Stimulated Raman scattering microscopic imaging system capable of simultaneously scanning double Raman frequency shift spectrums

Abstract

The invention relates to the technical field of microscopic imaging, and particularly provides a stimulated Raman scattering microscopic imaging system capable of simultaneously scanning double Raman frequency shift spectrums, which comprises a laser light source driving and modulating unit and a signal detecting and data processing unit, the laser light source driving and modulating unit comprises a dual-channel laser synchronous excitation module, a light beam polarization regulation and power distribution module, a pulse broadening and spectrum focusing module, a dual-channel dual-phase modulation module, a common-path different-time dual-frequency difference module and a laser scanning and telecentric focusing module; and the signal detection and data processing unit comprises a dual-channel orthogonal demodulation module and a scanning driving and signal synchronous acquisition module. Compared with the prior art, the dual-Raman frequency shift spectrum scanning stimulated Raman microimaging device has the advantages that dual-Raman frequency shift spectrum scanning stimulated Raman microimaging with higher acquisition speed, high resolution and wide spectrum coverage range is realized, and the problem of long time consumption for acquiring the complete spectrum of the to-be-tested sample by the traditional single-frequency resonance spectrum scanning stimulated Raman microimaging is solved.

Description

Technical Field

[0001] This invention relates to the field of microscopic imaging technology, and in particular to a stimulated Raman scattering microscopic imaging system with simultaneous dual Raman frequency shift spectral scanning. Background Technology

[0002] Stimulated Raman scattering (SRS) microscopy is a label-free optical imaging technique based on molecular vibrations. Its fundamental principle is based on the optical beat frequency (ω) between the pump light and the Stokes light. p -ω S The SRS spectral imaging technology excites Raman-active molecules in the sample to generate coherent vibrations, and each pixel contains a spectrum. By scanning the spectrum, information on the chemical composition of the sample can be obtained, and different molecules with overlapping Raman bands can be distinguished, resulting in better chemical specificity.

[0003] This imaging technique boasts advantages such as high molecular specificity, label-free operation, and quantitative analysis, enabling the visualization of complex biological processes. Furthermore, due to the addition of a spectral dimension and the proportionality of signal intensity to the number of chemical bonds being measured, it allows for the quantitative analysis of multiple chemical components in samples. Therefore, stimulated Raman scattering microscopy has found wide application in numerous fields, including pathological detection, biological metabolism, and drug delivery.

[0004] Traditional single-frequency resonance SRS technology uses a serial detection mode, which either adjusts the narrowband laser wavelength in each imaging region or repeatedly scans the zero point of the spectral focusing time to obtain a complete spectral image of the sample under test, which is very time-consuming. Summary of the Invention

[0005] The purpose of this invention is to provide a stimulated Raman scattering microscopy imaging system with simultaneous dual Raman frequency shift spectral scanning, so as to solve the problem that the traditional stimulated Raman microscopy imaging with single-frequency resonance spectral scanning takes a long time to acquire the complete spectrum of the sample under test.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A stimulated Raman scattering microscopy imaging system with simultaneous dual Raman frequency shift spectral scanning includes a laser source driving and modulation unit and a signal detection and data processing unit;

[0008] The laser source driving and modulation unit includes:

[0009] A dual-channel laser synchronous excitation module is used to generate two laser beams with different wavelengths, both of which are linearly polarized and have the same polarization direction.

[0010] The light beam polarization regulation and power distribution module is used for regulating the power of the two laser beams by regulating the polarization state of the two laser beams.

[0011] The pulse broadening and spectrum focusing module is used for making the frequency components of the two laser pulses have a certain distribution and time overlap on the time axis.

[0012] The double-channel double-phase modulation module is used for high-frequency intensity modulation of the two laser beams.

[0013] The common-path asynchronous double-frequency difference module is used for constructing two laser beams with horizontal and vertical polarizations in the common-path laser, so as to respectively match the specific Raman frequency shifts of two types of molecules.

[0014] The laser scanning and telecentric focusing module is used for focusing the laser beam to the sample plane and realizing two-dimensional scanning excitation of the sample to generate stimulated Raman signals, while effectively correcting the phase difference.

[0015] The signal detection and data processing unit comprises:

[0016] The double-channel quadrature demodulation module is used for realizing synchronous quadrature demodulation and amplitude recovery of the Raman frequency shift signals of different channels, and outputting two Raman frequency shift signals corresponding to two polarization states respectively.

[0017] The scanning driving and signal synchronous acquisition module is used for driving the laser scanning and telecentric focusing module, acquiring the demodulation signals output by the double-channel quadrature demodulation module, and realizing timing unification and automatic control of the system multi-optical and electronic units.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The simultaneous double-Raman frequency shift spectrum scanning stimulated Raman scattering microscopic imaging system provided by the present application can utilize spectrum focusing and double-channel quadrature modulation and demodulation technology, not only can simultaneously acquire Raman spectra of two types of molecules located in high and low beams, but also can splice the two molecular spectra by using spectrum splicing method, so as to obtain a complete spectral image of the detected sample, and can extract Raman frequency shift spectra from the double-channel Raman spectrum sequence image simultaneously, and splice the two-channel spectra by analogy. Compared with the traditional technology, the present application realizes double-Raman frequency shift spectrum scanning stimulated Raman microscopic imaging with faster acquisition speed, high resolution and wide spectral coverage, overcomes the problems of low acquisition efficiency and serious image drift in single-channel frequency-by-frequency scanning, and solves the problem of long time consumption in traditional single-frequency resonance spectrum scanning stimulated Raman microscopic imaging for acquiring a complete spectrum of a detected sample. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1The structure schematic diagram of the simultaneous dual-Raman-shift spectrum scanning stimulated Raman scattering microscopic imaging system disclosed by the embodiment of the present application is disclosed.

[0021] Figure 2 The structure schematic diagram of one of the specific embodiments of the simultaneous dual-Raman-shift spectrum scanning stimulated Raman scattering microscopic imaging system disclosed by the embodiment of the present application is disclosed.

[0022] Reference signs:

[0023] 101, dual-channel laser synchronous excitation module; 102, beam polarization regulation and power distribution module; 103, pulse expansion and spectral focusing module; 104, dual-channel dual-phase modulation module; 105, common-path asynchronous dual-frequency difference module; 106, laser scanning and telecentric focusing module; 201, dual-channel quadrature demodulation module; 202, scanning driving and signal synchronous acquisition module; 1, femtosecond laser; 2, first half-wave plate; 3, second half-wave plate; 4, first polarization beam splitter; 5, second polarization beam splitter; 6, first glass rod; 7, second glass rod; 8, third half-wave plate; 9, fourth half-wave plate; 10, quarter-wave plate; 11, electro-optical modulator; 12, high-precision displacement table; 13, dichroic mirror; 14, 4f system; 15, first birefringent crystal; 16, second birefringent crystal; 17, X-Y scanning galvanometer; 18, telecentric scanning lens; 19, sleeve lens; 20, microscopic objective lens; 21, sample; 22, condenser; 23, condenser lens; 24, filter; 25, third polarization beam splitter; 26, photodetector; 27, phase shifter; 28, adder; 29, lock-in amplifier; 30, signal generator; 31, voltage amplifier; 32, multifunctional acquisition card and controller. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0025] Please refer to Figure 1 The simultaneous dual-Raman-shift spectrum scanning stimulated Raman scattering microscopic imaging system comprises a laser light source driving and modulation unit A and a signal detection and data processing unit B. The laser light source driving and modulation unit A is used to generate and adjust signals. The signal detection and data processing unit B is used to demodulate stimulated Raman signals and reconstruct microscopic images.

[0026] In the embodiment, the laser source driving and modulation unit A comprises: a dual-channel laser synchronous excitation module 101, a beam polarization regulation and power distribution module 102, a pulse broadening and spectral focusing module 103, a dual-channel dual-phase modulation module 104, a common-path asynchronous dual-frequency difference module 105, and a laser scanning and telecentric focusing module 106. The dual-channel laser synchronous excitation module 101 is used to generate two laser beams with different wavelengths for exciting stimulated Raman signals. Both of the two laser beams are linearly polarized light with consistent polarization directions. The beam polarization regulation and power distribution module 102 is used to adjust the laser power of the two laser beams in the system. The pulse broadening and spectral focusing module 103 is used to make the frequency components of the two laser pulses have a certain distribution and time overlap on the time axis. The dual-channel dual-phase modulation module 104 is used to modulate the intensity of the two laser beams. The common-path asynchronous dual-frequency difference module 105 is used to construct two laser beams with horizontal and vertical polarizations in the laser common path, so as to match the specific Raman frequency shift of the two types of molecules, respectively. The laser scanning and telecentric focusing module 106 is used to focus the laser beams to the sample plane and realize two-dimensional scanning excitation of the sample to generate stimulated Raman signals, while effectively correcting the phase difference.

[0027] The signal detection and data processing unit B comprises a dual-channel quadrature demodulation module 201 and a scanning driving and signal synchronous acquisition module 202. The dual-channel quadrature demodulation module 201 is used to realize synchronous quadrature demodulation and amplitude recovery of Raman frequency shift signals in different channels, and output two Raman frequency shift signals corresponding to two polarization states, respectively. The scanning driving and signal synchronous acquisition module 202 is used to drive the laser scanning and telecentric focusing module 106, acquire the dual-channel demodulation signals output by the dual-channel quadrature demodulation module 201, and realize timing synchronization and automatic control of the system.

[0028] In the embodiment, the dual-channel quadrature demodulation module 201 and the dual-channel dual-phase modulation module 104 share the reference driving signal provided by the signal generator 30. The two modules receive modulation signals with the same frequency and phase, so as to ensure strict coherence and synchronization in the modulation and demodulation process. In addition, the scanning driving and signal synchronous acquisition module 202 is connected with the dual-channel quadrature demodulation module 201 and the laser scanning and telecentric focusing module 106 through electrical signals, respectively, to realize control instruction transmission and acquisition synchronization between the units of the system, and ensure the timing coordination and closed-loop linkage of the scanning execution, signal acquisition and demodulation process in the whole imaging process. Under the timing control of the scanning driving and signal synchronous acquisition module 202, the dual-frequency stimulated Raman signals output by the dual-channel quadrature demodulation module 201 are synchronously acquired and processed, so as to realize the reconstruction of the dual-frequency Raman image.

[0029] This embodiment provides a stimulated Raman scattering (SRS) microscopy imaging system with simultaneous dual Raman frequency shift spectral scanning. The system uses a pulse broadening and spectral focusing module 103 to control the dispersion of two laser pulses, causing their frequency components to broaden and partially overlap along the time axis. A high-precision displacement stage 12 is used to adjust the optical path length of the first laser beam, creating an adjustable frequency difference between the two beams. This frequency difference can match the vibrational modes of target molecules in the sample, thereby inducing stimulated Raman scattering signals.

[0030] Meanwhile, the common-path time-differential dual-frequency difference module 105 set in the system can construct dual laser paths with horizontal and vertical orthogonal polarization states in the common optical path, corresponding to different Raman frequency shift bands, thereby simultaneously exciting and detecting two types of target molecules in the high and low wavenumber ranges.

[0031] By simultaneously acquiring dual-channel data and performing spectral stitching, the complete Raman spectral image of the sample under test can be quickly reconstructed, realizing a stimulated Raman scattering microscopy imaging system with real-time performance, high spatial resolution, and wide-band Raman coverage, while simultaneously performing dual Raman frequency shift spectral scanning.

[0032] Please see Figure 2 In one embodiment, Figure 1 The dual-channel laser synchronous excitation module 101 is a femtosecond laser 1, which generates two femtosecond-level pulsed laser beams with different center wavelengths. These two laser beams are linearly polarized and have the same polarization direction. The two laser beams are then output to the beam polarization control and power distribution module 102 for subsequent modulation and power adjustment processing. The first laser beam is used as the pump light, and the second laser beam is used as a Stokes beam.

[0033] In one embodiment, the beam polarization control and power distribution module 102 includes: a first half-wave plate 2, a second half-wave plate 3, a first polarization beam splitter 4, and a second polarization beam splitter 5. This module is used to control the polarization state of two input laser beams and to dynamically control the power distribution of the two laser beams by changing the polarization direction of the two linearly polarized beams.

[0034] The first half-wave plate 2 and the second half-wave plate 3 are used to change the linear polarization angle of the first fractional-second laser pulse and the second femtosecond laser pulse, respectively. By rotating the first half-wave plate 2 and the second half-wave plate 3, the optical axis direction of the two beams is changed (i.e., the angle of the half-wave plates is adjusted), thereby changing the polarization direction of the two incident femtosecond laser pulses, but the total laser power remains unchanged. The two laser beams are linearly polarized before passing through the half-wave plates, but their linear polarization angles after passing through the two half-wave plates can be inconsistent or consistent.

[0035] The first polarization beam splitter 4 is used to split the first femtosecond pulse laser beam after changing its linear polarization angle into a transmission-reflection beam, thereby achieving power adjustment in the transmission direction. The second polarization beam splitter 5 is used to split the second femtosecond pulse laser beam after changing its linear polarization angle into a transmission-reflection beam, thereby achieving power adjustment in the transmission direction. The two laser beams after power adjustment are incident on the pulse broadening and spectral focusing module 103.

[0036] Two polarizing beam splitters divide the incident light beam into two beams based on the polarization direction of the light: one beam is transmitted light, and the other is reflected light. Therefore, by adjusting the angle of the half-wave plate, the amount of polarized light that can pass through the polarizing beam splitter in the transmission direction can be controlled, thereby enabling continuous and precise adjustment of the output light power (i.e., dynamic control).

[0037] Since the signal generated by this system is related to the intensity of the two input laser beams, the stronger the signal, power adjustment is necessary. However, higher power is not always better. Excessive laser power can cause optical damage to the sample being analyzed, preventing the collection of reliable data for analysis. Therefore, the laser power needs to be adjusted to a suitable range based on the light tolerance of different samples and experience from routine experiments, ensuring that the system generates a sufficiently strong Raman signal without causing optical damage to the sample.

[0038] The pulse broadening and spectral focusing module 103 includes a first glass rod 6 and a second glass rod 7. The first glass rod 6 is used to broaden the dispersion of the first femtosecond pulse laser beam after power adjustment, broadening it into a first picosecond beam. The second glass rod 7 is used to perform the same broadening process on the second femtosecond pulse laser beam after power adjustment, obtaining a second picosecond beam. The broadened first pulse laser beam is guided to the dual-channel biphase modulation module 104 via the first glass rod 6; the second pulse laser beam is similarly guided into the dual-channel biphase modulation module 104 via the second glass rod 7, thereby providing the necessary time-domain broadened pulse input for subsequent dual-channel modulation.

[0039] In this embodiment, the pulse broadening and spectral focusing module 103 is used to introduce linear chirp into the broadband femtosecond pulse after power adjustment, and by adjusting the parameters of the dispersion compensation unit (including the first glass rod 6 and the second glass rod 7), the first laser beam (pump light) and the second laser beam (Stokes light) obtain consistent linear chirp characteristics. Under this condition, the two laser pulses maintain a constant instantaneous frequency difference on the time axis. This makes the bandwidth of the difference frequency signal formed by the two beams much smaller than the difference in the Fourier-limited bandwidth of the two laser pulses, significantly improving the spectral resolution of the system and enabling high-resolution stimulated Raman scattering microscopy imaging.

[0040] After the light beam passes through the glass rod, the chirp effect—different frequency components propagate at different speeds due to their different refractive indices—broadens the spectral width along the time axis. Since the spectral focusing resolution is affected by the chirp parameter, if the chirp parameters of the two glass rods are different, the instantaneous frequency difference between the two beams after passing through the glass rods will be inconsistent in the time dimension, resulting in broadening of the resonant spectral width. The linear chirp parameter can be understood as the relative slope of the chirped pulse in the frequency and time domains. When the linear chirp parameters are consistent, i.e., two parallel lines, the instantaneous frequency difference width of the two beams is consistent, i.e., the distance between all points on the two parallel lines is the same, ensuring that the resonant spectral width is not affected by time.

[0041] In this embodiment, both the first glass rod 6 and the second glass rod 7 serve as dispersion devices to introduce linear chirp into the laser pulse. In other embodiments, the dispersion device can be replaced by other elements with linear dispersion characteristics, such as grating pairs, prism pairs, optical fibers, or other forms of linear chirp control devices, to achieve the same pulse broadening and frequency control functions as in this embodiment.

[0042] In one embodiment, the dual-channel dual-phase modulation module 104 includes: a third half-wave plate 8, a fourth half-wave plate 9, a quarter-wave plate 10, an electro-optic modulator 11, a high-precision displacement stage 12, a dichroic mirror 13, and a 4f system 14, used to achieve polarization control, frequency modulation, and wavelength beam combining of two laser beams. The functions of each component are as follows: the third half-wave plate 8 and the fourth half-wave plate 9 are used to adjust the linearly polarized light of the first picosecond beam and the second picosecond beam with a linear polarization angle of 45°, respectively; the high-precision displacement stage 12 is used to adjust the optical path length of the first picosecond beam (pump light), thereby controlling its overlap position with the second picosecond beam (Stokes light) on the chirped broadened time axis, and simultaneously achieving precise control of the instantaneous frequency difference between the two beams, matching it with the Raman frequency shift of the target molecule, thereby achieving selective excitation of the Raman signal; the quarter-wave plate 10 is used to convert the second picosecond beam before modulation from linearly polarized laser light to circularly polarized light; the electro-optic modulator... 11. High-frequency electrical modulation is applied to the second picosecond beam; the first femtosecond laser pulse is broadened by the first glass rod 6 to obtain the first picosecond beam, and then passes through the third half-wave plate 8 and the high-precision displacement stage 12 in sequence; the second femtosecond laser pulse is broadened by the second glass rod 7 to obtain the second picosecond beam, and then passes through the fourth half-wave plate 9, the quarter-wave plate 10 and the electro-optic modulator 11 in sequence; the two beams after the above path processing have the required time-domain chirp characteristics and polarization state, respectively, and are combined by the dichroic mirror 13 and transmitted coaxially; the combined double beams are collimated by the 4f system 14 and finally incident on the common-path time-difference dual-frequency difference module 105.

[0043] In this embodiment, the electro-optic modulator 11 is a resonant electro-optic modulator with an operating frequency of 10-20MHz. To achieve a high modulation depth under low driving voltage conditions, this system uses a fourth half-wave plate 9 and a quarter-wave plate 10 to modulate the polarization state of the second picosecond beam (Stokes light), converting it from linearly polarized to circularly polarized before it is incident on the electro-optic modulator 11. If the second picosecond beam input to the electro-optic modulator 11 is linearly polarized, a voltage of 0V to V is required to achieve complete modulation of the incident light amplitude. π (or -V) π The driving voltage ranges from -1 to 0V; however, the second picosecond beam input to the electro-optic modulator 11 in this application is circularly polarized light, requiring only an application of -1 / 2V. π up to 1 / 2V π The same modulation effect can be achieved with a driving voltage within the same range. This indicates that to obtain the same modulation depth or a larger range of modulation depth, i.e., a wider range of output light intensity variation, circularly polarized light requires a smaller external driving voltage and has higher energy utilization. This significantly reduces the system's requirement for high-voltage driving and improves modulation efficiency and system stability.

[0044] In one embodiment, the common-path time-differential dual-frequency difference module 105 includes a first birefringent crystal 15 and a second birefringent crystal 16. The first birefringent crystal 15 is used to separate the polarization of the combined laser beam into a horizontal polarization state and a vertical polarization state, and introduces a phase difference to achieve beam path control of the horizontal and vertical polarization states. The second birefringent crystal 16 is used as an optical delay line to introduce a time delay between the two polarized beams, thereby realizing dual-frequency timing control.

[0045] The combined laser beam from the 4f system 14 is incident on the first birefringent crystal 15. Inside the crystal, the horizontally polarized and vertically polarized components are separated into horizontally polarized light and vertically polarized light due to the phase delay caused by the optical path difference. Subsequently, the two polarized light states pass sequentially through the second birefringent crystal 16, and after a specific time delay, enter the laser scanning and telecentric focusing module 106.

[0046] In this embodiment, the first birefringent crystal 15 is used for polarization separation in the dual-channel dual-phase modulation module 104. By introducing a phase difference into the laser beam, the combined laser beam is separated into horizontally polarized light and vertically polarized light. The intensity change of the horizontally polarized Stokes light is sin(ωt+α), and the intensity change of the vertically polarized pump light is -sin(ωt+α), where ω = 2πf, f is the modulation frequency, and α is the initial phase. The pump light after beam splitting is consistent with the Stokes light that propagates along the same path in polarization state. Therefore, the influence of the polarization depolarization ratio of the Raman signal does not need to be considered during signal detection, which helps to improve system stability and modulation signal-to-noise ratio.

[0047] In this embodiment, by controlling the optical path difference introduced into the first picosecond beam (pump beam) by the high-precision displacement stage 12, the overlap position of the first picosecond beam (Stokes beam) with the second picosecond beam (Stokes beam) on the chirped broadened time axis is controlled. Simultaneously, precise control of the instantaneous frequency difference between the two beams is achieved, matching it with the Raman frequency shift of the target molecule, thereby achieving selective excitation of the Raman signal. Then, through the first birefringent crystal 15 and the second birefringent crystal 16 in the common-path time-difference dual-frequency difference module 105, the common-path combined laser beam is separated into horizontally polarized light and vertically polarized light. The resonant frequencies of the Stokes beam and the pump beam are locked in the two molecular channels located in the high and low beams of the sample under test. Therefore, by controlling the position of the high-precision displacement stage 12 and introducing time delay control, the spectra of molecules in the high and low beams of the sample under test can be simultaneously scanned. Then, by using a spectral stitching method, the two spectra are stitched together to obtain a complete spectral image of the sample under test. Furthermore, in Raman imaging, the same method can be used to acquire Raman spectral sequence images, extract the Raman frequency shift spectrum from the image, and then stitch the two channel spectra together.

[0048] In one embodiment, the laser scanning and telecentric focusing module 106 includes an XY scanning galvanometer 17, a telecentric scanning lens 18, a sleeve lens 19, and a microscope objective 20. The XY scanning galvanometer 17 controls the scanning position of the laser beam on the sample plane, achieving two-dimensional scanning of the sample in the XY direction. The telecentric scanning lens 18 ensures the beam enters the sleeve lens in a collimated state and corrects the off-axis phase difference introduced by the scanning galvanometer. The sleeve lens 19 further optimizes the optical path, keeping the focused spot size of the laser on the sample plane constant throughout the entire field of view, while avoiding astigmatism or spot distortion at the edges of the field of view. The microscope objective 20 focuses the laser beam.

[0049] The laser beam, after being processed by the common-path time-varying dual-frequency difference module 105, is transmitted through the XY scanning galvanometer 17, the telecentric scanning lens 18, the sleeve lens 19, and the microscope objective 20 in sequence to achieve two-dimensional scanning excitation of the sample 21. The stimulated Raman signal generated by this excitation is then detected and analyzed by the dual-channel orthogonal demodulation module 201.

[0050] In one embodiment, the dual-channel orthogonal demodulation module 201 includes a condenser lens 22, a focusing lens 23, a filter 24, a polarization beam splitter 25, a dual-channel photodetector 26, a phase shifter 27, an adder 28, and a lock-in amplifier 29; wherein, the condenser lens 22 is used to collect the forward scattered signal passing through the sample; the focusing lens 23 is used to focus the signal beam; the filter 24 is used to filter out other beams besides the signal beam; the polarization beam splitter 25 is used to separate the signal beam into horizontally polarized signal beam and vertically polarized signal beam; the dual-channel photodetector 26... The detector 26 is used to detect two polarized signal beams and convert them into a first voltage signal and a second voltage signal. The phase shifter 27 is used to add π / 2 phase to the first voltage signal and modulate its intensity. The adder 28 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. The lock-in amplifier 29 is used to orthogonally demodulate the linearly superimposed horizontal polarized signal and the vertical polarized signal, and output two Raman frequency shift signals corresponding to the two polarization states.

[0051] In this embodiment, the forward-scattered stimulated Raman signal generated by sample 21 is collected by condenser lens 22, focused by focusing lens 23, and then passed through filter 24 to filter out the excitation light background. The filtered signal light is then incident on polarization beam splitter 25 and separated into two signals: a horizontally polarized state and a vertically polarized state. The two beams are received by dual-channel photodetector 26 and converted into a first voltage signal and a second voltage signal, respectively. The first voltage signal is given a π / 2 phase delay by phase shifter 27 and then linearly superimposed with the second voltage signal in adder 28. Finally, the superimposed signal is input to lock-in amplifier 29 to complete the orthogonal demodulation and extraction of the dual-polarization Raman signal.

[0052] In this embodiment, after the second laser beam (Stokes beam) is scattered by sample 21, its signal portion is effectively filtered out by a bandpass filter 24 with an optical density (OD) greater than 6, retaining only the pump light signal for subsequent detection. The first laser beam (pump light), after being separated by polarization beam splitter 25, is then incident on a dual-channel photodetector 26 for detection. The photodetector 26 contains two photosensitive elements with a photosensitive area of ​​1×1 cm². 2A photodiode is used, and a -24V reverse bias voltage is applied through an external DC power supply to improve its response sensitivity. The photocurrent signal output by the diode is converted into a voltage signal by a transimpedance amplifier with a bandwidth of 100MHz. One of the voltage signals is subjected to a π / 2 phase delay by a phase shifter 27, changing its modulation form to -sin(ωt+α+π / 2), i.e., -cos(ωt+α). This signal is then linearly superimposed with the voltage signal output from the other channel in an adder 28, and both are input to a lock-in amplifier 29 for quadrature demodulation. After demodulation, the system obtains two mutually orthogonal stimulated Raman loss (SRL) signals, which can be output as the X and Y components of the demodulation result, respectively, thereby realizing the extraction of the target Raman signal.

[0053] In this embodiment, the two orthogonal biphase SRL signals can be expressed as formula (1):

[0054]

[0055] Where β is the phase of the reference signal provided to the lock-in amplifier, f is the modulation frequency of the electro-optic modulator when modulating the intensity of the second laser beam, t is time, and I SRL (t) represents the energy change caused by the first laser beam passing through the sample, which is the stimulated Raman loss signal; I(Ω1) and I(Ω2) correspond to the vibrational signals of the two molecules, respectively;

[0056] The signal generator 30 provides a reference signal to the lock-in amplifier 29, with its phase set to β, to demodulate the in-phase (X) and quadrature (Y) components of the two SRL signals, thereby obtaining the vibrational signals I(Ω1) and I(Ω2) of the two types of molecules respectively. Compared with the customized dual-channel lock-in amplifier scheme, the detection circuit used in this embodiment is simpler in terms of design, printing and welding, and has a lower cost. It is also easy to integrate and deploy in various spectral focusing stimulated Raman scattering microscopy imaging systems, and has good system compatibility and scalability.

[0057] The dual-channel quadrature demodulation module 201 and the dual-channel biphase modulation module 104 share a reference drive signal provided by the signal generator 30. Both modules receive modulation signals of the same frequency and phase to ensure strict coherence and synchronization in the modulation and demodulation processes. Furthermore, the scan drive and signal synchronization acquisition module 202 is electrically connected to the dual-channel quadrature demodulation module 201 and the laser scanning and telecentric focusing module 106, respectively, to achieve synchronized transmission and acquisition of control commands between system units, ensuring timing coordination and closed-loop linkage of the scanning execution, signal acquisition, and demodulation processes throughout the imaging process. Under the timing control of the scan drive and signal synchronization acquisition module 202, the dual-frequency stimulated Raman signal output by the dual-channel quadrature demodulation module 201 is synchronously acquired and processed, thereby achieving the reconstruction of the dual-frequency Raman image.

[0058] In one embodiment, the signal acquisition and scanning synchronization module 202 includes: a signal generator 30, a multi-functional acquisition card, and a controller 32; wherein...

[0059] Signal generator 30 is used to simultaneously input two analog signals with the same parameters to electro-optic modulator 11 and lock-in amplifier 29;

[0060] The multi-functional acquisition card and controller 32 are used to drive the scanning galvanometer 17 in the laser scanning and telecentric focusing module 106, and to acquire the two orthogonal voltage signals output by the dual-channel orthogonal demodulation module 201, so as to realize the synchronous control of signal output and acquisition; and to control the movement of the high-precision displacement stage 12 and sample stage in the pulse broadening and spectral focusing module 103, as well as the focusing and displacement adjustment of the microscope objective 20 in the laser scanning and telecentric focusing module 106.

[0061] In addition, voltage amplifier 31 is used to provide the required voltage to signal generator 30 and electro-optic modulator.

[0062] In all embodiments of this application, to facilitate laser transmission, the propagation path of the laser can be adjusted by setting several reflectors F, such as... Figure 2 As shown, this enables effective control of the laser direction.

[0063] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0064] It should be understood that although the terms first, second, third, etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, first information may also be referred to as second information without departing from the scope of this invention, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to a determination."

[0065] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A system for simultaneous dual-Raman-shift spectroscopy scanning stimulated Raman scattering microscopy, comprising a laser light source driving and modulating unit, a signal detecting and data processing unit, characterized in that, the laser light source driving and modulating unit comprises: a dual-channel laser synchronous excitation module for generating two laser beams with different wavelengths, both of which are linearly polarized light with consistent polarization direction; a beam polarization control and power distribution module for adjusting the power of the two laser beams by controlling the polarization state of the two laser beams; a pulse broadening and spectral focusing module for making the frequency components of the two laser beam pulses have a certain distribution and time overlap on the time axis; a dual-channel dual-phase modulation module for high-frequency intensity modulation of the two laser beams; a common-path asynchronous dual-frequency difference module for constructing two laser beams with horizontal and vertical polarizations in the laser common path, thereby respectively matching the specific Raman shift of two types of molecules; a laser scanning and telecentric focusing module for focusing the laser beam to the sample plane and realizing two-dimensional scanning excitation of the sample to generate stimulated Raman signals, while effectively correcting the phase difference; the signal detecting and data processing unit comprises: a dual-channel quadrature demodulation module for realizing synchronous quadrature demodulation and amplitude recovery of Raman shift signals in different channels, and outputting two Raman shift signals corresponding to two polarization states, respectively; a scanning driving and signal synchronous acquisition module for driving the laser scanning and telecentric focusing module, acquiring the demodulation signals output by the dual-channel quadrature demodulation module, and realizing the timing unification and automatic control of the system multi-optical and electronic units.

2. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 1, wherein, The two laser beams include a first femtosecond pulsed laser used as pump light and a second femtosecond pulsed laser used as Stokes light; the beam polarization control and power distribution module comprises: a first half-wave plate for changing the linear polarization angle of the first femtosecond pulsed laser; a second half-wave plate for changing the linear polarization angle of the second femtosecond pulsed laser; a first polarization beam splitter for transmitting-reflection splitting of the first femtosecond pulsed laser after changing the linear polarization angle, to realize power adjustment in the transmission direction; a second polarization beam splitter for transmitting-reflection splitting of the second femtosecond pulsed laser after changing the linear polarization angle, to realize power adjustment in the transmission direction; The two laser beams after power adjustment are incident to the pulse broadening and spectral focusing module.

3. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 2, wherein, The pulse broadening and spectral focusing module comprises: a first glass rod for broadening the first femtosecond pulsed laser after power adjustment into a first picosecond beam and incident to the dual-channel dual-phase modulation module; a second glass rod for broadening the second femtosecond pulsed laser after power adjustment into a second picosecond beam and incident to the dual-channel dual-phase modulation module.

4. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 3, wherein, The dual-channel dual-phase modulation module comprises: a third half-wave plate for adjusting the linear polarization angle of the first picosecond beam to 45 degrees; a fourth half-wave plate for changing the linear polarization angle of the second picosecond beam to 45 degrees; A high-precision displacement stage is configured to adjust an optical path of the first picosecond laser beam, so that the spectra of the two laser beams are overlapped on the time axis after being chirped, and the frequency difference between the two laser beams is changed by changing the optical path of the first picosecond laser beam, so that the Raman signal of a specific frequency molecule is collected. A quarter-wave plate is configured to convert the polarization state of the second picosecond laser beam from linear polarization to circular polarization. An electro-optical modulator is configured to perform high-frequency intensity modulation on the circularly polarized second picosecond laser beam. A dichroic mirror is configured to combine the linearly polarized first picosecond laser beam and the circularly polarized second picosecond laser beam. A 4f system is configured to collimate the combined laser beam and then input the collimated laser beam into the common-path asynchronous dual-frequency difference module.

5. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 4, wherein, The electro-optical modulator is a resonant electro-optical modulator with a frequency of 10-20 MHz.

6. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 4, wherein, The common-path asynchronous dual-frequency difference module comprises: A first birefringent crystal is configured to separate the combined laser beam into horizontally polarized light and vertically polarized light that are transmitted in a common path. A second birefringent crystal is configured to set an optical delay line, so that the horizontal polarization and the vertical polarization of the two laser beams satisfy the Raman frequency shift of two types of molecules, and then the laser beam is input into a laser scanning and telecentric focusing module.

7. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 1, wherein, The laser scanning and telecentric focusing module comprises: An X-Y scanning galvanometer is configured to control the scanning position of the laser beam on the sample plane, so as to realize two-dimensional scanning of the sample in the X-Y direction. A telecentric scanning lens is configured to collimate the laser beam and correct the off-axis phase difference introduced by the scanning galvanometer. A sleeve lens is configured to keep the size of the focused laser spot on the sample plane constant in the entire field of view, while avoiding astigmatism or spot distortion in the edge field of view. A microscope objective is configured to focus the laser beam to generate a stimulated Raman signal.

8. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 7, wherein, The dual-channel quadrature demodulation module comprises: A condenser is configured to collect the forward scattering signal that passes through the sample. A focusing lens is configured to focus the signal beam. A filter is configured to filter out other light beams except the signal light. A polarization beam splitter is configured to separate the signal light into horizontally polarized signal light and vertically polarized signal light. A dual-channel photodetector is configured to detect the horizontally polarized signal light and the vertically polarized signal light, and convert the two polarized light beams into a first voltage signal and a second voltage signal. A phase shifter is configured to increase the phase of the first voltage signal by π / 2, and modulate the intensity of the first voltage signal. An adder is configured to linearly superimpose the first voltage signal output by the phase shifter and the second voltage signal output by the dual-channel photodetector. A lock-in amplifier is configured to quadrature demodulate the input linearly superimposed horizontally polarized signal and vertically polarized signal, and output two Raman frequency shift signals corresponding to the two polarized states.

9. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 7, wherein, The two quadrature stimulated Raman loss signals demodulated by the dual-channel quadrature demodulation module are shown in the following formula: β is the phase of the reference signal provided to the lock-in amplifier, f is the modulation frequency of the electro-optical modulator for intensity modulation of the second laser beam, and t is time. I SRL (t) is the energy change caused by the first laser beam passing through the sample, i.e. the stimulated Raman loss signal; I(Ω1) and I(Ω2) correspond to two molecular vibration signals, respectively.

10. The simultaneous dual Raman-shifted optical spectrum scanning stimulated Raman scattering microscopy system of claim 8, wherein, The scanning driving and signal synchronous acquisition module comprises: A signal generator is configured to simultaneously input two analog signals with the same parameters to the electro-optical modulator and the lock-in amplifier. The multifunctional acquisition card and the controller are used for collecting the voltage signals of the two-way orthogonal output in the X-Y scanning galvanometer and the double-channel orthogonal demodulation module; and realize the motion control of the high-precision displacement table, the sample carrier table and the microscope objective, and the synchronous control of the output signal acquisition.

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