints between imaging speed, field of view, resolution and integration time. In this case, it is often necessary to sacrifice a certain parameter to meet other requirements, making it difficult for the current technical solution to meet the application requirements of large-field-of-view and high-speed spectral imaging of living tissues. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a stimulated Raman synchronous excitation line scanning spectrum imaging system and method.

[0005] The first object of the present invention is to provide a stimulated Raman synchronous excitation line scanning spectral imaging system, comprising: a pulse modulation module, a stimulated Raman signal excitation module and a stimulated Raman signal detection and acquisition module;

[0006] 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 laser light that are synchronized but have different center frequencies, with the frequencies being denoted as ω1 and ω2, respectively, one beam being the modulated light and the other being 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 modulated light is divided into two paths by the beam splitter, one path generates multiple deflected light beams through the acousto-optic deflector, and the other path interferes with the deflected light after frequency shifting by the acousto-optic frequency shifter, and is then transmitted to the dichroic mirror after being combined; the second optical path adjustment group includes an optical delay line and a binary diffraction element; after the detection light is adjusted in optical path by the optical delay line and modulated by the binary diffraction element, it interferes with and merges with the modulated light at the dichroic mirror to achieve spatiotemporal synchronization;

[0007] The stimulated Raman signal excitation module includes two groups of 4-f lenses, a one-dimensional scanning galvanometer, an objective lens, and a two-dimensional translation stage. The three synchronized light beams output by the pulse modulation module are projected onto the reflective mirror plane of the one-dimensional scanning galvanometer after passing through a group of 4-f lenses. The light beams are then coupled to the objective lens through another group of 4-f lenses. The objective lens focuses the light beams onto the sample plane on the two-dimensional translation stage, forming a row of equally spaced focused light spots. The one-dimensional scanning galvanometer is used to perform linear scanning on the sample plane, with the scanning direction perpendicular to the beam arrangement direction.

[0008] The stimulated Raman signal detection and acquisition module includes a condenser, 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. The collected signal is filtered out by the filter to remove the modulated light with the original center frequency of ω1. The photodiode detector detects the intensity of the excitation light with a center frequency of ω2, and after converting the collected light signal into an electrical signal, the computer quickly records the signal, completing the first-line excitation and sampling of the sample's stimulated Raman signal.

[0009] Preferably, 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 spectroscopy imaging system is changed by changing the value of ω2;

[0010] When the modulated light is pump light, the detection light is Stokes light; or when the modulated light is Stokes light, the detection light is pump light.

[0011] 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;

[0012] A laser beam with a center frequency of ω1 is incident on the first beam splitter and is split into two beams of equal intensity. One beam is reflected by the first reflector and enters the acousto-optic deflector, which causes the incident laser to produce different deflection angles at the same time, and the beam is divided into N beams. The other excitation light that passes through the first beam splitter enters the acousto-optic frequency shifter and is modulated by the first binary diffraction element, causing the laser to produce N beams with the same angle θ as the light that passes through the acousto-optic deflector. i The deflected light, i = 1, 2, ..., N; the two groups of deflected light beams passing through the acousto-optic deflector and the first binary diffraction element interfere with each other at the beam splitting plane of the second beam splitter, overlap in space, and continue to be transmitted to the dichroic mirror; at this time, the intensity of each transmission light after interference is as follows:

[0013] ;

[0014] Where: I represents the intensity of the beam after interference; a is the amplitude of the two lasers; t is the time; ω si represents the i-th modulation frequency in the modulation frequency comb signal of the acousto-optic deflector, and the deflection angle is θ i The frequency of light produced ω si The offset of ω L Represents a single frequency signal of the acousto-optic frequency shifter. The light passing through the acousto-optic frequency shifter will generate ω L The frequency offset of si -ω L represents the frequency difference between the i-th deflected beam and the frequency-shifted beam; after interference, θ i The frequency of the beam intensity at the deflection angle is ω si -ω L The corresponding intensity is periodically modulated.

[0015] Preferably, the second optical path adjustment group includes a reflector, an optical delay line, and a second binary diffraction element; a path of light with a center frequency of ω2 emitted by the laser is reflected by the reflector, and then adjusted by the optical delay line, and then reflected by the reflector and modulated by the second binary diffraction element, and is divided into angles of θ i N beams of light; i=1, 2, ..., N;

[0016] The light beam output by the second light path adjustment group and the intensity modulated light beams with a center frequency of ω1 output by the first light path adjustment group respectively generate reflected light and transmitted light after passing through the dichroic mirror, and are spatially overlapped.

[0017] Preferably, the beam splitter is a 50:50 beam splitter;

[0018] The objective lens is mounted on a piezoelectric displacer, which drives the objective lens to perform submicron-level fine movement along the optical axis to achieve stimulated Raman spectroscopy imaging at different axial position planes.

[0019] Preferably, in the stimulated Raman signal detection and acquisition module, the specific method of the primary line excitation and sampling of the stimulated Raman signal of the sample is as follows:

[0020] The molecules at each focused spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by a condenser. The collected signals are filtered out by a filter to remove the modulated light with a center frequency of ω1. A photodiode detector is used to detect and collect the sum of the detection light signals with a center frequency of ω2. After the optical signal is converted into an electrical signal, the electrical signal contains the frequency of ω si -ω L Then perform Fourier transform on the electric signal to extract all ω si -ω L The signal intensity value corresponding to the frequency, the stimulated Raman signal corresponding to the N position of the spot in the column and ω si -ω L The signal intensity under the frequency corresponds one to one, and the image is reconstructed according to the position to obtain a column of image signals, where i = 1, 2, ..., N;

[0021] The calculation method of the electrical signal sampling time t is shown in the following formula:

[0022] ;

[0023] Where: k is a positive integer, ω sN and ω s1 are the maximum and minimum frequency values of the frequency comb signal input to the acousto-optic deflector, and N is the number of generated focused light spots.

[0024] A second object of the present invention is to provide a stimulated Raman synchronous excitation line scanning spectral imaging method, which uses a stimulated Raman synchronous excitation line scanning spectral imaging system and specifically includes the following steps:

[0025] S1. The laser generates two synchronized pulsed laser beams at different frequencies: the modulated light and the detection light. The modulated light is split into two paths by a beam splitter. One path passes through an acousto-optic deflector to generate multiple deflected light beams. The other path is frequency-shifted by an acousto-optic frequency shifter, where it interferes with the deflected light beams. The combined beams are then transmitted to a dichroic mirror. The detection light, after its optical path is adjusted by an optical delay line, interferes with and merges with the modulated light beam at the dichroic mirror, achieving spatiotemporal synchronization.

[0026] S2. The three synchronized beams pass through the first 4-f lens group and are projected onto the reflective mirror plane of the one-dimensional scanning galvanometer. The beams are then coupled to the objective lens through the second 4-f lens group. The objective lens focuses the beams onto the sample plane on the two-dimensional translation stage, forming a series of equally spaced focused spots.

[0027] S3. The molecules at each focused spot react with the excitation light to produce stimulated Raman scattering. The scattered signal is collected by a condenser and then filtered through a filter to remove the modulated light with a center frequency of ω1. The optical signal is then received by a photodiode detector and converted into an electrical signal. The electrical signal is rapidly recorded by an acquisition card on a computer, completing the primary linear excitation and sampling of the sample's stimulated Raman signal.

[0028] S4. Perform Fourier transform on the collected electrical signal, or use a multi-channel lock-in amplifier to process the electrical signal and extract all frequencies ω si -ω L The signal intensity under the condition of ω si -ω L The signal intensity under the frequency corresponds one to one, and the image is reconstructed according to the position to obtain a column of image signals; i=1,2,...,N;

[0029] S5. Use a one-dimensional scanning galvanometer to scan along the Y direction, gradually completing stimulated Raman line scan spectroscopy imaging of the two-dimensional image. Use a two-dimensional translation stage to move the sample along the XY direction to complete field of view switching and stitching, achieving planar large field of view imaging. Use a piezoelectric shifter to drive the objective lens along the Z axis to achieve stimulated Raman spectroscopy imaging at different axial positions.

[0030] S6. Gradually scan and stitch the field of view to finally achieve label-free three-dimensional stimulated Raman line scan imaging of the sample.

[0031] Preferably, step S1 specifically includes the following sub-steps:

[0032] S101. Use a laser to generate two synchronized pulsed laser beams with different center frequencies, namely, the modulating light and the detection light, with frequencies ω1 and ω2, respectively; where ω1 is a fixed value and ω2 is tunable; the modulating light is frequency modulated, and the detection light is used for subsequent signal detection;

[0033] S102. Modulation of the optical path: A laser beam with a center frequency of ω1 is incident on the first beam splitter and split into two beams of equal intensity. One beam enters the acousto-optic deflector (AOD). Driven by an equally spaced frequency comb signal, the AOD causes the incident laser to produce different deflection angles θi, splitting the beam into N beams. The other ω1 excitation light beam, which passes through the first beam splitter, enters the AOF shifter (AOF), which, driven by a single frequency signal, shifts the frequency of the incident laser to ω1+ω. L The deflected light beam is modulated by the first binary diffraction element, causing the laser to generate N deflected light beams with the same angle θi as the light passing through the acousto-optic deflector; the two groups of deflected light beams interfere at the second beam splitter, causing the transmitted light and the reflected light to overlap in space and continue to be transmitted to the dichroic mirror;

[0034] S103. Detection of optical path adjustment: A beam of light with a central frequency of ω2 emitted by the laser is reflected by a second reflector and then passes through an optical delay line for optical path adjustment. The adjusted beam is modulated by a second binary diffraction element and split into N beams of light with angles θi. The beams output by the second optical path adjustment group interfere with the beams output by the first optical path adjustment group at the dichroic mirror and merge into a single beam that continues to propagate; where i = 1, 2, ..., N.

[0035] Preferably, the objective lens is mounted on a piezoelectric displacer, and the piezoelectric displacer drives the objective lens to perform submicron-level fine movement along the optical axis to achieve stimulated Raman spectroscopy imaging at different axial position planes;

[0036] In step S3, the electrical signal is quickly recorded by an acquisition card on the computer.

[0037] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0038] The imaging system and method of the present invention, in microscopic stimulated Raman testing, uses an acousto-optic deflector driven by an equally spaced frequency comb signal to cause the modulated light to produce different deflection angles and frequency shifts.

[0039] And through the interference of two beams with similar frequencies, the periodic modulation of the specific frequency of the excitation light intensity of different deflection angle beams is achieved. Different deflection angles have different modulation frequencies, and the spatial synchronization of the intensity modulated beam and the other excitation light is achieved at the same time. The role of the acousto-optic frequency shifter is to L The frequency of the incident light is modulated by a single frequency signal to produce ω L A row of uniformly spaced light spots is generated on the sample image plane to stimulate the stimulated Raman signal at each point in the row on the sample. The spectrum information of each point is demodulated by Fourier transform of the obtained overall signal, thus realizing line scanning detection of stimulated Raman spectroscopy imaging.

[0040] Compared with the traditional stimulated Raman scattering spectroscopy imaging system that stimulates the signal of each point on the image plane point by point and detects it point by point, the present invention can increase the speed of stimulated Raman spectroscopy imaging by one to two orders of magnitude. Only one scan of the one-dimensional scanning galvanometer is required to obtain two-dimensional image information of the sample surface. It is expected that the speed of single-field stimulated Raman label-free imaging can be increased to hundreds of frames / second to one thousand frames / second. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is an optical path diagram of a stimulated Raman synchronous excitation line scanning spectral imaging system provided according to an embodiment of the present invention.

[0042] Figure 2It is an enlarged view of the interference of each light beam and the beam combining light path of the second light path adjustment group in a stimulated Raman synchronous excitation line scanning spectral imaging system provided according to an embodiment of the present invention.

[0043] Figure 3 Schematic diagram of focused light spots distributed at equal intervals according to an embodiment of the present invention.

[0044] Reference numerals:

[0045] 1. Laser;

[0046] 2. The first beam splitter;

[0047] 3. First reflector;

[0048] 4. Acousto-optic deflector;

[0049] 401. Arbitrary waveform generator; 402. First power amplifier;

[0050] 5. Second beam splitter;

[0051] 6. Dichroic mirror;

[0052] 7. Acousto-optic frequency shifter;

[0053] 701. Signal generator; 702. Second power amplifier;

[0054] 8. First binary diffraction element;

[0055] 9. The first 4-f lens group;

[0056] 10. One-dimensional scanning galvanometer;

[0057] 11. Second 4-f lens group;

[0058] 12. Piezoelectric displacement device;

[0059] 13. Objective lens;

[0060] 14. Two-dimensional translation stage;

[0061] 15. Condenser;

[0062] 16. Filter;

[0063] 17. Photodiode detector;

[0064] 18. Computer;

[0065] 19. Second reflector;

[0066] 20. Optical delay line;

[0067] 21. The third reflector;

[0068] 22. The fourth reflector;

[0069] 23. Second binary diffraction element;

[0070] 24. Fifth reflector;

[0071] 25. Excitation light focused spot. DETAILED DESCRIPTION

[0072] 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.

[0073] 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.

[0074] The present invention provides a stimulated Raman synchronous excitation line scanning spectrum imaging system (see Figure 1-Figure 3 ), including: a pulse modulation module, a stimulated Raman signal excitation module and a stimulated Raman signal detection and acquisition module;

[0075] The pulse modulation module is capable of generating a set of modulated lights with different spatial deflection angles, and the excitation light corresponding to each angle has different frequency, intensity, and period modulation; at the same time, it generates detection light that spatially overlaps with the modulated light. The pulse modulation module includes: a laser 1, a first optical path adjustment group, a second optical path adjustment group, and a dichroic mirror 6;

[0076] Laser 1 is configured to output two pulsed laser beams (of frequencies ω1 and ω2) that are synchronized but have different center frequencies; one beam is a modulated light beam and the other is a detection light beam; when the modulated light beam is a pump light beam, the detection light beam is a Stokes light beam; or when the modulated light beam is a Stokes light beam, the detection light beam is a pump light beam;

[0077] In the present invention, the modulated light is Stokes light and the detection light is pump light. Stokes light is used as the modulated light, and its frequency is recorded as ω1; pump light is used as the detection light, and its frequency is recorded as ω2. Where ω1 is a fixed value, and ω2 can be tuned. By changing the value of ω2, the Raman frequency shift |ω2-ω1| detected by the system can be changed.

[0078] 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;

[0079] Specifically, the first beam splitter 2 and the second beam splitter 5 are both 50:50 beam splitters;

[0080] A laser beam with a center frequency of ω1 is incident on the first beam splitter 2 and is split into two beams of equal intensity. One beam is reflected by the first reflector 3 and enters the acousto-optic deflector 4. Driven by an equally spaced frequency comb signal composed of multi-frequency sinusoidal signals, the acousto-optic deflector 4 causes the incident laser to simultaneously produce different deflection angles θi (i=1,2,…,N), and the beam is split into N beams. The center 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 constant value, n=2, 3...N; and the frequency levels in the frequency comb are in the hundreds of MHz level, which is much smaller than the excitation light frequencies ω1 and ω2 of the terahertz level. Each modulation frequency ω si The incident light produces θ i The deflection angle of the laser beam is changed to ω1+ω si , i=1,2……N;

[0081] The other ω1 excitation light passing through the first beam splitter 2 is reflected by the fifth reflector 24 and enters the acousto-optic frequency shifter 7. L Driven by a single frequency signal, the frequency shift of the incident laser is ω1+ω L Then, the laser beam passes through the first binary diffraction element 8 and modulates the phase of the excitation light to produce the same N beams as the light passing through the acousto-optic deflector 4 with an angle of θ. i (i=1, 2...N) deflected light; the two groups of deflected light beams passing through the acousto-optic deflector 4 and the first binary diffraction element 8 are symmetrical with respect to the beam splitting plane of the second beam splitter 5, and the two groups of light beams interfere with each other in the beam splitting plane. Then, 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 overlapped and continue to be transmitted to the dichroic mirror 6; at this time, the intensity of each transmission light after interference is expressed as follows (1):

[0082] (1)

[0083] Where: I represents the intensity of the beam after interference; a is the amplitude of the two lasers; t is the time; ω si It represents the i-th modulation frequency in the frequency comb signal, which is a component of the multi-frequency sinusoidal signal and is used to drive the acousto-optic deflector 4 to produce an angular deflection. The deflection angle is θ i The frequency of light produced ω si The offset of ω LRepresents a single frequency signal of the acousto-optic frequency shifter 7, which shifts the frequency of the incident laser. The light passing through the acousto-optic frequency shifter 7 will generate ω L The frequency offset of si -ω L represents the frequency difference between the i-th deflected beam and the frequency-shifted beam. This frequency difference determines the periodic modulation frequency of the interference light intensity. After interference, θ i The frequency of the beam intensity at the deflection angle is ω si -ω L The corresponding intensity of is periodically modulated;

[0084] The second optical path adjustment group includes an optical delay line 20 and a second binary diffraction element 23. The light of the center frequency ω2 emitted by the laser 1 is reflected by the second reflector 19, and then passes through the optical delay line 20 for optical path adjustment (the purpose is to synchronize and coincide with the pulse of the center frequency ω1). It is reflected by the third reflector 21 and the fourth reflector 22 in sequence and modulated by the second binary diffraction element 23, and is divided into two parts with an angle of θ. i N beams of light (i=1, 2, ...N);

[0085] The light beam output by the second light path adjustment group and the intensity modulated light beams with a center frequency of ω1 output by the first light path adjustment group respectively generate reflected light and transmitted light after passing through the dichroic mirror 6 and spatially overlap.

[0086] The enlarged diagram of the light path of each light beam interfering with and combining the light beams after passing through the second beam splitter 5 and the dichroic mirror 6 is shown in FIG. Figure 2 As shown. Figure 2 In the embodiment, the driving signal of the acousto-optic frequency shifter 7 comes from the single frequency (ω L ) signal is output after being amplified by the second power amplifier 702; the driving signal of the acousto-optic deflector 4 comes from the equally spaced frequency comb signal (ω generated by the arbitrary waveform generator 401 si ) is the output signal after being amplified by the first power amplifier 402. L The function is to adjust the amplitude modulation frequency ω of the ω1 beam after interference si -ω L , so that the frequency is reduced to 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 ω L The setting range is 0<ω L <2ω s1 -ω sN Among them, ω s1 is the starting frequency of the equally spaced frequency comb, ω sN is the stop frequency of the equally spaced frequency comb.

[0087] like Figure 2 As shown, at the interference plane of the second beam splitter 5, the light is deflected by the acousto-optic deflector 4 and has a value of θ i The light beam with the deflection angle has the same θ as that after passing through the first binary diffraction element 8. i The light beams corresponding to the deflection angles are symmetrical about the mirror plane of the second beam splitter 5 ; the two beams interfere at the second beam splitter 5 , and the transmitted light and the reflected light after interference overlap in space and are combined into one beam.

[0088] At the dichroic mirror 6, the light beam with a center frequency of ω2 is deflected by the second binary diffraction element 23 to produce a deflection angle of θ i The light beam of ω1 is symmetrical with the light beam of the corresponding deflection angle in the ω1 light beam about the mirror plane of the dichroic mirror 6. The two light beams interfere with each other at the dichroic mirror 6. The reflected light output by the second optical path adjustment group and the transmitted light of the ω1 light beam of the first optical path adjustment group overlap in space and merge into one light beam to continue propagating. The spatiotemporal synchronization of the pulses is achieved through the adjustment of the optical delay line 20.

[0089] The stimulated Raman signal excitation module is used to focus the excitation light and excite the sample to generate stimulated Raman scattering signals. 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.

[0090] The direction of the one-dimensional scanning galvanometer 10 is perpendicular to the direction of light beam arrangement;

[0091] The objective lens 13 is mounted on the piezoelectric displacer 12, so that the objective lens 13 can be moved in the submicron level along the optical axis. By driving the objective lens 13 to move axially by the piezoelectric displacer 12, stimulated Raman spectroscopy imaging can be achieved at different axial position planes.

[0092] The pulse modulation module outputs three synchronized light beams, which are projected onto the reflective mirror plane of the one-dimensional scanning galvanometer 10 after passing through the first 4-f lens group 9 composed of two lenses. Then, the light beams are coupled to the objective lens 13 through the second 4-f lens group 11, and are focused on the sample plane on the two-dimensional translation stage 14 through the objective lens 13 to form the following Figure 3 Shown is a row of equally spaced focused light spots.

[0093] Stimulated Raman signal detection and acquisition module, used to collect, filter, convert and process stimulated Raman scattering signals to complete image reconstruction and imaging; the stimulated Raman signal detection and acquisition module includes a condenser 15, a filter 16, a photodiode detector 17 and a computer 18;

[0094] The molecules at each focused spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by the condenser 15. The collected signals are filtered out by the filter 16 to remove the modulated light with the original center frequency ω1. Only the intensity of the excitation light with the center frequency ω2 is detected by the photodiode detector 17. After the collected light signals are converted into electrical signals, the acquisition card on the computer 18 quickly records the signals, completing the first-line excitation and sampling of the sample stimulated Raman signal. The calculation method of the column signal sampling time t is shown in the following formula:

[0095] (2)

[0096] Where k is a positive integer, ω sN is the maximum frequency of 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 beams after deflection, which is equal to Figure 3 The number of focused spots generated in .

[0097] like Figure 3 As shown, the deflection angle θ i After the beam passes through the objective lens, corresponding spots i are generated on the image plane (i=1, 2...N), namely the excitation light focused spots 25. These spots contain frequencies of ω1+ω si (i=1, 2, ... N) and the excitation light signal of ω2, ω si It is much smaller than ω1 and ω2, so it can be ignored when calculating the Raman frequency shift corresponding to each light spot. The Raman frequency shift signal 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 frequency of ω. si -ω L The periodic change of the frequency of ω2 will cause the excitation light of ω2 to produce a frequency of ω si -ω L The periodic intensity change signal of the frequency signal corresponds to the spatial position of the light spot it generates. In this way, after using only a single photodiode detector 17 to collect the sum of the excitation light signals at all positions ω2, the electrical signal contains the frequency ω si -ω L After obtaining the signal of time period t as shown in formula (2), the electric signal is Fourier transformed to extract all ω si -ω L The signal strength corresponding to the frequency (i=1, 2...N) is Figure 3The stimulated Raman signal intensity corresponding to the light spot at each position in the column (i=1, 2, ...N) is reconstructed according to the position to obtain the image signal of the column.

[0098] After acquiring the image signal of the column, use the one-dimensional scanning galvanometer 10 to scan along the Figure 3 Scanning is performed in the Y direction shown in the figure, that is, in a direction perpendicular to the distribution direction of the linear spot. Fourier transform is gradually used to solve the signal, and the stimulated Raman line scan spectroscopy imaging of the two-dimensional image can be completed, completing the imaging of the field of view. The sample is moved in the XY direction using the two-dimensional translation stage 14. After completing the field of view switching, the image is repeatedly scanned by the stimulated Raman image line. Then, the stitched image is reconstructed according to the field of view switching sequence to achieve planar large field of view imaging. After completing the planar imaging, the objective lens 13 is driven axially by the piezoelectric displacer 12 to achieve stimulated Raman spectroscopy imaging of planes at different axial positions. This step-by-step field of view scanning and stitching can ultimately achieve label-free three-dimensional stimulated Raman line scan imaging of the sample. Subsequently, by adjusting the center frequency of ω2, stimulated Raman spectroscopy imaging with different Raman frequency shifts (|ω2-ω1|) can be achieved.

[0099] Based on the above system (see Figure 1-Figure 3 ), provides a stimulated Raman synchronous excitation line scanning spectroscopy imaging method, specifically comprising the following steps:

[0100] S1. Generation and modulation of pulsed lasers: Laser 1 is used to generate two synchronized pulsed laser beams with different frequencies, namely, pump light and Stokes light. The Stokes light is split into two paths by a beam splitter. One path passes through an acousto-optic deflector 4 to generate multiple deflected light beams. The other path is frequency-shifted by an acousto-optic frequency shifter 7, where it interferes with the deflected light beams. The combined beams are then transmitted to a dichroic mirror 6. The Stokes light beam, after adjusting its optical path length by an optical delay line 20, interferes with and merges with the Stokes light beams at the dichroic mirror 6 to achieve spatiotemporal synchronization. This process specifically includes the following sub-steps:

[0101] S101. Use laser 1 to generate two synchronized pulsed laser beams, namely, pump light and Stokes light, with frequencies ω1 and ω2, respectively. ω1 is fixed, and ω2 is tunable. The Stokes light (ω1) is used for frequency modulation, and the pump light (ω2) is used for subsequent signal detection. The pump light and Stokes light are used together to excite the stimulated Raman signal.

[0102] S102. Stokes optical path adjustment: A laser beam with a center frequency of ω1 is incident on the first beam splitter 2 and split into two beams of equal intensity. One beam is reflected by the first reflector 3 and enters the acousto-optic deflector 4. Driven by an equally spaced frequency comb signal composed of multi-frequency sinusoidal signals, the acousto-optic deflector 4 causes the incident laser to produce different deflection angles θi (i = 1, 2, ..., N), thus splitting the beam into N beams. The other excitation beam of ω1, which passes through the first beam splitter 2, is reflected by the fifth reflector 24 and enters the acousto-optic frequency shifter 7. The acousto-optic frequency shifter 7 generates a single frequency (ω L ) signal drives the incident laser to shift its frequency by ω1+ω L The deflected light 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 beams of deflected light with the same angle θi (i=1,2,...,N) as the light passing through the acousto-optic deflector 4; the two groups of deflected light beams interfere at the second beam splitter 5, and the transmitted light and the reflected light overlap in space and continue to be transmitted to the dichroic mirror 6.

[0103] S103. Pump optical path adjustment: A light beam with a center frequency of ω2 emitted by laser 1 is reflected by the second reflector 19 and then passes through the optical delay line 20 for optical path adjustment to achieve time synchronization and spatial overlap with the pulse with a center frequency of ω1; the adjusted light beam is sequentially reflected by the third reflector 21 and the fourth reflector 22 and modulated by the second binary diffraction element 23, and is then divided into N light beams with angles of θi (i=1,2,...,N); the light beam output by the second optical path adjustment group and the light beam output by the first optical path adjustment group spatially overlap at the dichroic mirror 6, and the reflected light and the transmitted light spatially overlap and merge into one light beam for continued propagation.

[0104] S2. Stimulation of stimulated Raman signals: The three synchronized light beams output by the pulse modulation module are projected onto the reflective mirror plane of the one-dimensional scanning galvanometer 10 after passing through the first 4-f lens group 9. The light beams are then coupled to the objective lens 13 through the second 4-f lens group 11. The objective lens 13 focuses the light beams onto the sample plane on the two-dimensional translation stage 14, forming a row of equally spaced focused light spots.

[0105] The direction of the one-dimensional scanning galvanometer 10 is perpendicular to the direction of the light beam arrangement; the objective lens 13 is mounted on the piezoelectric displacer 12, and the piezoelectric displacer 12 drives the objective lens 13 to perform submicron-level fine movement along the optical axis to achieve stimulated Raman spectroscopy imaging in different axial position planes.

[0106] S3. Detection and collection of stimulated Raman signals: The molecules at each focused spot generate stimulated Raman scattering with the excitation light, and the scattered signals are collected by the condenser 15. The collected signals are filtered out by the filter 16 to remove the modulated light with a center frequency of ω1 (only retaining the intensity of the excitation light with a center frequency of ω2). The photodiode detector 17 then receives the light signal and converts it into an electrical signal. The electrical signal is quickly recorded by the acquisition card on the computer 18, completing the primary line excitation and sampling of the sample's stimulated Raman signal.

[0107] S4. Perform Fourier transform on the collected electrical signal, or use a multi-channel lock-in amplifier to process the electrical signal and extract all frequencies ω si -ω L (i=1,2,...,N) under the signal intensity value; the stimulated Raman signal corresponding to the N positions of the spot in the column and ω si -ω L The signal intensity under the frequency corresponds one to one, and is reconstructed according to the position. The demodulation obtains the stimulated Raman signal of the pixel at the corresponding position of the column pixel in the image, that is, the image signal of the column;

[0108] S5. Use the one-dimensional scanning galvanometer 10 to scan along the Y direction, gradually completing the stimulated Raman line scan 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, realizing planar large field of view imaging; use the piezoelectric displacer 12 to drive the objective lens 13 to move along the axial direction (Z axis) to realize stimulated Raman spectral imaging of different axial position planes;

[0109] S6. Gradually scan and stitch the field of view to achieve label-free 3D stimulated Raman line scan imaging of the sample.

[0110] In steps S5 and S6, rapid spectral imaging of a two-dimensional field of view can be achieved by simply using a one-dimensional galvanometer to push and scan and repeating the above-mentioned excitation demodulation process. After acquiring the field of view image, the horizontal field of view is switched by cooperating with a two-dimensional translation stage, and the axial field of view is switched by a piezoelectric objective lens driver. Then, by splicing the corresponding fields of view, three-dimensional large-field-of-view high-resolution stimulated Raman label-free spectral imaging can be achieved.

[0111] In the present invention, the binary diffraction element can be replaced by a cylindrical mirror, but the cylindrical mirror cannot realize discrete θi angle beam splitting; at the same time, when demodulating the signal, 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 signal is used to demodulate the information of each pixel.

[0112] The stimulated Raman synchronous excitation line-scanning spectroscopy imaging system of the present invention enables rapid, high-resolution imaging of samples. The imaging method modulates the center frequency of ω2 to change the Raman frequency shift (|ω2 - ω1|) detected by the system, enabling stimulated Raman spectroscopy imaging of different Raman frequency shifts. This method combines fine modulation of the pulsed laser, precise focusing and scanning of the beam, efficient signal collection and processing, and image reconstruction and splicing, ultimately achieving label-free three-dimensional stimulated Raman spectroscopy imaging of samples.

[0113] In a microscopic stimulated Raman test, the imaging system and method of the present invention use an acousto-optic frequency shifter driven by an equally spaced frequency comb signal to cause the stimulated Raman excitation light to produce different deflection angles and frequency shifts, and achieve periodic modulation of the specific frequency of the excitation light intensity of the light beams with different deflection angles through dual-beam interference with similar frequencies. Different deflection angles correspond to different modulation frequencies, and spatial synchronization of the intensity modulated light beam with another channel of excitation light is achieved. A column of uniformly equally spaced light spots is generated on the sample image plane, and the stimulated Raman signal at each point in the column on the sample is stimulated at the same time. The spectral information of each point is demodulated by Fourier transform on the obtained overall signal, thereby achieving line scanning detection of stimulated Raman spectral imaging. Compared to traditional stimulated Raman scattering (SRS) spectroscopy systems, this method, which excites and detects signals at each point on the image plane point by point, can increase the speed of SRS imaging by one to two orders of magnitude. Only a single scan of the one-dimensional scanning galvanometer is required to acquire two-dimensional image information of the sample surface. It is expected that the speed of single-field SRS label-free imaging can be increased to hundreds to a thousand frames per second, a level previously unattainable by traditional methods. In addition to its application in rapid imaging of biological tissues, this method can also be applied to high-speed molecular Raman spectroscopy imaging applications such as large-area material inspection and defect analysis.

[0114] 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.

[0115] 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. A stimulated Raman synchronous excitation line scanning spectroscopy imaging system, characterized by: include: Pulse modulation module, stimulated Raman signal excitation module and 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 synchronized pulsed laser beams with different center frequencies, with frequencies denoted as ω1 and ω2, respectively. One beam is modulated light and the other is 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 modulated light is split into two paths by the beam splitter. One path generates multiple deflected light beams through the acousto-optic deflector, and the other path interferes with the deflected light after frequency shifting by the acousto-optic frequency shifter. The combined light is then transmitted to the dichroic mirror. The second optical path adjustment group includes an optical delay line and a binary diffraction element. After the detection light is adjusted in optical path length by the optical delay line and modulated by the binary diffraction element, it interferes with and merges with the modulated light at the dichroic mirror to achieve spatiotemporal synchronization. The stimulated Raman signal excitation module includes two groups of 4-f lenses, a one-dimensional scanning galvanometer, an objective lens, and a two-dimensional translation stage. The three synchronized light beams output by the pulse modulation module are projected onto the reflective mirror plane of the one-dimensional scanning galvanometer after passing through a group of 4-f lenses. The light beams are then coupled to the objective lens through another group of 4-f lenses. The objective lens focuses the light beams onto the sample plane on the two-dimensional translation stage, forming a row of equally spaced focused light spots. The one-dimensional scanning galvanometer is used to perform linear scanning on the sample plane, with the scanning direction perpendicular to the beam arrangement direction. The stimulated Raman signal detection and acquisition module includes a condenser, 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. The collected signal is filtered out by the filter to remove the modulated light with the original center frequency of ω1. The photodiode detector detects the intensity of the excitation light with a center frequency of ω2, and after converting the collected light signal into an electrical signal, the computer quickly records the signal, completing the first-line excitation and sampling of the sample's stimulated Raman signal.

2. The stimulated Raman synchronous excitation line scanning spectroscopy imaging system according to claim 1, characterized in that: 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 spectroscopy imaging system is changed by changing the value of ω2; When the modulated light is pump light, the detection light is Stokes light; or when the modulated light is Stokes light, the detection light is pump light.

3. The stimulated Raman spectroscopy imaging system according to claim 2, characterized in that: 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 center frequency of ω1 is incident on the first beam splitter and is split into two beams of equal intensity. One beam is reflected by the first reflector and enters the acousto-optic deflector, which causes the incident laser to produce different deflection angles at the same time, and the beam is divided into N beams. The other excitation light that passes through the first beam splitter enters the acousto-optic frequency shifter and is modulated by the first binary diffraction element, causing the laser to produce N beams with the same angle θ as the light that passes through the acousto-optic deflector. i The deflected light, i = 1, 2, ..., N; the two groups of deflected light beams passing through the acousto-optic deflector and the first binary diffraction element interfere with each other at the beam splitting plane of the second beam splitter, overlap in space, and continue to be transmitted to the dichroic mirror; at this time, the intensity of each transmission light after interference is as follows: ； Where: I represents the intensity of the beam after interference; a is the amplitude of the two lasers; t is the time; ω si represents the i-th modulation frequency in the modulation frequency comb signal of the acousto-optic deflector, and the deflection angle is θ i The frequency of light produced ω si The offset of ω L Represents a single frequency signal of the acousto-optic frequency shifter. The light passing through the acousto-optic frequency shifter will generate ω L The frequency offset of si -ω L represents the frequency difference between the i-th deflected beam and the frequency-shifted beam; after interference, θ i The frequency of the beam intensity at the deflection angle is ω si -ω L The corresponding intensity is periodically modulated.

4. The stimulated Raman spectroscopy 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 path of light with a center frequency of ω2 emitted by the laser is reflected by the reflector, and then adjusted by the optical delay line, and then reflected by the reflector and modulated by the second binary diffraction element, and is divided into two paths with an angle of θ i N beams of light; i=1,2,...,N; The light beam output by the second light path adjustment group and the intensity modulated light beams with a center frequency of ω1 output by the first light path adjustment group respectively generate reflected light and transmitted light after passing through the dichroic mirror, and are spatially overlapped.

5. The stimulated Raman synchronous excitation line scanning spectroscopy imaging system according to claim 1, characterized in that: The beam splitter is a 50:50 beam splitter; The objective lens is mounted on a piezoelectric displacer, which drives the objective lens to perform submicron-level fine movement along the optical axis to achieve stimulated Raman spectroscopy imaging at different axial position planes.

6. The stimulated Raman spectroscopy imaging system according to claim 1, characterized in that: In the stimulated Raman signal detection and acquisition module, the specific method of primary line excitation and sampling of the sample stimulated Raman signal is as follows: The molecules at each focused spot generate stimulated Raman scattering with the excitation light. The scattered signals are collected by a condenser. The collected signals are filtered out by a filter to remove the modulated light with a center frequency of ω1. A photodiode detector is used to detect and collect the sum of the detection light signals with a center frequency of ω2. After the optical signal is converted into an electrical signal, the electrical signal contains the frequency of ω si -ω L Then perform Fourier transform on the electric signal to extract all ω si -ω L The signal intensity value corresponding to the frequency, the stimulated Raman signal corresponding to the N position of the spot in the column and ω si -ω L The signal intensity under the frequency corresponds one to one, and the image is reconstructed 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: ； Where: k is a positive integer, ω sN and ω s1 are the maximum and minimum frequency values of the frequency comb signal input to the acousto-optic deflector, and N is the number of generated focused light spots.

7. A stimulated Raman synchronous excitation line scanning spectral imaging method, using the stimulated Raman synchronous excitation line scanning spectral imaging system according to claim 1, characterized in that: The specific steps include: S1. The laser generates two synchronized pulsed laser beams at different frequencies: the modulated light and the detection light. The modulated light is split into two paths by a beam splitter. One path passes through an acousto-optic deflector to generate multiple deflected light beams. The other path is frequency-shifted by an acousto-optic frequency shifter, where it interferes with the deflected light beams. The combined beams are then transmitted to a dichroic mirror. The detection light, after its optical path is adjusted by an optical delay line, interferes with and merges with the modulated light beam at the dichroic mirror, achieving spatiotemporal synchronization. S2. The three synchronized beams pass through the first 4-f lens group and are projected onto the reflective mirror plane of the one-dimensional scanning galvanometer. The beams are then coupled to the objective lens through the second 4-f lens group. The objective lens focuses the beams onto the sample plane on the two-dimensional translation stage, forming a series of equally spaced focused spots. S3. The molecules at each focused spot react with the excitation light to produce stimulated Raman scattering. The scattered signal is collected by a condenser and then filtered through a filter to remove the modulated light with a center frequency of ω1. The optical signal is then received by a photodiode detector and converted into an electrical signal. The electrical signal is rapidly recorded by an acquisition card on a computer, completing the primary linear excitation and sampling of the sample's stimulated Raman signal. S4. Perform Fourier transform on the collected electrical signal, or use a multi-channel lock-in amplifier to process the electrical signal and extract all frequencies ω si -ω L The signal intensity under the condition of ω si -ω L The signal intensity under the frequency corresponds one to one, and the image is reconstructed according to the position to obtain a column of image signals; i=1,2,...,N; S5. Use a one-dimensional scanning galvanometer to scan along the Y direction, gradually completing stimulated Raman line scan spectroscopy imaging of the two-dimensional image. Use a two-dimensional translation stage to move the sample along the XY direction to complete field of view switching and stitching, achieving planar large field of view imaging. Use a piezoelectric shifter to drive the objective lens along the Z axis to achieve stimulated Raman spectroscopy imaging at different axial positions. S6. Gradually scan and stitch the field of view to finally achieve label-free three-dimensional stimulated Raman line scan imaging of the sample.

8. The stimulated Raman spectroscopy imaging method according to claim 7, characterized in that: The step S1 specifically includes the following sub-steps: S101. Use a laser to generate two synchronized pulsed laser beams with different center frequencies, namely, the modulating light and the detection light, with frequencies ω1 and ω2, respectively; where ω1 is a fixed value and ω2 is tunable; the modulating light is frequency modulated, and the detection light is used for subsequent signal detection; S102. Modulation of the optical path: A laser beam with a center frequency of ω1 is incident on the first beam splitter and split into two beams of equal intensity. One beam enters the acousto-optic deflector (AOD). Driven by an equally spaced frequency comb signal, the AOD causes the incident laser to produce different deflection angles θi, splitting the beam into N beams. The other ω1 excitation light beam, which passes through the first beam splitter, enters the AOF shifter (AOF), which, driven by a single frequency signal, shifts the frequency of the incident laser to ω1+ω. L The deflected light beam is modulated by the first binary diffraction element, causing the laser to generate N deflected light beams with the same angle θi as the light passing through the acousto-optic deflector; the two groups of deflected light beams interfere at the second beam splitter, causing the transmitted light and the reflected light to overlap in space and continue to be transmitted to the dichroic mirror; S103. Detection of optical path adjustment: A beam of light with a central frequency of ω2 emitted by the laser is reflected by a second reflector and then passes through an optical delay line for optical path adjustment. The adjusted beam is modulated by a second binary diffraction element and split into N beams of light with angles θi. The beams output by the second optical path adjustment group interfere with the beams output by the first optical path adjustment group at the dichroic mirror and merge into a single beam that continues to propagate; where i = 1, 2, ..., N.

9. The method for stimulated Raman spectroscopy imaging according to claim 7, wherein: The objective lens is mounted on a piezoelectric displacer, which drives the objective lens to perform submicron-level fine movement along the optical axis to achieve stimulated Raman spectroscopy imaging at different axial position planes; In step S3, the electrical signal is quickly recorded by an acquisition card on the computer.

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