A dual-frequency stimulated Raman scattering microscopic imaging system

Abstract

This invention discloses a dual-frequency stimulated Raman scattering (SERS) microscopy imaging system, comprising a signal generation and modulation device and an imaging processing terminal. The signal generation and modulation device includes: a pulse chirp and delay module for ensuring that the spectral components of the two beams of light have a certain temporal distribution and overlap; a dual-phase modulation module for high-frequency intensity modulation of the two excitation beams; a dual-difference frequency module for ensuring that the horizontal and vertical polarizations of the two excitation beams satisfy the Raman frequency shifts of the two types of molecules; and a scanning and focusing module for focusing the excitation light onto the sample and achieving point scanning. The imaging processing terminal includes: an orthogonal demodulation module for synchronously orthogonally demodulating the detection signal passing through the sample; and a signal acquisition and synchronization control module for synchronously controlling the scanning system drive and demodulation result acquisition.

Description

Technical Field

[0001] This invention relates to the field of nonlinear optical microscopy, and more particularly to a dual-frequency stimulated Raman scattering microscopy. Background Technology

[0002] Stimulated Raman scattering microscopy (SRS) is a nonlinear optical imaging technique based on molecular vibrations. It primarily utilizes the difference frequency between two laser beams to resonate with the vibrations of Raman-active molecules, thereby obtaining molecular chemical characteristic maps of static and dynamic living systems. This enables highly sensitive, precise, and specific analysis. This non-invasive imaging method can directly visualize complex biological processes and is therefore widely used in the biomedical field, such as assessing peripheral nerve degeneration, identifying tumor margins in fresh, unprocessed tissue, and discovering biomarkers and therapeutic targets for invasive cancers.

[0003] Stimulated Raman scattering (SRS) microscopy based on spectral focusing can efficiently utilize femtosecond pulse energy and is compatible with other nonlinear optical imaging systems. However, spectral focusing SRS microscopy typically performs spectral scanning frame-by-frame, which can take several minutes to acquire a complete spectrum on unlabeled biological samples (without Raman probes). Other SRS spectral imaging schemes, such as wavelength-tunable narrowband lasers, pulse shaping, Fourier or compressed domain spectral encoding, and parallel detection, often require optical equipment with higher parameter requirements or complex optical path structures that are space-consuming and difficult to operate. Furthermore, in terms of speed and signal-to-noise ratio, they have not yet met the needs of the life sciences for studying dynamic biological processes and for rapid, high-quality imaging.

[0004] Dual-frequency stimulated Raman scattering microscopy can simultaneously acquire images of two molecules (lipids and proteins). As a label-free histological tool, it has the potential to identify tumor tissues, obtain diagnostic results similar to those obtained by hematoxylin and eosin (H&E) staining, and can perform real-time imaging of fresh, unstained tissues without cumbersome staining steps. Summary of the Invention

[0005] The main objective of this invention is to provide a dual-frequency stimulated Raman scattering microscopy imaging system, which aims to achieve a low-cost and highly compatible dual-frequency stimulated Raman microscopy imaging technology with real-time, high signal-to-noise ratio and high spatial resolution, thus solving the problem of long information acquisition time in spectral focusing stimulated Raman microscopy imaging systems.

[0006] To achieve the above objectives, this invention provides a dual-frequency stimulated Raman scattering (SERS) microscopy imaging system, comprising a signal generation and modulation device and an imaging processing terminal. The signal generation and modulation device includes: a pulse chirp and delay module for ensuring that the spectral components of the two beams of light have a certain temporal distribution and overlap; a dual-phase modulation module for high-frequency intensity modulation of the two excitation beams; a dual-difference frequency module for ensuring that the horizontal and vertical polarizations of the two excitation beams satisfy the Raman frequency shifts of the two types of molecules; and a scanning and focusing module for focusing the excitation light onto the sample and achieving point scanning. The imaging processing terminal includes: an orthogonal demodulation module for synchronously orthogonally demodulating the detection signal passing through the sample; and a signal acquisition and synchronization control module for synchronously controlling the scanning system drive and demodulation result acquisition.

[0007] The system also includes: a femtosecond laser, a first half-wave plate, a first polarization beam splitter, a second half-wave plate, and a second polarization beam splitter. The femtosecond laser is used to synchronously output two femtosecond pulses. The first laser beam passes through the first half-wave plate to change the linear polarization angle of the laser. The output light passes through the first polarization beam splitter to output a horizontally polarized beam. The laser power of this beam can be adjusted by combining the first half-wave plate. The second laser beam passes through the second half-wave plate to change the linear polarization angle of the laser. The output light passes through the second polarization beam splitter. The laser power of the second beam can be adjusted by combining the second half-wave plate. The two excitation beams are then simultaneously incident on the pulse chirp and delay module.

[0008] The pulse chirping and delay module includes: a first glass rod for chirping and broadening another laser beam split from the first polarization beam splitter into a picosecond beam; a second glass rod for chirping and broadening a laser beam passing through the second polarization beam splitter into a picosecond beam; and a first time delay unit for adjusting the optical path of the first laser beam so that the two beams can overlap in time after pulse chirping; the first beam passes through the first glass rod and then through the first time delay unit; the second beam passes through the second glass rod and is incident on the biphasic modulation module.

[0009] The dual-phase modulation module includes: a third half-wave plate for changing the linear polarization angle of the laser beam; a first quarter-wave plate for changing the polarization state of the excitation beam; and an electro-optic modulator for intensity modulation of the excitation beam.

[0010] The light beam passing through the second glass rod becomes linearly polarized light with a polarization angle of 45° after passing through the third half-wave plate, and then becomes circularly polarized light after passing through the first quarter-wave plate. The output light is intensity modulated by the electro-optic modulator. This light beam and the light beam passing through the first time delay unit are wavelength coupled by the first dichroic mirror, and then enter the dual-frequency difference module after passing through the first 4f system.

[0011] The dual-frequency difference module includes: a third polarization beam splitter for polarization separation and polarization combining of the beam; a first hollow roof prism reflector for setting optical delay lines; and a second hollow roof prism reflector for setting optical delay lines. The laser beam from the first 4f system is incident on the third polarization beam splitter and separated into a horizontal polarization state and a vertical polarization state. The horizontal polarization state is incident on the scanning and focusing module, and the vertical polarization state is coupled with the horizontal polarization state by the third polarization beam splitter after passing through the first and second hollow roof prism reflectors in sequence, and then incident on the scanning and focusing module.

[0012] The optical delay line, which is set by the first hollow roof prism reflector and the second hollow roof prism reflector, enables the optical path difference between the horizontally polarized and vertically polarized optical paths separated by the third polarization beam splitter to match the Raman frequency difference corresponding to the two molecules in the tissue.

[0013] The scanning and focusing module includes: an XY scanning galvanometer for two-dimensional scanning of the sample in the XY direction; a telecentric scanning lens and a sleeve lens for generating a constant spot size across the entire field of view on the sample plane, while effectively correcting aberrations; and a microscope objective for exciting and focusing the beam. The laser beam, after passing through the dual-frequency difference module, sequentially passes through the XY scanning galvanometer, the telecentric scanning lens and the sleeve lens, and the microscope objective to achieve two-dimensional scanning excitation of the sample. The signal light is detected and analyzed by the orthogonal demodulation module.

[0014] The orthogonal demodulation module includes: a condenser lens for collecting transmitted signal light; a focusing lens for focusing the signal beam; a filter for filtering out other beams besides the probe beam; a fourth polarization beam splitter for separating the signal light into a vertically polarized state and a horizontally polarized state; a dual-channel photodetector for converting the horizontally polarized signal light and the vertically polarized signal light into first and second voltage signals, respectively; a phase shifter for performing a π / 2 phase shift on the first voltage signal; an adder for linearly superimposing the first voltage signal output by the phase shifter with the second voltage signal output by the dual-channel photodetector; and a lock-in amplifier for achieving orthogonal demodulation of the horizontally polarized signal light and the vertically polarized signal light.

[0015] The forward-scattered signal after passing through the sample is collected by the condenser lens and focused by the focusing lens. The focused beam is then separated into horizontally polarized signal light and vertically polarized signal light by the fourth polarization beam splitter. The two polarized signal beams are detected by the dual-channel photodetector and converted into first and second voltage signals. The first voltage signal is phase-shifted by π / 2 by the phase shifter and linearly superimposed with the second voltage signal by the adder. The signal is then connected to a lock-in amplifier for quadrature demodulation.

[0016] The biphase modulation module and quadrature demodulation module further include: a signal generator; the signal generator is used to simultaneously output two analog signals with the same parameters; one is used to drive the electro-optic modulator, and the other is used as a reference signal for the lock-in amplifier; the signal input terminal of the lock-in amplifier is a linear superposition of the horizontal polarization state signal and the vertical polarization state signal, and the demodulation of the lock-in amplifier selects XY quadrature output, which corresponds to the Raman frequency shift signals corresponding to the two polarization states respectively.

[0017] The signal acquisition and control system includes: a multi-functional acquisition card and a controller; the multi-functional acquisition card is used to drive the acquisition of voltage signals from the scanning galvanometer in the scanning and focusing module and the two orthogonal output voltage signals in the quadrature demodulation module, and to realize the synchronous control of the above signal output and acquisition; the controller is used to control the first delay unit in the pulse chirp and delay module, the stage motion control, and the objective lens motion control in the scanning and focusing module.

[0018] In this design, both the pulse chirp and the first and second glass rods in the delay module are dispersive devices. These dispersive devices can also be grating pairs, prism pairs, optical fibers, or other linearly chirped devices.

[0019] This invention provides a dual-frequency stimulated Raman scattering (SMR) microscopy imaging system. Its advantages include: the ability to simultaneously modulate the high-frequency intensity of two excitation beams with different polarization states using a dual-phase modulation module, followed by polarization separation via a polarization beam splitter, and combined with a dual-difference frequency module to ensure that the horizontal and vertical polarizations of the two excitation beams satisfy the Raman frequency shifts of the two types of molecules; the dual-frequency SMR signals can be simultaneously demodulated by an orthogonal demodulation module, ultimately achieving a real-time, high signal-to-noise ratio, and high spatial resolution low-cost, highly compatible dual-frequency SMR microscopy imaging system. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic block diagram of the structure of the multimodal nonlinear microscopic imaging system according to an embodiment of this application;

[0022] Figure 2 This is a schematic diagram of the structure of the multimodal nonlinear microscopic imaging system according to an embodiment of this application. Detailed Implementation

[0023] To make the objectives, features, and advantages of this invention more apparent and understandable, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] Please see Figure 1 This is a dual-frequency stimulated Raman microscopy imaging system, comprising: a signal generation and modulation device and a processing terminal. The signal generation and modulation device is used for signal generation and signal modulation, and the processing terminal is used for demodulation of stimulated Raman signals and image reconstruction.

[0025] The signal generation and modulation device includes: a pulse chirp and delay module 101, a biphase modulation module 102, a dual-frequency difference module 103, and a scanning and focusing module 104; the pulse chirp and delay module 101 is used to ensure that the spectral components of the two beams of light have a certain distribution and overlap in time; the biphase modulation module 102 is used to perform high-frequency intensity modulation on the two excitation beams; the dual-frequency difference module 103 is used to ensure that the horizontal and vertical polarizations of the two excitation beams satisfy the Raman frequency shift of the two types of molecules; the scanning and focusing module 104 is used to focus the excitation light onto the sample and realize point scanning; the imaging processing terminal includes: an orthogonal demodulation module 105 and a signal acquisition and synchronization control module 106; the orthogonal demodulation module 105 is used to synchronously orthogonally demodulate the detection signal passing through the sample; the signal acquisition and synchronization control module 106 is used to realize the synchronous control of the scanning system drive and demodulation result acquisition.

[0026] In this embodiment, the reference signal of the quadrature demodulation module 105 and the driving signal of the biphase modulation module 102 are the same signal; the signal acquisition and synchronization control module 106, the quadrature demodulation module 105, and the scanning and focusing module 104 are all connected and transmitted by electrical signals; according to the logical synchronization of the signal acquisition and synchronization control module 106, the dual-frequency stimulated Raman signal output by the quadrature demodulation module 105 is reconstructed into an image.

[0027] The dual-frequency stimulated Raman microscopy system provided in this embodiment can simultaneously perform high-frequency intensity modulation on two excitation beams with different polarization states using a dual-phase modulation module 102, and then perform polarization separation through a polarization beam splitter. Combined with a dual difference frequency module 103, the horizontal and vertical polarizations of the two excitation beams can satisfy the Raman frequency shifts of the two types of molecules. The dual-frequency stimulated Raman signals can be simultaneously demodulated by an orthogonal demodulation module 105, ultimately realizing a real-time, high signal-to-noise ratio, and high spatial resolution low-cost, highly compatible dual-frequency stimulated Raman microscopy system.

[0028] Please see Figure 2 In one embodiment, the system further includes: a femtosecond laser 1, a first half-wave plate 2, a first polarization beam splitter 3, a second half-wave plate 4, and a second polarization beam splitter 5; the femtosecond laser 1 is used to synchronously output two femtosecond pulses, the first laser beam passes through the first half-wave plate 2 to change the linear polarization angle of the laser, the output light passes through the first polarization beam splitter 3 to output a horizontally polarized beam, and the laser power of the beam can be adjusted by combining the first half-wave plate 2; the second laser beam passes through the second half-wave plate 4 to change the linear polarization angle of the laser, the output light passes through the second polarization beam splitter 5, and the laser power of the second beam can be adjusted by combining the second half-wave plate 4, and the two excitation beams are then simultaneously incident on the pulse chirp and delay module 101;

[0029] In one embodiment, the pulse chirping and delay module 101 includes: a first glass rod 6, a second glass rod 7, and a first time delay unit 8. The first glass rod 6 is used to chirp and broaden another laser beam split from the first polarization beam splitter into a picosecond beam; the second glass rod 7 is used to chirp and broaden a laser beam passing through the second polarization beam splitter into a picosecond beam; the first time delay unit 8 is used to adjust the optical path of the first laser beam so that the two beams can achieve time overlap after pulse chirping; the first beam passes through the first glass rod 6 and then through the first time delay unit 8; the second beam passes through the second glass rod 7 and is incident on the biphasic modulation module 102.

[0030] In this embodiment, the pulse chirp and delay module 101 can introduce linear chirp into the broadband femtosecond beam output by the femtosecond laser 1, and by adjusting the parameters of the corresponding dispersion compensation device, the first beam (pump beam) and the second beam (Stokes beam) have the same linear chirp parameters. During this process, the instantaneous frequency difference between the two beams is constant, and the difference frequency spectrum bandwidth is much narrower than the difference frequency bandwidth of the Fourier transform-limited spectrum of the two, which can realize high-spectral-resolution stimulated Raman scattering microscopy imaging.

[0031] In this embodiment, both the first glass rod 6 and the second glass rod 7 are dispersion devices; in other embodiments, the dispersion device may be a grating pair, a prism pair, an optical fiber, or other linear chirped devices to replace the first glass rod 6 and the second glass rod 7 in this embodiment.

[0032] In one embodiment, the dual-phase modulation module 102 includes: a third half-wave plate 9, a first quarter-wave plate 10, and an electro-optic modulator 11; wherein, the third half-wave plate 9 is used to change the linear polarization angle of the laser beam; the first quarter-wave plate 10 is used to change the polarization state of the excitation beam; the electro-optic modulator 11 is used to intensity modulate the excitation beam; the beam passing through the second glass rod 7 becomes linearly polarized light with a polarization angle of 45° after passing through the third half-wave plate 9, and then becomes circularly polarized light after passing through the first quarter-wave plate 10. The output light is intensity modulated by the electro-optic modulator 11. This beam and the beam passing through the first time delay unit 8 are wavelength coupled by the first dichroic mirror 12, and then incident on the dual-frequency difference module 103 after passing through the first 4f system 13.

[0033] In this embodiment, the second beam of light (Stokes beam) is intensity modulated by a 20MHz resonant electro-optic modulator 11. To increase the modulation depth at a lower driving voltage, a third half-wave plate 9 and a first quarter-wave plate 10 are used to convert the Stokes beam from a linearly polarized state to a circularly polarized state. If the input to the electro-optic modulator 11 is linearly polarized light, the driving voltage signal must change from 0V to V. π (or from -V) π To achieve complete modulation of the incident light amplitude, a voltage of -1 / 2V is required (up to 0V). When the input to the electro-optic modulator is circularly polarized light, complete modulation of the incident light amplitude only requires -1 / 2V. π up to 1 / 2V π The driving voltage.

[0034] In one embodiment, the dual-frequency difference module 103 includes: a third polarization beam splitter 14, a first hollow roof prism reflector, and a second hollow roof prism reflector 15; wherein, the third polarization beam splitter 14 is used for polarization separation and polarization combining of the beam; the first hollow roof prism reflector and the second hollow roof prism reflector 15 are used to set optical delay lines; the laser beam from the first 4f system 13 is incident on the third polarization beam splitter 14 and separated into a horizontal polarization state and a vertical polarization state, the horizontal polarization state is incident on the scanning and focusing module, and the vertical polarization state is coupled with the horizontal polarization state by the third polarization beam splitter after passing through the first hollow roof prism reflector 14 and the second hollow roof prism reflector 15 in sequence, and is incident on the scanning and focusing module 104;

[0035] In this embodiment, the third polarization beam splitter 14 is used for polarization detection of the dual-phase modulation module 103, and splits the combined beam into two excitation beams: horizontally polarized and vertically polarized. The intensity change of the horizontally polarized Stokes beam is sin(ωt+α), and the intensity change of the vertically polarized Stokes beam is -sin(ωt+α), where ω = 2πf, f is the modulation frequency, and α is the initial phase. The polarization states of the two pump beams after beam splitting are consistent with the polarization state of their collinear Stokes beams, therefore, the influence of the Raman depolarization ratio does not need to be considered.

[0036] In this embodiment, the optical delay lines set by the first and second hollow roof prism reflectors 15 enable the optical path difference between the horizontally polarized and vertically polarized light paths separated by the third polarization beam splitter to match the Raman frequency difference corresponding to the two molecules in the tissue; in this process, the vertically polarized excitation light is re-beamed by the same polarization beam splitter.

[0037] In one embodiment, the scanning and focusing module 104 includes: an XY scanning galvanometer 16, a telecentric scanning lens 17 and a sleeve lens 18, and a microscope objective 19; wherein, the XY scanning galvanometer 16 is used to realize two-dimensional scanning of the sample in the XY direction; the telecentric scanning lens 17 and the sleeve lens 18 are used to generate a constant spot size in the entire field of view on the sample plane, while effectively correcting aberrations; the microscope objective 19 is used to excite and focus the beam. The laser beam passing through the dual-frequency difference module 103 passes sequentially through the XY scanning galvanometer 16, the telecentric scanning lens 17 and the sleeve lens 18, and the microscope objective 19 to realize two-dimensional scanning excitation of the sample 20, and the signal light is detected and analyzed by the orthogonal demodulation module 105.

[0038] In one embodiment, the quadrature demodulation module 105 includes: a condenser lens 21, a focusing lens 22, a filter 23, a fourth polarization beam splitter 24, a dual-channel photodetector 25, a phase shifter 26, an adder 27, and a lock-in amplifier 28; wherein, the condenser lens 21 is used to collect transmitted signal light; the focusing lens 22 is used to focus the signal beam; the filter 23 is used to filter out other beams besides the probe beam; the fourth polarization beam splitter 24 is used to separate the signal light into a vertical polarization state and a horizontal polarization state; the dual-channel photodetector 25 is used to convert the horizontally polarized signal light and the vertically polarized signal light into first and second voltage signals, respectively; the phase shifter 26 is used to perform a π / 2 phase shift on the first voltage signal; the adder 27 is used to linearly superimpose the first voltage signal output by the phase shifter and the second voltage signal output by the dual-channel photodetector; and the lock-in amplifier 28 is used to achieve quadrature demodulation of the horizontally polarized signal light and the vertically polarized signal light;

[0039] In this embodiment, the forward-scattered signal after passing through sample 20 is collected by the condenser lens 21 and focused by the focusing lens 22. The focused beam is then separated into horizontally polarized signal light and vertically polarized signal light by the fourth polarization beam splitter 24 after passing through the filter 23. The two polarized signal beams are detected by the dual-channel photodetector 25 and converted into first and second voltage signals. The first voltage signal is phase-shifted by π / 2 by the phase shifter 26 and linearly superimposed with the second voltage signal by the adder 27, and then connected to the lock-in amplifier 28 for quadrature demodulation.

[0040] In this embodiment, the scattering signal of the second excitation beam (Stokes beam) from sample 20 is filtered out by a bandpass filter 23 with an optical density (OD) greater than 6. The pump beam is separated by a fourth polarization beam splitter 24 and detected by a dual-channel phase-shifting detector 25. The detector has two photosensitive areas of 1×1 cm². 2 The photodiode is subjected to a reverse bias voltage of -24V through a DC power supply to improve its detection sensitivity. The photocurrent signal of the diode is converted into a voltage signal by a transimpedance amplifier with a bandwidth of 100MHz. A phase shifter 26 adds π / 2 phase to the first voltage signal, making its modulation intensity -sin(ωt+α+π / 2), i.e., -cos(ωt+α). After being linearly superimposed with the second voltage signal by an adder 27, the signal is input to a lock-in amplifier 28, thereby demodulating two stimulated Raman loss (SRL) signals. At this time, the two SRL demodulated signals are orthogonal and can be output as the XY components of the demodulated amplitude, respectively.

[0041] In this embodiment, the two orthogonal biphase SRL signals can be represented as follows:

[0042]

[0043] By setting an appropriate reference signal phase β, the two SRL signals can be demodulated in phase (X) and quadrature (Y) to obtain two molecular vibrational signals I(Ω1) and I(Ω2). Compared to custom-designed dual-channel demodulated lock-in amplifiers, this detector is inexpensive to design, print, and solder, and is easy to deploy in any spectrally focused stimulated Raman scattering imaging system.

[0044] In one embodiment, the biphase modulation module 102 and the quadrature demodulation module 105 further include: a signal generator 29, used to simultaneously output two analog signals with the same parameters; one for driving the electro-optic modulator and the other for a reference signal for the lock-in amplifier;

[0045] In one embodiment, the signal acquisition and control system 106 includes: a multi-function acquisition card and a controller 30; wherein, the multi-function acquisition card is used to drive the acquisition of voltage signals from the scanning galvanometer in the scanning and focusing module and the two orthogonal output voltage signals in the quadrature demodulation module, and to realize the synchronous control of the above signal output and acquisition; the controller is used to control the first delay unit in the pulse chirp and delay module, the stage motion control, and the objective lens motion control in the scanning and focusing module;

[0046] In all embodiments of this application, a reflector can be used to change the direction of the laser in order to facilitate laser transmission.

[0047] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0048] The above is a description of a dual-frequency stimulated Raman scattering microscopy imaging system provided by the present invention. For those skilled in the art, based on the ideas of the embodiments of the present invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A dual-frequency stimulated Raman scattering microscopy imaging system, characterized in that, include: Signal generation and modulation device and imaging processing terminal; The signal generation and modulation device includes: The pulse chirp and delay module is used to ensure that the spectral components of the two lasers have a certain temporal distribution and overlap. A dual-phase modulation module is used for high-frequency intensity modulation of two excitation beams; The dual difference frequency module is used to ensure that the horizontal and vertical polarizations of the two excitation beams satisfy the Raman frequency shifts of the two types of molecules. The scanning and focusing module is used to focus the excitation light onto the sample and achieve point scanning; The imaging processing terminal includes: The quadrature demodulation module is used to synchronously orthogonally demodulate the detection signal passing through the sample; The signal acquisition and synchronization control module is used to realize the synchronous control of the scanning system drive and demodulation result acquisition; The system also includes: a femtosecond laser, a first half-wave plate, a first polarization beam splitter, a second half-wave plate, and a second polarization beam splitter; The femtosecond laser is used to simultaneously output two femtosecond pulses. The first laser pulse passes through a first half-wave plate to change the linear polarization angle of the laser. The output light passes through a first polarization beam splitter to output a horizontally polarized beam. The laser power of this beam can be adjusted by combining the first half-wave plate. The second laser pulse passes through a second half-wave plate to change the linear polarization angle of the laser. The output light passes through a second polarization beam splitter. The laser power of the second beam can be adjusted by combining the second half-wave plate. The two excitation beams are then simultaneously incident on the pulse chirp and delay module. The pulse chirp and delay module includes: The first glass rod is used to chirp-broaden the first laser beam split by the first polarizing beam splitter into a picosecond beam; The second glass rod is used to chirp-broaden the second laser beam passing through the second polarizing beam splitter into a picosecond beam; The first time delay unit is used to adjust the optical path of the first laser beam so that the two beams can overlap in time after pulse chirping. The first laser beam passes through the first glass rod and then through the first time delay unit; the second laser beam passes through the second glass rod and is incident on the biphase modulation module. The biphase modulation module includes: The third half-wave plate is used to change the linear polarization angle of the laser beam; The first quarter-wave plate is used to change the polarization state of the excitation beam; Electro-optic modulators are used to modulate the intensity of an excitation beam. The light beam passing through the second glass rod becomes linearly polarized light with a polarization angle of 45° after passing through the third half-wave plate, and then becomes circularly polarized light after passing through the first quarter-wave plate. The output light is intensity modulated by the electro-optic modulator. This light beam and the light beam passing through the first time delay unit are wavelength coupled by the first dichroic mirror, and then enter the dual-frequency difference module after passing through the first 4f system. The dual-frequency difference module includes: The third polarization beam splitter is used for polarization separation and polarization combining of the beam; The first hollow roof prism reflector is used to set the optical delay line; The second hollow roof prism reflector is used to set the optical delay line; The laser beam from the first 4f system is incident on the third polarization beam splitter, which separates it into a horizontal polarization state and a vertical polarization state. The horizontal polarization state is incident on the scanning and focusing module, and the vertical polarization state is sequentially incident on the first hollow roof prism reflector and the second hollow roof prism reflector before being coupled with the horizontal polarization state by the third polarization beam splitter and incident on the scanning and focusing module. The optical delay line set by the first hollow roof prism reflector and the second hollow roof prism reflector can make the optical path difference between the horizontally polarized and vertically polarized optical paths separated by the third polarization beam splitter match the Raman frequency difference corresponding to the two molecules in the tissue.

2. The dual-frequency stimulated Raman scattering microscopy imaging system according to claim 1, characterized in that, The scanning and focusing module includes: XY scanning galvanometer is used to achieve two-dimensional scanning of the sample in the XY direction; Telecentric scanning lenses and sleeve lenses are used to generate a constant spot size across the entire field of view on the sample plane, while effectively correcting aberrations. Microscope objectives are used to excite and focus light beams; The laser beam passing through the dual-frequency difference module sequentially passes through the XY scanning galvanometer, the telecentric scanning lens and the sleeve lens and the microscope objective, realizing two-dimensional scanning excitation of the sample. The signal light is detected and analyzed by the orthogonal demodulation module.

3. The dual-frequency stimulated Raman scattering microscopy imaging system according to claim 1, characterized in that, The orthogonal demodulation module includes: A condenser lens is used to collect transmitted signal light; A focusing lens is used to focus a signal beam. A filter is used to filter out light beams other than the probe beam. The fourth polarization beam splitter is used to separate the signal light into vertical and horizontal polarization states; A dual-channel photodetector is used to convert horizontally polarized signal light and vertically polarized signal light into first and second voltage signals, respectively. A phase shifter is used to shift the first voltage signal by π / 2. An adder is used to linearly superimpose the first voltage signal output by the phase shifter with the second voltage signal output by the dual-channel photodetector; A lock-in amplifier is used to achieve orthogonal demodulation of horizontally polarized signal light and vertically polarized signal light; The forward-scattered signal after passing through the sample is collected by the condenser lens and focused by the focusing lens. The focused beam is then separated into horizontally polarized signal light and vertically polarized signal light by the fourth polarization beam splitter. The two polarized signal beams are detected by the dual-channel photodetector and converted into first and second voltage signals. The first voltage signal is phase-shifted by π / 2 by the phase shifter and linearly superimposed with the second voltage signal by the adder. The signal is then connected to a lock-in amplifier for quadrature demodulation.

4. The dual-frequency stimulated Raman scattering microscopy imaging system according to claim 3, characterized in that, The biphase modulation module and quadrature demodulation module further include: a signal generator; The signal generator is used to simultaneously output two analog signals with the same parameters; one is used to drive the electro-optic modulator, and the other is used as a reference signal for the lock-in amplifier. The signal input of the lock-in amplifier is a linear superposition of the horizontal polarization signal and the vertical polarization signal. The demodulation of the lock-in amplifier selects XY quadrature output, which corresponds to the Raman frequency shift signals corresponding to the two polarization states.

5. The dual-frequency stimulated Raman scattering microscopy imaging system according to claim 1, characterized in that, The signal acquisition and synchronization control module includes: a multi-functional acquisition card and a controller; The multi-functional acquisition card is used to drive the acquisition of the scanning galvanometer in the scanning and focusing module and the two orthogonal output voltage signals in the quadrature demodulation module, and to realize the synchronous control of the above signal output and acquisition. The controller is used to control the first time delay unit in the pulse chirp and delay module, the stage motion control, and the objective lens motion control in the scanning and focusing module.

6. The dual-frequency stimulated Raman scattering microscopy imaging system according to claim 2, characterized in that, The first and second glass rods in the pulse chirp and delay module are both dispersive devices; The dispersion device can also be a grating pair, a prism pair, an optical fiber, or other linear chirped devices.

Images