Background signal suppression system of coherent Raman scattering microscope

Abstract

The invention provides a background signal suppression system of a coherent Raman scattering microscope, and a laser light source module of the background signal suppression system can provide a laser light source comprising first Stokes light and first pump light; the laser adjusting module can be used for respectively adjusting the first stokes light and the first pump light to obtain second stokes light and second pump light; the frequency modulation module can perform frequency modulation processing and laser excitation processing on the second stokes light and the second pump light respectively to obtain a modulation signal comprising a background signal and a resonance signal; the filtering module can remove the background signal to obtain a resonance signal. Through combination of the frequency modulation module and the chirped femtosecond laser, modulation of the detection frequency is realized through a simple structure, then real-time elimination of background signals is realized, and the detection sensitivity and imaging quality of the system are greatly improved.

Description

Technical Field

[0001] The present application relates to the field of optical imaging technology, and in particular to a background signal suppression system for a coherent Raman scattering microscope. Background Art

[0002] Coherent Raman Scattering Microscopy (CRS) is a microscopic imaging technology that has developed rapidly in recent years and is widely used in biology, materials science and other fields. However, this technology still faces the problem of signal background interference in practical applications, especially in coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) imaging.

[0003] The non-resonant background signals in CARS signals will affect signal sensitivity and interfere with spectrum analysis, while the SRS signals contain background signals such as transient absorption, photothermal and cross-phase modulation signals.

[0004] Currently, the equipment for suppressing and eliminating background signals is complex and the clarity and quality of imaging are low. Summary of the invention

[0005] Based on this, it is necessary to provide a background signal suppression system for a coherent Raman scattering microscope that can improve imaging clarity and quality and has a simple device structure in order to address the above technical problems.

[0006] In a first aspect, the present application provides a background signal suppression system for a coherent Raman scattering microscope, the background signal suppression system comprising a laser light source module, a laser adjustment module, a frequency modulation module and a filter module;

[0007] A laser light source module, used to provide a laser light source; the laser light source includes a first Stokes light and a first pump light;

[0008] A laser adjustment module, used for adjusting the first Stokes light and the first pumping light respectively to obtain the second Stokes light and the second pumping light;

[0009] A frequency modulation module is used to perform frequency modulation processing and laser excitation processing on the second Stokes light and the second pump light respectively to obtain a modulation signal; the modulation signal includes a background signal and a resonance signal;

[0010] The filtering module is used to remove the background signal and obtain the resonance signal.

[0011] In one embodiment, the frequency modulation module includes a pump light adjustment module, a Stokes light adjustment module, a chirp broadening module and a microscope module;

[0012] A pump light adjustment module, used for performing time delay processing on the second pump light to obtain a third pump light;

[0013] A Stokes light adjustment module is used to adjust the polarization state and laser irradiation position of the second Stokes light, and to filter the adjusted second Stokes light to obtain a third Stokes light and a fourth Stokes light;

[0014] A chirp stretching module is used to combine the third pump light, the third Stokes light, and the fourth Stokes light, and to stretch the pulse width of the combined first combined light to obtain a second combined light;

[0015] The microscope module is used for performing excitation processing on the second combined light to obtain a modulated signal.

[0016] In one embodiment, the pump light adjustment module includes a plurality of first reflectors, each of which is used to change the propagation direction of the second pump light and perform time delay processing on the second pump light.

[0017] In one embodiment, the Stokes light adjustment module comprises a polarization beam splitter cube, a quarter wave plate, a high-speed multi-faceted scanning mirror, a light screening device and a second reflector arranged in sequence;

[0018] A polarization beam splitter cube is used to split the second Stokes light to obtain parallel polarized light and vertical polarized light;

[0019] A quarter wave plate is used to adjust the polarization state of parallel polarized light to obtain one of circular polarized light and linear polarized light;

[0020] a high-speed multi-faceted scanning mirror for scanning the circularly polarized light to the light screening device and receiving the reflected light reflected by the second reflector;

[0021] A light screening device, used for screening the circularly polarized light to obtain a first polarized light and a second polarized light;

[0022] The second reflector is used for reflecting the first polarized light and the second polarized light to obtain the third Stokes light and the fourth Stokes light.

[0023] In one embodiment, the second reflector includes a plane reflector and a large-size ruled grating.

[0024] In one embodiment, the optical screening device comprises a lens and a mask plate which are stacked;

[0025] A lens, used for receiving circularly polarized light of multiple angles transmitted by the high-speed multi-faceted scanning mirror, and transmitting each circularly polarized light to the mask plate;

[0026] The mask plate is used to screen each circularly polarized light to obtain the first polarized light and the second polarized light.

[0027] In one embodiment, the chirp broadening module comprises a dichroic mirror, a glass rod and a third reflector arranged in sequence;

[0028] A dichroic mirror is used for combining the third pump light, the third Stokes light, and the fourth Stokes light to obtain a first combined light;

[0029] A glass rod, used for performing chirp broadening processing on the first combined light to obtain a second combined light;

[0030] The third reflecting mirror is used to transmit the second combined light to the microscope module.

[0031] In one embodiment, the microscope module comprises a first optical element group, a stage, and a second optical element group arranged in sequence;

[0032] A first optical element group is used for focusing the second combined light to obtain focused light, and focusing the focused light onto a sample on the stage;

[0033] A stage for carrying a sample; the sample is used to react with the focused light to generate a modulated signal;

[0034] The second optical element group is used to transmit the modulated signal to the filtering module.

[0035] In one embodiment, the laser adjustment module comprises a half wave plate, a polarization beam splitter cube and a lens group arranged in sequence;

[0036] A half wave plate is used to adjust the polarization direction of the Stokes light to obtain adjusted Stokes light;

[0037] A polarization beam splitter cube is used to perform power adjustment and screening processing on the adjusted Stokes light to obtain screened Stokes light;

[0038] The lens group is used to expand the screened Stokes light to obtain the second Stokes light.

[0039] In one embodiment, the filtering module includes a bandpass filter and a signal detector;

[0040] A bandpass filter is used to filter the background signal to obtain a filtered light signal;

[0041] The signal detector is used to perform photoelectric conversion processing on the filtered optical signal to obtain a resonance signal.

[0042] In one embodiment, the background signal suppression system further includes a phase-locked amplifier, which is used to perform phase locking and demodulation processing on the modulated signal to obtain a demodulation result.

[0043] The background signal suppression system of the above-mentioned coherent Raman scattering microscope, the laser light source module of the background signal suppression system can provide a laser light source including a first Stokes light and a first pump light; the laser adjustment module can adjust the first Stokes light and the first pump light respectively to obtain a second Stokes light and a second pump light; the frequency modulation module can perform frequency modulation processing and laser excitation processing on the second Stokes light and the second pump light respectively to obtain a modulated signal including a background signal and a resonance signal; the filtering module can remove the background signal to obtain a resonance signal. The present application realizes the modulation of the detection frequency with a simple structure through the combination of the frequency modulation module and the chirped femtosecond laser, thereby realizing the real-time elimination of the background signal, greatly improving the detection sensitivity and imaging quality of the system. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of a background signal suppression system in one embodiment;

[0045] Figure 2 is a schematic diagram of a frequency modulation module in one embodiment;

[0046] Figure 3 is a schematic diagram of a pump light adjustment module in one embodiment;

[0047] Figure 4 is a schematic diagram of a Stokes light adjustment module in one embodiment;

[0048] Figure 5 is a schematic diagram of a high-speed resonant galvanometer in one embodiment;

[0049] Figure 6 A schematic diagram of a large-size ruled grating in one embodiment;

[0050] Figure 7 is a schematic diagram of a chirp stretching module in one embodiment;

[0051] Figure 8 is a schematic diagram of a microscope module in one embodiment;

[0052] Fig. 9 is a schematic diagram of a laser adjustment module in one embodiment;

[0053] Fig.10 is a schematic diagram of a filtering module in one embodiment;

[0054] Fig.11is an overall schematic diagram of a background signal suppression system in one embodiment;

[0055] Fig.12 FIG. 1 is a schematic diagram of chirp broadening of a femtosecond laser in one embodiment;

[0056] Fig.13 It is a schematic diagram of the technical principle in one embodiment. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0058] First of all, before specifically introducing the technical solution of the embodiments of the present application, the technical background based on the embodiments of the present application is introduced.

[0059] Coherent Raman scattering (CRS) microscopy technology images biological samples by measuring the vibration of chemical bonds of molecules. It has the characteristics of being label-free, non-invasive, and highly chemically specific. CRS microscopy uses two beams of excitation light to illuminate the sample, namely, the pump light ( ) and Stokes light ( ). When the two laser beams interact with the sample being detected, When the corresponding Raman shift matches the molecular vibration mode of the detected sample, a CRS signal will be generated. CRS includes two types of imaging: coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). For CARS signals, an anti-Stokes light signal with a frequency of . For SRS, the intensity of the pump light will attenuate while the intensity of the Stokes light will gain. The SRS signal can be obtained by detecting the attenuation of the intensity of the pump light. Since the main factor affecting molecular vibration is the chemical bond, and the vibration modes and frequencies of different chemical bonds are relatively fixed, the CRS microscope can perform label-free imaging of samples and has strong chemical specificity. Its detection sensitivity is also stronger than that of the spontaneous Raman microscope. However, there is a background signal in the CRS signal, which affects its detection sensitivity and detection imaging effect.

[0060] There will be a non-resonant background signal in the CARS signal, which does not carry any chemical specific information and will distort and mask the resonant signal that needs to be detected. The presence of non-resonant background signals will also greatly limit the detection sensitivity of the CARS microscope and cause great difficulties in spectral analysis. There will be background signals such as transient absorption signals, photothermal signals, and cross-phase modulation signals in the SRS signal, which will cause great interference to SRS imaging and affect its detection sensitivity and chemical specificity.

[0061] Currently, the technologies used to suppress or eliminate the background signal of CRS microscope are mainly divided into: polarization direction resolution method (applied to CARS), time resolution method (applied to CARS), reverse detection method (applied to CARS) and frequency modulation method (applied to CARS and SRS).

[0062] The CARS microscope that distinguishes polarization direction uses the inconsistency between the polarization direction of the resonant signal and the polarization direction of the non-resonant background signal to suppress the non-resonant background signal. Usually, a polarizer is added in front of the signal detector to make its polarization direction perpendicular to the polarization direction of the non-resonant background signal, thereby filtering out the non-resonant background signal. However, due to the birefringence of the optical devices in the optical path and the interference with the polarization direction at the tight focus, a part of the non-resonant background signal will leak. This method will weaken the resonant signal while suppressing the non-resonant background signal.

[0063] When the pump light and Stokes light excite the sample at the same time, non-resonant background signals will also be generated at the same time. The generation and disappearance of non-resonant background signals is an instantaneous process, while the resonant signal usually lasts for several picoseconds. By delaying the time when the detection beam reaches the sample relative to the pump light and Stokes light, time-resolved CARS microscopy can reduce the impact of non-resonant background signals on CARS signals. However, this technology still has limitations. The resonant signal decays exponentially with time, resulting in a lower signal level. The detection of the signal also depends on the phase shift time of the detected Raman mode.

[0064] The reverse signal intensity of the reverse detection CARS microscope is much smaller than the forward signal intensity. And due to the phase matching condition of CARS, only objects smaller than the wavelength of the incident light will produce reverse signals, which limits its scope of use.

[0065] The frequency modulation method can effectively suppress or even eliminate the background signal, and can eliminate the background signal in real time and ensure that the required CRS signal is not affected. However, the existing frequency modulation method is too complicated to implement. Usually, two different pump lights or Stokes lights are obtained by adding lasers and redesigning the laser resonant cavity to modulate the detection frequency, or the detection frequency is modulated by combining polarization modulation elements with hyperspectral technology. This increases the complexity of the system, makes the calibration of the optical path and the use of the equipment more difficult, and greatly limits the application of frequency modulation schemes.

[0066] Based on this, the present application provides a background signal suppression system for a coherent Raman scattering microscope, aiming to solve the above technical problems.

[0067] In one embodiment, Figure 1 As shown, the present application provides a background signal suppression system for a coherent Raman scattering microscope, and the background signal suppression system includes a laser light source module 1, a laser adjustment module 2, a frequency modulation module 3 and a filtering module 4.

[0068] The laser light source module 1 is used to provide a laser light source, wherein the laser light source includes a first Stokes light and a first pump light.

[0069] The laser light source module 1 is mainly responsible for providing a laser light source to excite molecules in the sample to generate a CRS signal.

[0070] Laser: Select an appropriate laser type (such as solid-state laser or fiber laser) to generate the first pump light and the first Stokes light with specific wavelengths.

[0071] Wavelength selection: The first pump light usually has a short wavelength to efficiently excite the sample, while the first Stokes light is relatively long to facilitate the acquisition of CRS signals.

[0072] The laser adjustment module 2 is used to adjust the first Stokes light and the first pumping light respectively to obtain the second Stokes light and the second pumping light.

[0073] Specifically, the first Stokes light and the first pump light are optically adjusted to improve the quality and effectiveness of the laser.

[0074] The composition may include:

[0075] Lens system: Lenses focus, expand or converge the light beam to ensure that the incident light is concentrated on the sample surface.

[0076] Adjustment device: including mechanical devices such as angle adjustment and pitch adjustment, which can dynamically adjust the beam path to adapt to different sample characteristics.

[0077] The frequency modulation module 3 is used to perform frequency modulation processing and laser excitation processing on the second Stokes light and the second pump light respectively to obtain a modulation signal, wherein the modulation signal includes a background signal and a resonance signal.

[0078] Components can include frequency modulators, high-speed scanning mirrors, etc., which control the phase and amplitude of the signal by adjusting the frequency of light.

[0079] Laser excitation processing: Through excitation processing operations (such as pulse excitation), the CRS signal of a specific wavelength is enhanced.

[0080] The filtering module 4 is used to remove the background signal to obtain the resonance signal.

[0081] The composition may include:

[0082] Optical filters: such as bandpass filters or longpass filters, can selectively pass signals within a specific wavelength range and remove unnecessary background light.

[0083] Digital signal processing unit: The signal is further processed through digital filtering algorithms (such as fast Fourier transform) to improve the sensitivity and accuracy of the system.

[0084] The background signal suppression system of the above-mentioned coherent Raman scattering microscope, the laser light source module of the background signal suppression system can provide a laser light source including a first Stokes light and a first pump light; the laser adjustment module can adjust the first Stokes light and the first pump light respectively to obtain a second Stokes light and a second pump light; the frequency modulation module can perform frequency modulation processing and laser excitation processing on the second Stokes light and the second pump light respectively to obtain a modulated signal including a background signal and a resonance signal; the filtering module can remove the background signal to obtain a resonance signal. The present application realizes the modulation of the detection frequency with a simple structure through the combination of the frequency modulation module and the chirped femtosecond laser, thereby realizing the real-time elimination of the background signal, greatly improving the detection sensitivity and imaging quality of the system.

[0085] In an exemplary embodiment, based on the above embodiment, see Figure 2 The frequency modulation module 3 of the embodiment of the present application includes a pump light adjustment module 31 , a Stokes light adjustment module 32 , a chirp broadening module 33 and a microscope module 34 .

[0086] The pump light adjustment module 31 is used to perform time delay processing on the second pump light to obtain a third pump light.

[0087] The pump light adjustment module 31 performs time delay processing on the second pump light to generate a third pump light. By adjusting the pulse delay of the pump light, the interaction between the two light sources can be optimized, thereby enhancing the generation efficiency of the CRS signal.

[0088] The Stokes light adjustment module 32 is used to adjust the polarization state and laser irradiation position of the second Stokes light, and to filter the adjusted second Stokes light to obtain the third Stokes light and the fourth Stokes light.

[0089] The Stokes light adjustment module 32 adjusts the polarization state and laser irradiation position of the second Stokes light. By precisely controlling the polarization angle and irradiation position, the optimal interaction with the pump light can be ensured. In addition, the module also screens the adjusted second Stokes light to separate the third Stokes light and the fourth Stokes light suitable for detection. This process ensures the quality of the signal and its corresponding optical properties.

[0090] The chirp stretching module 33 is used to combine the third pump light, the third Stokes light and the fourth Stokes light, and to stretch the pulse width of the combined first combined light to obtain the second combined light.

[0091] The chirp stretching module 33 combines the third pump light, the third Stokes light and the fourth Stokes light, and stretches the pulse width of the combined first combined light to obtain the second combined light. By increasing the pulse width, the interaction time between the light and the sample can be effectively increased, thereby enhancing the detection sensitivity of the CRS signal.

[0092] The microscope module 34 is used to perform excitation processing on the second combined light to obtain a modulated signal.

[0093] The microscope module 34 performs excitation processing on the second combined light to generate a modulation signal. The signal includes both a background signal and a resonance signal. The design of the microscope module ensures effective light signal transmission, improves the resolution of imaging and the selectivity of the target signal.

[0094] In the embodiment of the present application, the optimal interaction between the pump light and the Stokes light is ensured through time delay processing and polarization adjustment, thereby improving the intensity of the CRS signal. The chirp broadening technology is used to extend the excitation time, thereby increasing the chance of exciting the target molecules and improving the signal-to-noise ratio. The excitation processing of the microscope module ensures high-quality CRS image capture.

[0095] In an exemplary embodiment, based on the above embodiment, see Figure 3The pump light adjustment module of the embodiment of the present application includes a plurality of first reflectors M, each of which is used to change the propagation direction of the second pump light and perform time delay processing on the second pump light.

[0096] The pump light adjustment module performs direction adjustment and time delay processing on the second pump light to optimize its interaction with the Stokes light, thereby improving the intensity and imaging quality of the CRS signal.

[0097] The pump light adjustment module is composed of a plurality of first reflectors. Each reflector performs the following functions in this module:

[0098] Changing the propagation direction: The mirror refracts the laser beam to change the propagation direction of the pump light so that it can be accurately aligned with other light sources (such as Stokes light) in the sample. This process can achieve flexible optical path design to meet different experimental needs.

[0099] Time delay processing: Under a specific configuration, multiple reflections using multiple first reflectors can effectively control the propagation path of light, thereby introducing time delay. By precisely controlling the time delay, it is possible to ensure that the second pump light collides with the adjusted Stokes light at the best time, thereby improving the excitation efficiency.

[0100] Specific implementation details may include:

[0101] Reflector angle: The angle of each first reflector is designed according to the transmission requirements of the light beam, usually including different reflection angle settings to achieve adjustment of the light path in different experimental environments.

[0102] High-reflection coating: The mirrors can be equipped with a high-reflection coating, which ensures that the energy loss of the light beam is reduced, thus increasing the power of the pump light.

[0103] Implementation of time delay:

[0104] Path Length Control: Adjusting the position of mirrors or increasing the path length in a multi-mirror configuration can introduce a precise time delay by delaying the time it takes the beam to reach the sample.

[0105] The design of the pump light adjustment module in the embodiment of the present application optimizes the propagation direction and time delay of the pump light through the use of multiple first reflectors, laying a solid foundation for subsequent CRS signal detection.

[0106] In an exemplary embodiment, based on the above embodiment, see Figure 4The Stokes light adjustment module of the present application includes a polarization beam splitter cube PBS, a quarter wave plate QWP, a high-speed multi-faceted scanning mirror PS, a light screening device and a second reflector M which are arranged in sequence. The high-speed scanning mirror can also be used as follows Figure 5 The second reflector M can be placed in a variety of ways.

[0107] The polarization beam splitter cube is used to split the second Stokes light to obtain parallel polarized light and vertical polarized light.

[0108] When the second Stokes light enters the polarization beam splitter cube at a fixed incident angle, the optical properties of the material are used to effectively split the light beam into two paths according to the polarization direction of the incident light: one is parallel polarized light and the other is vertical polarized light. This beam splitting method lays the foundation for subsequent polarization adjustment.

[0109] A quarter wave plate is used to adjust the polarization state of parallel light to obtain one of circularly polarized light and linearly polarized light.

[0110] Specifically, if the polarization state is parallel light, after passing through a quarter-wave plate, it can be converted into left-handed or right-handed circularly polarized light after appropriate phase delay to enhance the interaction with the sample.

[0111] The high-speed multi-faceted scanning mirror is used to scan the circularly polarized light to the light screening device and receive the reflected light reflected by the second reflector.

[0112] The high-speed multi-faceted scanning mirror can be driven by a motor to rotate rapidly, so that the excitation light illuminates the sample at different angles, thereby achieving full-field scanning imaging. The high speed of the rotating mirror ensures that multiple data points can be obtained in a short time, which is suitable for dynamic imaging.

[0113] The optical screening device is used for screening circularly polarized light to obtain first polarized light and second polarized light.

[0114] The optical screening device can be an interference filter, a lens, a mask, etc., which can select light of a specific wavelength by adjusting its central wavelength and bandwidth, thereby achieving effective separation of the first polarized light (such as linearly polarized light) and the second polarized light (such as linearly polarized light in another direction), thereby improving the selectivity and quality of the signal.

[0115] The second reflector is used for reflecting the first polarized light and the second polarized light to obtain the third Stokes light and the fourth Stokes light.

[0116] The second reflector reflects the screened light at a specific reflection angle to ensure that no additional phase delay is introduced in the light path, thereby obtaining high-quality third Stokes light and fourth Stokes light. These light sources will be used for subsequent signal processing and imaging.

[0117] In an exemplary embodiment, based on the above embodiment, the second reflector may include a plane reflector M and a large-size ruled grating G. The large-size ruled grating G is as follows: Figure 6 shown.

[0118] Among them, the plane reflector is usually made of glass material with a high reflective coating to ensure the lowest light reflection loss. It can guide the light beam quickly and effectively. It is suitable for conventional imaging in a static environment.

[0119] Large-size ruled gratings can be used as plane reflectors by increasing the scale of the ruled lines of the grating. This allows light beams at different positions to be focused onto different ruled lines after screening, thereby controlling their optical path length.

[0120] The embodiment of the present application significantly improves the performance of the coherent Raman scattering microscope system by introducing a plane reflector and a large-size ruled grating as a second reflector.

[0121] In an exemplary embodiment, based on the above embodiments, the optical screening device of the embodiment of the present application includes a lens and a mask plate Mask that are stacked.

[0122] The lens is used to receive circularly polarized light of multiple angles transmitted by the high-speed multi-faceted scanning mirror and transmit each circularly polarized light to the mask plate.

[0123] The lens generally adopts an aspherical design to ensure that light at different incident angles can be effectively focused and reduce optical distortion. The lens material usually chooses optical glass with high light transmittance to ensure good transmittance for circularly polarized light at multiple wavelengths.

[0124] For example, a set of aspheric lenses with a focal length of about 45mm can effectively receive circularly polarized light from 0° to 30°. By precisely designing the lens surface, nearly 40% of the light can be kept in the transmission path.

[0125] The mask plate is used to screen each circularly polarized light to obtain the first polarized light and the second polarized light.

[0126] The mask plate is usually composed of multiple layers of optical films, each layer of which has a specific refractive index and optical properties. It can effectively selectively transmit and reflect light of different wavelengths and polarization states. Apertures of different shapes can be set to guide and filter light of a specific polarization state.

[0127] For example, the central area of ​​the mask is designed as a rectangular aperture to allow the first polarized light to pass through, and the material therein can be a polarizing film. The outer area is coated to reflect the second polarized light, thereby achieving effective light separation.

[0128] In an exemplary embodiment, based on the above embodiment, see Figure 7 The chirp broadening module of the embodiment of the present application includes a dichroic mirror DM, a glass rod and a third reflector M which are arranged in sequence.

[0129] The dichroic mirror DM is used to combine the third pump light, the third Stokes light and the fourth Stokes light to obtain the first combined light.

[0130] The dichroic mirror DM uses the reflection and transmission characteristics of light of different wavelengths to accurately introduce three light sources separately and merge them. By reasonably selecting the reflection and transmission angles, the light of different wavelengths is merged into the first merged light at the output end, ensuring the balance of light intensity between the light sources and providing a uniform beam for subsequent processing.

[0131] The glass rod is used to perform chirp broadening processing on the first combined light to obtain the second combined light.

[0132] Among them, the selection and design of the glass rod is crucial. Glass rods of appropriate size and refractive index produce different group velocity effects through multiple refractions of light, causing the light signal to produce a chirp broadening effect during propagation. The broadening process expands the originally relatively concentrated light beam, reduces the instantaneous fluctuation of light intensity in space, and thus improves the overall stability of imaging.

[0133] The third reflecting mirror M is used to transmit the second combined light to the microscope module.

[0134] The third reflector plays the role of guiding light and adjusting direction in the optical path design, ensuring that the widened light beam is smoothly and accurately transmitted to the optical system of the microscope. By reasonably setting the angle of the reflector, the light loss during the transmission process is minimized to achieve efficient imaging.

[0135] The embodiments of the present application significantly improve the performance of the coherent Raman scattering microscope by effectively merging and broadening the light beams.

[0136] In an exemplary embodiment, based on the above embodiment, see Figure 8 The microscope module of the embodiment of the present application includes a first optical element group, a stage, and a second optical element group arranged in sequence. The first optical element group may include a galvanometer SU, a lens L, a reflector M, and an objective lens arranged in sequence; the second optical element group may include a condenser Condenser, a reflector M, and a lens L arranged in sequence.

[0137] The first optical element group is used to focus the second combined light to obtain focused light, and to focus the focused light onto the sample on the stage. The second optical element group may include optical elements such as lenses and reflectors.

[0138] Among them, the galvanometer is used to control the path of light, change the incident angle of the light beam, and adapt to different positions of the sample.

[0139] Lens: Used to focus light and ensure that the light enters the objective lens at the appropriate inspection angle.

[0140] Reflector: changes the direction of light propagation and adjusts the light path.

[0141] Objective lens: Finally, the focused light is further focused onto the sample on the stage to achieve imaging of the sample.

[0142] The stage is used to carry the sample, wherein the sample is used to react with the focused light to generate a modulated signal.

[0143] The stage can be equipped with a fine-tuning structure that can move up and down, left and right to precisely adjust the position of the sample and facilitate observation of different areas.

[0144] The second optical element group is used to transmit the modulated signal to the filter module. It can include a condenser, a reflector and a lens. It is specifically used to transmit the signal modulated by the sample to the filter module to ensure the effective transmission of the signal. The condenser here converges the light signal emitted by the sample to provide a stronger signal intensity for the subsequent steps.

[0145] In an exemplary embodiment, based on the above embodiment, see Fig. 9 The laser adjustment module of the embodiment of the present application includes a half wave plate HWP, a polarization beam splitter cube PBS and a lens group L which are arranged in sequence.

[0146] The half wave plate HWP is used to adjust the polarization direction of the Stokes light to obtain the adjusted Stokes light. The polarization direction of the Stokes light is adjusted to make it more compatible with the operation of subsequent optical elements.

[0147] The polarization beam splitter cube PBS is used to perform power adjustment and screening processing on the adjusted Stokes light to obtain screened Stokes light and improve the signal-to-noise ratio of the system.

[0148] The lens group L is used to expand the Stokes light after screening to obtain the second Stokes light. The beam expansion process increases the diameter of the light beam to ensure the uniformity of the signal.

[0149] In an exemplary embodiment, based on the above embodiment, see Fig.10The filtering module of the embodiment of the present application includes a bandpass filter Filter and a signal detector Detector.

[0150] The bandpass filter is used to filter the background signal to obtain a filtered optical signal. The background signal is filtered to reduce the influence of noise and ensure that only the optical signal within the target signal frequency band is retained. For example, a bandpass filter that can filter a specific wavelength range (such as 550 nm to 600 nm) is selected to enhance the strength of a specific CRS signal.

[0151] The signal detector is used to perform photoelectric conversion on the filtered optical signal to obtain the CRS signal. The optical signal after filtering is photoelectrically converted to generate an electrical signal for subsequent analysis.

[0152] In an exemplary embodiment, based on the above embodiment, the background signal suppression system of the embodiment of the present application further includes a phase-locked amplifier, which is used to phase-lock and demodulate the modulation signal to obtain a demodulation result. The phase-locked amplifier is used to phase-lock and demodulate the modulation signal formed by the output resonance signal and the background signal, thereby effectively suppressing the background signal and improving the contrast and stability of the signal. A high-performance phase-locked amplifier is used, and the operating frequency is set to match the modulation signal frequency to achieve the best signal extraction effect.

[0153] The overall system diagram is as follows: Fig.11 As shown, the overall workflow is as follows:

[0154] The laser provides two synchronized femtosecond laser beams, one of which is a Stokes beam ( ), wavelength 1045nm, pulse width 220fs. The other beam is used as pump light ( ), wavelength tunable range 680nm-1300nm, pulse width 120fs. Laser repetition frequency 80MHz.

[0155] The pump light exits the laser and passes through a half-wave plate (HWP) to adjust its polarization direction. At the same time, the polarization beam splitter prism only allows the vertical or horizontal component of the laser to pass through, and the polarization beam splitter prism (PBS) is used to adjust its laser power. After exiting the polarization beam splitter prism, it enters the lens group to expand the pump light so that it can cover the rear light inlet of the objective lens when it reaches the objective lens of the microscope. After beam expansion, the pump light passes through the time delay line, and the time delay state of the pump light and the Stokes light is adjusted so that the two laser beams reach the sample at the same time. After passing through the time delay line, the pump light reaches the dichroic mirror and merges with the Stokes light.

[0156] Stokes light exits the laser, passes through a half wave plate (HWP) to adjust its polarization direction, and is matched with a polarization beam splitter (PBS) to adjust its laser power. After exiting the polarization beam splitter, it enters the lens group to expand the Stokes light so that it can cover the rear light entrance of the objective lens when it reaches the objective lens of the microscope. It then passes through the PBS again, and passes through a quarter wave plate (QWP) to become circularly polarized light before entering the high-speed scanning galvanometer. The high-speed scanning galvanometer allows the Stokes light to be incident on the lens at different angles. The mask behind the lens will select the S1 and S2 lights that meet the experimental conditions. The two beams will be focused on the plane reflector and returned by the original path. After passing through the QWP again, it changes from circularly polarized light to linearly polarized light, and its polarization direction changes from the incident vertical polarization to horizontal polarization and is reflected by the PBS. It reaches the dichroic mirror and merges with the pump light, and is spatially collinear. The time delay of the S1 and S2 lights can be adjusted by adjusting the scanning amplitude of the high-speed scanning galvanometer and the opening distance of the mask. At the same time, the S1 and S2 lights will appear alternately according to the scanning frequency of the galvanometer.

[0157] After passing through the glass rod, it enters the microscope system and is focused on the sample through the objective lens. The excited CRS signal and background signal are collected by the condenser, enter the signal detector through the bandpass filter, and finally enter the phase-locked amplifier to demodulate the signal.

[0158] This application utilizes a high-speed scanning galvanometer, a lens, and a plane mirror, combined with a chirped femtosecond laser, to achieve modulation of the detection frequency with a simpler and more reliable solution. The high-speed scanning galvanometer can incident Stokes light onto the lens position at different angles and at an extremely high scanning frequency. At this time, the galvanometer is located on the optical axis of the lens and is at the focal position. Adding a mask after the lens can screen out Stokes light with a fixed incident angle. Among them, S1 is incident along the optical axis of the lens, then focused onto the plane mirror, and then returned by the reflector along the original path. S2 is incident on the lens at a certain angle, then focused onto the plane mirror, and then returned by the reflector along the original path. Before passing through the lens, the optical path lengths experienced by S2 and S1 are different, which will cause a time delay. , and S2 and S1 will appear alternately at the scanning frequency of the galvanometer. The wavelength distribution of the laser can be broadened in the time domain by using an optical glass rod, such as Fig.12 As shown, the schematic diagram of the chirp broadening (Chirp) of the femtosecond laser (Pump and Stokes) by the selected optical glass rod;

[0159] Using the galvanometer to adjust the incident angle of S2 can make the pump light and Stokes light form different time delays, and obtain different state. Thus, S1 and S2 form different frequency combinations with the pump light. and , completing the modulation of the detection frequency. The principle diagram of the technical principle of separating the background signal from the resonance signal is shown in the figure Fig.13 shown.

[0160] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0161] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A background signal suppression system for a coherent Raman scattering microscope, characterized in that: The background signal suppression system includes a laser light source module, a laser adjustment module, a frequency modulation module and a filter module; The laser light source module is used to provide a laser light source; the laser light source includes a first Stokes light and a first pump light; The laser adjustment module is used to adjust the first Stokes light and the first pump light respectively to obtain the second Stokes light and the second pump light; The frequency modulation module is used to perform frequency modulation processing and laser excitation processing on the second Stokes light and the second pump light respectively to obtain a modulation signal; the modulation signal includes a non-resonance background signal and a resonance signal; The filtering module is used to remove the background signal to obtain the resonance signal.

2. The background signal suppression system according to claim 1, characterized in that: The frequency modulation module includes a pump light adjustment module, a Stokes light adjustment module, a chirp broadening module and a microscope module; The pump light adjustment module is used to perform time delay processing on the second pump light to obtain a third pump light; The Stokes light adjustment module is used to adjust the polarization state and laser irradiation position of the second Stokes light, and to filter the adjusted second Stokes light to obtain a third Stokes light and a fourth Stokes light; The chirp stretching module is used to combine the third pump light, the third Stokes light, and the fourth Stokes light, and to stretch the pulse width of the combined first combined light to obtain a second combined light; The microscope module is used to perform excitation processing on the second combined light to obtain the modulated signal.

3. The background signal suppression system according to claim 2, characterized in that: The pump light adjustment module includes a plurality of first reflectors, each of which is used to change the propagation direction of the second pump light and perform time delay processing on the second pump light.

4. The background signal suppression system according to claim 2, characterized in that: The Stokes light adjustment module includes a polarization beam splitter cube, a quarter wave plate, a high-speed multi-faceted scanning mirror, a light screening device and a second reflector which are arranged in sequence; The polarization beam splitter cube is used to split the second Stokes light to obtain parallel polarized light and vertical polarized light; The quarter wave plate is used to adjust the polarization state of the parallel polarized light to obtain one of circularly polarized light and linearly polarized light; The high-speed multi-faceted scanning mirror is used to scan the circularly polarized light to the light screening device and receive the reflected light reflected by the second reflector; The light screening device is used to screen the circularly polarized light to obtain the first polarized light and the second polarized light; The second reflector is used to reflect the first polarized light and the second polarized light to obtain the third Stokes light and the fourth Stokes light.

5. The background signal suppression system according to claim 4, characterized in that: The second reflector includes a plane reflector and a large-size ruled grating.

6. The background signal suppression system according to claim 4, characterized in that: The optical screening device includes a lens and a mask plate which are stacked; The lens is used to receive circularly polarized lights of multiple angles transmitted by the high-speed multi-faceted scanning mirror, and transmit each circularly polarized light to the mask plate; The mask plate is used to screen each of the circularly polarized lights to obtain the first polarized light and the second polarized light.

7. The background signal suppression system according to claim 2, characterized in that: The chirp broadening module comprises a dichroic mirror, a glass rod and a third reflector which are arranged in sequence; The dichroic mirror is used to combine the third pump light, the third Stokes light, and the fourth Stokes light to obtain the first combined light; The glass rod is used to perform chirp broadening processing on the first combined light to obtain the second combined light; The third reflecting mirror is used to transmit the second combined light to the microscope module.

8. The background signal suppression system according to claim 2, characterized in that: The microscope module includes a first optical element group, a stage, and a second optical element group which are arranged in sequence; The first optical element group is used to focus the second combined light to obtain focused light, and focus the focused light onto the sample on the stage; The stage is used to carry the sample; The sample is used to react with the focused light to generate the modulated signal; The second optical element group is used to transmit the modulated signal to the filtering module.

9. The background signal suppression system according to claim 1, characterized in that: The laser adjustment module includes a half wave plate, a polarization beam splitter cube and a lens group arranged in sequence; The half wave plate is used to adjust the polarization direction of the Stokes light to obtain adjusted Stokes light; The polarization beam splitter cube is used to perform power adjustment and screening processing on the adjusted Stokes light to obtain screened Stokes light; The lens group is used to perform beam expansion processing on the screened Stokes light to obtain the second Stokes light.

10. The background signal suppression system according to claim 1, characterized in that: The filtering module includes a bandpass filter and a signal detector; The bandpass filter is used to filter the background signal to obtain a filtered optical signal; The signal detector is used to perform photoelectric conversion processing on the filtered optical signal to obtain the resonant signal and the non-resonant signal.

11. The background signal suppression system according to any one of claims 1 to 10, characterized in that: The background signal suppression system further comprises a phase-locked amplifier, which is used to perform phase locking and demodulation processing on the modulated signal to obtain a demodulation result.

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