A method and system for Brillouin scattering microscopy imaging beyond the diffraction limit

Abstract

The present invention discloses a super-diffraction-limited Brillouin scattering microscopy method and system, which uses the pump-probe method to excite stimulated Brillouin scattering on the surface or inside of a sample, and based on the stimulated Brillouin scattering mechanism, uses optical field modulation to change the spatial distribution of Brillouin gain within the detection spot, and realizes the unity of spatial super-diffraction imaging and Brillouin scattering spectrum measurement through point spread function modification, structured light illumination or high-order Brillouin scattering, and finally realizes non-contact, super-diffraction-limited Brillouin scattering microscopy imaging of the sample.

Description

Technical Field

[0001] The present invention belongs to the technical field of Brillouin spectroscopy measurement and imaging, and particularly relates to a method and system for Brillouin scattering microscopy with super-diffraction limit. Background Art

[0002] Brillouin scattering microscopy is a technique based on the interaction between light and acoustic waves in a medium, and has the following remarkable characteristics: label-free, without the need for chemical dyes or fluorescent labels, suitable for imaging of living cells and tissues, and avoiding interference that may be introduced during the labeling process; specificity, capable of directly measuring the mechanical properties of samples (such as elastic modulus and viscosity), providing information that cannot be obtained by traditional optical microscopy techniques; non-contact, using optical methods for measurement, not causing physical contact or damage to the sample, and suitable for the study of living samples and sensitive samples.

[0003] Although Brillouin scattering microscopy has advantages in label-free, specific, and non-contact imaging, the existing technology still has limitations in terms of resolution. The resolution of traditional Brillouin microscopes is usually at the sub-micron level, showing a significant gap compared with other super-resolution imaging techniques. This technical bottleneck limits the wide application of Brillouin scattering microscopy in the study of fine structures. Summary of the Invention

[0004] In view of the above, the purpose of the present invention is to provide a method and system for Brillouin scattering microscopy with super-diffraction limit, which uses the pump-probe method to excite stimulated Brillouin scattering on the surface or inside of the sample, and based on the stimulated Brillouin scattering mechanism, uses optical field modulation to change the spatial distribution of Brillouin gain within the detection spot, and realizes the unification of spatial super-diffraction imaging and Brillouin scattering spectrum measurement through point spread function modification, structured light modulation, or high-order Brillouin scattering, and finally realizes non-contact, super-diffraction limit Brillouin scattering microscopy of the sample.

[0005] To achieve the above-mentioned invention purpose, a super-diffraction limit Brillouin scattering microscopy system provided by an embodiment includes:

[0006] A light source module, which is used to generate two excitation light beams as detection light and pump light;

[0007] An optical field modulation module, which is used to perform optical parameter modulation on the excitation light beam to realize optical field modulation, change the spatial distribution of Brillouin gain within the detection spot, and achieve super-diffraction limit modulation;

[0008] A microscopy module, which is used to focus the detection light and pump light after optical field modulation on the sample, cause stimulated Brillouin scattering on the surface or inside of the sample, generate a super-resolution Brillouin spatial spot, capture the detection light and transmit it to the signal acquisition and processing module;

[0009] A signal acquisition and processing module, which is used to filter, acquire, and process the detection light to obtain the single-point Brillouin scattering spectrum information beyond the diffraction limit.

[0010] A scanning and imaging module, which is used to control the working of the light source module, the light field modulation module, and the microscopy module, perform point scanning, and also realize multi-dimensional Brillouin scattering microscopy imaging beyond the diffraction limit based on the single-point Brillouin scattering spectrum information.

[0011] Preferably, the linewidth of the excitation beam generated by the light source module is lower than 100 kHz, and the optical parameters modulated by the light field modulation module include at least one of wavelength (frequency), polarization, wave vector, and phase, where the relationship between wavelength (λ) and frequency (f) is f = vλ, where v is the wave velocity.

[0012] Alternatively preferably, the modulation beyond the diffraction limit includes: modifying the point spread function based on the additional pump light. At this time, the light source module also generates an additional beam as the additional pump light. Among them, the process of modifying the point spread function based on the additional pump light is as follows:

[0013] Introduce a ring-shaped additional pump light to deplete the phonons or energy around the detection spot to reduce the range of the point spread function. Specifically, the frequencies of the pump light, the detection light, and the ring-shaped additional pump light satisfy f P -f S = f S -f L = f B , where, f P , f S , f L are the frequencies of the pump light, the detection light, and the ring-shaped additional pump light respectively, and f B is the Brillouin frequency shift of the sample at the focal point. Based on the stimulated Brillouin scattering mechanism, the energy of the pump light spot is transferred to the detection light spot, and the energy around the detection light spot will be further transferred to the ring-shaped additional pump light spot, thereby reducing the effective signal area of the detection light spot, realizing the modification of the point spread function, and obtaining a detection light spot beyond the diffraction limit.

[0014] Alternatively preferably, the modulation beyond the diffraction limit includes: modifying the point spread function based on the additional detection light. At this time, the light source module also generates an additional beam as the additional detection light. Among them, the process of modifying the point spread function based on the additional detection light is as follows:

[0015] Introduce a ring-shaped additional probe light to induce phonon depletion or form destructive interference at the periphery of the probe spot, thereby reducing the range of the point spread function. Specifically, the frequency of the ring-shaped additional probe light is the same as that of the probe light but the phase is opposite to achieve destructive interference and reduce the range of the point spread function; or specifically design the ring-shaped additional probe light to satisfy the Brillouin scattering condition with the pump light to deplete the phonons at the periphery of the Airy disk, while ensuring that the Gaussian probe light satisfies the Brillouin scattering condition with the pump light to maintain the phonon effect in the central region of the Airy disk, so that there is a slight difference in the wave vectors of the ring-shaped additional probe light and the Gaussian probe light to form an energy competition mode, and facilitate optical signal separation, ultimately reducing the range of the point spread function and obtaining a detection spot with super-diffraction limit.

[0016] Preferably in parallel, the super-diffraction limit modulation includes: structured light illumination based on polarization control. Specifically, by adjusting the polarization parameters, the spatial polarization distributions of the pump spot and the probe spot are changed, and the structured light is focused on the sample through the microscopic module, so that the Brillouin gain in the high polarization matching region within the focal point is enhanced, and the Brillouin gain in the low polarization matching region is suppressed, realizing the spatial selective enhancement of stimulated Brillouin scattering within the probe spot and obtaining the detection spot information with super-diffraction limit.

[0017] Preferably in parallel, the super-diffraction limit modulation includes: structured light modulation based on phase modulation. Specifically, periodic structured light with different phases and directions is generated by adjusting the spatial frequency, phase parameters, and direction parameters, and the structured light is focused on the sample through the microscopic module, obtaining the high spatial frequency Brillouin scattering signal within the focal point, encoding the high spatial frequency information that originally exceeds the resolution limit of the optical system into the observable low frequency range, and obtaining the detection spot information with super-diffraction limit.

[0018] Preferably in parallel, the super-diffraction limit modulation includes: high-order Brillouin modulation. Specifically, the frequencies and spot distributions of the probe light and the pump light are adjusted to match the excitation conditions of the high-order Brillouin scattering process, and the modulated light beam is focused on the sample through the microscopic module, inducing high-order acoustic modes within the focal point, obtaining the high-order Brillouin scattering signal containing the microscopic structure information of the sample, and obtaining the detection spot information with super-diffraction limit.

[0019] Preferably, the microscopic module includes a first mirror, a first microscopic objective lens, a second mirror, a beam splitter, a second microscopic objective lens, and a three-dimensional motorized displacement stage. The pump light after optical field modulation sequentially passes through the first mirror, the beam splitter, and the first microscopic objective lens and is focused into the sample on the three-dimensional motorized displacement stage. The probe light after optical field modulation sequentially passes through the second mirror and the second microscopic objective lens and is focused into the sample on the three-dimensional motorized displacement stage to excite stimulated Brillouin scattering inside or on the surface of the sample.

[0020] To achieve the above-mentioned invention objectives, an embodiment of the present invention provides a Brillouin scattering microscopy imaging method with super diffraction limit. Using the above-mentioned Brillouin scattering microscopy imaging system, the method includes the following steps:

[0021] Generate two excitation light beams as the probe light and the pump light using the light source module and output them;

[0022] Use the light field modulation module to modulate the optical parameters of the excitation light beam, thereby achieving light field modulation, changing the spatial distribution of Brillouin gain within the probe spot, and realizing super diffraction limit modulation;

[0023] Use the microscopy module to focus the probe light and the pump light after light field modulation on the sample, causing stimulated Brillouin scattering to occur on the surface or inside of the sample, generating a super-resolution Brillouin spatial spot, capturing the probe light, and transmitting it to the signal acquisition and processing module;

[0024] Use the signal acquisition and processing module to filter, acquire, and process the probe light to obtain the single-point Brillouin scattering spectrum information with super diffraction limit;

[0025] Use the scanning and imaging module to control the operation of the light source module, the light field modulation module, and the microscopy module, and perform point scanning to achieve multi-dimensional Brillouin scattering microscopy imaging with super diffraction limit based on the single-point Brillouin scattering spectrum information.

[0026] Compared with the prior art, the beneficial effects of the present invention at least include:

[0027] 1. Compared with the traditional stimulated Brillouin scattering microscopy imaging technology, the present invention is based on the energy competition mechanism and uses light field modulation to change the spatial distribution of Brillouin gain within the probe spot, and can achieve non-contact spatial super diffraction limit Brillouin scattering microscopy imaging;

[0028] 2. There are no special requirements for the type of the sample. It can be either a labeled sample or an unlabeled sample, and can be either a high Brillouin gain sample or an ordinary sample. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a schematic structural diagram of the Brillouin scattering microscopy imaging system with super diffraction limit provided by the present invention;

[0031] Figure 2Frequency domain intensity map and spatial domain intensity map of the interaction of three focused light spots in Example 1;

[0032] Figure 3 Schematic diagram of three light spots in Example 2;

[0033] Figure 4 Schematic diagram of polarization pattern in Example 3;

[0034] Figure 5 Schematic diagram of structured light mask in Example 4;

[0035] Figure 6 High-order Brillouin scattering spectrum diagram in Example 5;

[0036] In the figure: 1 - light source module, 2 - light field regulation module, 3 - microscopy module, 4 - sample, 5 - signal acquisition and processing module, 6 - scanning and imaging module, 301 - first reflector, 302 - beam splitter, 303 - first microscope objective, 304 - three-dimensional electric displacement stage, 305 - second microscope objective, 306 - second reflector. Detailed implementation manners

[0037] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific implementation manners described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0038] As Figure 1As shown in the figure, the super-diffraction-limited Brillouin scattering microscopy system provided by the embodiment includes a light source module 1, an optical field modulation module 2, a microscopy module 3, a signal acquisition and processing module 5, and a scanning and imaging module 6. The light source module 1 is used to generate an excitation light beam with a narrow linewidth. The laser beam includes a probe light and a pump light, etc. The linewidths of the beams are all lower than 100 kHz to ensure high coherence and high frequency resolution. The optical field modulation module 2 is used to modulate optical parameters such as wavelength (frequency), polarization, wave vector, and phase of the excitation light beam, so as to change the spatial distribution of Brillouin gain within the detection spot and achieve super-diffraction-limited modulation. The microscopy module 3 is used to focus the light beam after optical field modulation on the sample, causing stimulated Brillouin scattering on the surface or inside of the sample to generate a super-resolution Brillouin spatial spot, capture the probe light signal and transmit it to the signal acquisition and processing module 5. The key components in the microscopy module 3 include a first mirror 301, a beam splitter 302, a first microscopy objective 303, a three-dimensional motorized displacement stage 304, a second microscopy objective 305, and a second mirror 306. The pump light after optical field modulation sequentially passes through the first mirror 301, the beam splitter 302, and the first microscopy objective 303 and is focused into the sample 4 on the three-dimensional motorized displacement stage 304. The probe light after optical field modulation sequentially passes through the second mirror 306 and the second microscopy objective 305 and is focused into the sample 4 on the three-dimensional motorized displacement stage 304 to stimulate stimulated Brillouin scattering inside or on the surface of the sample. The signal acquisition and processing module 5 is used for filtering, acquisition, and data processing of the probe light to obtain a super-diffraction-limited single-point Brillouin scattering image. The scanning and imaging module 6 is used to control the light source module 1, the optical field modulation module 2, and the three-dimensional motorized displacement stage 304 to perform point scanning and achieve super-diffraction-limited multi-dimensional Brillouin scattering microscopy imaging based on the single-point Brillouin scattering spectral information.

[0039] The method steps for performing Brillouin scattering microscopy imaging using the above super-diffraction-limited Brillouin scattering microscopy system are as follows:

[0040] (1) Beam generation: Use the light source module to generate at least two excitation light beams as the pump light and the probe light based on the pump-probe method. Optionally, a third additional light beam is also generated to modify the point spread function. The linewidth of each light beam is lower than 100 kHz to ensure high coherence and high frequency resolution.

[0041] (2) Super-diffraction-limited modulation: Use the optical field modulation module to modulate the optical parameters of the excitation light beam generated by the light source module. By changing the spatial distribution of Brillouin gain within the detection spot, the gain in some regions is kept unchanged or enhanced, and the gain in other regions is lost / suppressed, achieving super-diffraction-limited modulation.

[0042] (3) Stimulated Brillouin excitation: The beam modulated by the optical field modulation module is focused on the sample through the microscopic module. The pump light and the probe light satisfy the stimulated Brillouin scattering conditions, so as to stimulate stimulated Brillouin scattering inside or on the surface of the sample. During the scattering process, the energy of the high-frequency pump light is transferred to the low-frequency probe light to achieve specific energy conversion and acoustic wave excitation;

[0043] (4) Signal acquisition and processing: The signal acquisition and processing module is used to filter, acquire, and process the probe light, and extract the single-point Brillouin scattering spectrum information beyond the diffraction limit;

[0044] (5) Scanning imaging: The scanning and imaging module is used to scan the sample point by point, process the spectral signals, and perform multi-dimensional image reconstruction to achieve multi-dimensional, non-contact, and super-diffraction-limited Brillouin scattering microscopy imaging of the sample.

[0045] The occurrence of stimulated Brillouin scattering in the sample needs to satisfy the stimulated Brillouin scattering conditions, that is, the momentum matching and energy matching formulas among the pump light, the probe light, and the generated acoustic wave:

[0046]

[0047] In the formula, k P , k S , and q are the wave vectors of the pump light, the probe light, and the acoustic wave respectively; f P , f S are the frequencies of the pump light and the probe light respectively; f B is the frequency of the acoustic wave, also known as the Brillouin frequency shift, which is related to the optical wavelength, the scattering angle, and the characteristics of the sample itself. According to the wave equation and the photoacoustic coupling equation, the optical field and the acoustic field can be correlated through the nonlinear coupling term, and the momentum matching and energy matching directly affect the efficiency of stimulated Brillouin scattering. Therefore, by changing the optical field parameters, the spatial distribution of Brillouin gain can be dynamically regulated to generate a super-resolution Brillouin spatial spot.

[0048] In the embodiment, the super-diffraction-limited modulation methods of the optical field modulation module include point spread function modification, structured light modulation, and high-order Brillouin scattering. Each method will be described in detail below.

[0049] Embodiment 1

[0050] The point spread function modification based on the additional pump light is adopted as the super-diffraction-limited modulation method in this embodiment. The core is to introduce a ring-shaped additional pump light to deplete the phonons or energy around the probe spot, thereby reducing the range of the point spread function.

[0051] During the stimulated Brillouin scattering process, the energy transfer form is from high-frequency light to low-frequency light. In this embodiment, the frequencies of the pump light, the probe light, and the ring-shaped additional pump light are made to satisfy f P-f S = f S -f L = f B , where f L is the frequency of the annular additional pump light.

[0052] As shown in (a) of Figure 2 , when there is no annular additional pump light and the pump light and the probe light satisfy the stimulated Brillouin scattering condition, the energy of the pump light is transferred to the probe light, the pump light generates a Brillouin loss spectrum, and the probe light generates a Brillouin gain spectrum;

[0053] As shown in (b) of Figure 2 , when the pump light, the probe light, and the annular additional pump light exist simultaneously, the pump light and the probe light satisfy the stimulated Brillouin scattering condition. Therefore, the energy of the pump light is transferred to the probe light, and the probe light and the annular additional pump light also satisfy the stimulated Brillouin scattering condition. Therefore, the energy of the probe light is transferred to the annular additional pump light, resulting in a depression in the frequency-domain intensity map of the probe light at the frequency f S and generating a Brillouin gain-loss spectrum.

[0054] As shown in 2(c) of the figure, since the pump light and the probe light are circular spots, while the additional pump light is an annular spot, only the pump light and the probe light interact at the center of the probe spot. The frequency-domain intensity map of the central region of the probe light is the Brillouin gain spectrum; the annular region of the probe spot interacts with the pump light and the additional pump light simultaneously, and the frequency-domain intensity map of the probe light here is the Brillouin gain-loss spectrum. Therefore, the spatial size of the probe light signal with a frequency of f S is the size of the circular probe spot minus the size of the annular additional pump spot. Since the probe spot reaches the diffraction limit under the focusing of the microscopic module, after subtracting the annular additional pump spot, the remaining central spot is the spatial spot that breaks through the optical diffraction limit.

[0055] Embodiment 2

[0056] The super-diffraction limit modulation method in this embodiment adopts the transformation of the point spread function based on the additional probe light. The core lies in introducing a beam of annular additional probe light to induce the depletion of phonons around the probe spot, or forming destructive interference by setting the condition of opposite phases, thereby reducing the range of the point spread function.

[0057] Specifically, the key to realizing phonon depletion is to control the wave vector mismatch amount Δk (Δk = k P - k s - q) between the pump light and the probe light. The theoretical relationship between the Brillouin gain g B and the wave vector mismatch amount is:

[0058]

[0059] In the formula, a is the damping constant related to the characteristics of the medium phonons, which reflects the mechanical and optical properties of the sample. When Δk is 0, the Brillouin gain reaches the maximum value, and the energy transfer efficiency from the pump light to the probe light is the highest; when |Δk| increases, the Brillouin gain decreases significantly. Therefore, by regulating the wave vectors of the two probe lights to change the wave vector mismatch, an energy competition can be formed between the Gaussian probe light and the annular additional probe light, thereby modifying the point spread function and achieving the effect beyond the diffraction limit.

[0060] Specifically, as Figure 3 shown, the wave vector of the annular additional probe light is adjusted so that the wave vector mismatch Δk I = 0, that is, the Brillouin scattering condition is satisfied, so as to deplete the phonons around the Airy disk to the greatest extent; at the same time, the wave vector of the Gaussian probe light is adjusted so that the wave vector mismatch Δk s ≈ 0, that is, the Brillouin scattering condition is satisfied, and the Gaussian probe light will also undergo stimulated Brillouin scattering with the pump light, but it is at a disadvantage in the energy competition with the annular additional probe light. Therefore, the phonons in the peripheral region of the Airy disk will interact with the annular additional probe light and be depleted, and the phonons in the central region will interact with the Gaussian probe light, ultimately reducing the range of the point spread function and generating a spatial light spot that breaks through the optical diffraction limit.

[0061] Example 3

[0062] The structured light illumination based on polarization modulation is adopted as the super-diffraction limit modulation method in this example. In this example, by regulating the polarization states of the pump light and the probe light, the super-diffraction limit Brillouin scattering microscopy imaging is realized by using the influence of the polarization matching degree on the Brillouin gain. By optimizing the polarization distribution of the light, the intensity and resolution of the signal can be regulated. The specific implementation scheme is as follows:

[0063] (1) Polarization illumination: The spatial light modulator is used to control the polarization states of the pump light and the probe light at different spatial positions respectively. For this purpose, a preset polarization pattern is designed to cover the entire imaging area. In the high polarization matching region, the polarization directions of the pump light and the probe light are kept the same or approximate as much as possible to maximize the Brillouin gain and thus enhance the signal intensity; in the low polarization matching region, the polarization states of the pump light and the probe light are set to be orthogonal or have significant differences to reduce the Brillouin gain and suppress the signal intensity; a gradual polarization distribution is set between the high and low polarization matching regions to ensure the smooth transition of the light field and avoid the generation of edge effects or artifacts. By controlling the dynamic adjustment of the spatial polarization distribution of the pump light spot and the probe light spot, the gain enhancement and high-resolution imaging of a specific region can be realized. As Figure 4 shown is a schematic diagram of the polarization pattern of a probe light spot and a pump light spot.

[0064] (2) Signal acquisition: Structured light with different polarization modulation patterns is focused on the sample through the microscopic module. Under each polarization pattern condition, the super-diffraction-limited single-point stimulated Brillouin scattering spectral signal generated by the sample is collected to form a series of image stacks, and these images reflect the intensity distribution of the scattering spectral signals under different polarization matching conditions. Specifically, a highly sensitive detector can be used in the signal acquisition and processing module to ensure that the changes in the scattering spectral signals under each polarization condition can be accurately captured.

[0065] (3) Scanning imaging: The obtained image stacks are processed to extract and synthesize super-resolution information, and the super-resolution information under each polarization matching condition is fused to finally generate a multi-dimensional Brillouin scattering microscopic image with super-diffraction limit.

[0066] Example 4

[0067] The structured light illumination modulation based on phase modulation is adopted as the super-diffraction limit modulation method in this example. This example uses the structured light illumination technology to improve the spatial resolution of Brillouin scattering imaging through the Moiré fringe effect. Specifically, periodic structured light with different phases and directions is generated by adjusting the phase parameter and the direction parameter. The structured light illumination can encode the high-frequency information beyond the resolution limit of the optical system into the observable low-frequency range, so as to achieve super-resolution in the subsequent image processing. The specific implementation process is as follows:

[0068] (1) Structured light illumination: Use a digital micromirror device or a spatial light modulator to generate various structured light patterns, such as Figure 5 shown. According to the experimental requirements, these structured light patterns can be stripes, grids or other periodic structures. The structured light patterns are projected onto the sample through the microscopic module, and by changing the spatial frequency, phase and direction of the light, a controllable Moiré fringe effect is generated. During this process, accurately adjusting the light pattern can optimize the illumination of the sample to achieve the best signal encoding effect.

[0069] (2) Frequency encoding: Different spatial frequency structured light patterns are superimposed on the sample to generate Moiré fringes. These Moiré fringes can transfer the high-frequency information existing in the sample to the low-frequency range detectable by the optical system. Through structured light modulation, the high-frequency features of the sample can be effectively encoded, enabling the decoding and recovery of information in the subsequent signal processing;

[0070] (3) Data acquisition and reconstruction: Illuminate the sample with structured light at different phases and directions, collect the super-diffraction-limited single-point stimulated Brillouin scattering spectral signals generated by the sample, and form multiple sets of Moiré fringe images; the classical three-step phase-shifting method or five-step phase-shifting method can be used to obtain image data at different phases; perform Fourier transform on each set of image data to extract the encoded high-frequency information; combine the high-frequency information with the low-frequency information to finally reconstruct the spatially super-resolved Brillouin scattering image.

[0071] The optical modulation strategy includes the following methods: a. Probe light structuring: By performing structured light modulation on the probe light, enhance the detection and analysis capabilities of the Brillouin scattering signal, which helps improve the signal-to-noise ratio and resolution of the signal; b. Pump light structuring: Perform structured light modulation on the pump light, directly affect the generation process of acoustic waves, enhance the interaction with the sample, and improve the stimulated Brillouin scattering efficiency; c. Combined modulation: Perform combined modulation on the pump light and the probe light to achieve rich information encoding and higher resolution, optimize the overall performance of the system, and provide more detailed imaging information.

[0072] Example 5

[0073] Adopt high-order Brillouin modulation as the super-diffraction-limited modulation method in this example. Generate multi-order Stokes signals through high-order nonlinear optical processes to achieve super-resolution imaging. As Figure 6 shown, in the process of stimulated Brillouin scattering, in addition to the fundamental frequency Stokes signal, a series of high-order Stokes signals with frequencies higher than the fundamental frequency will also be generated. The frequency intervals between these signals are equal to integer multiples of the Brillouin frequency shift. The high-order Stokes signals provide additional spectral information, which helps to distinguish finer structural features in the sample. In addition, the high-order Brillouin scattering process has a compression effect on the point spread function of the system, especially suppressing its tail, thereby reducing the effective point spread function of the optical system and achieving a super-resolved Brillouin spatial spot. This example specifically includes the following steps:

[0074] (1) High-order Brillouin excitation: Use the optical field control module combined with wavelength tuning and spatial modulation techniques to adjust the frequencies, phases, and spatial distributions of the pump light and the probe light to match the excitation conditions of the high-order Brillouin scattering process. Focus the modulated pump light and probe light into the sample interior through a high numerical aperture microscope objective to induce the generation of high-order acoustic modes. These modes are excited through multi-photon processes (such as fifth-order or seventh-order nonlinear effects).

[0075] (2) Signal detection: In the signal acquisition and processing module, a highly sensitive photodetector is used in combination with an ultra-narrowband filter to selectively detect the single-point high-order Brillouin scattering spectral information beyond the diffraction limit. The ultra-narrowband filter is designed to isolate the high-order signal frequency band, thereby suppressing the non-resonant background signal and improving the signal-to-noise ratio. The lock-in amplifier or coherent detection technology is used to further improve the detection sensitivity and accuracy of the spectral information.

[0076] (3) Data processing and reconstruction: Extract the microscopic structure information and characteristic information of the sample. Through the joint analysis of multi-band signals, and using the additional spectral information provided by the single-point high-order Brillouin scattering spectral information, achieve Brillouin scattering microscopy imaging beyond the diffraction limit.

[0077] The specific embodiments described above have elaborated on the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A super-diffraction-limited Brillouin scattering microscopy system, characterized in that: include: A light source module, which is used to generate two excitation light beams as detection light and pump light, wherein the line width of the excitation light beams generated by the light source module is less than 100 kHz; A light field control module is used to control the optical parameters of the excitation light beam to achieve light field control, change the spatial distribution of the Brillouin gain in the detection spot, and achieve super-diffraction limit modulation, wherein the optical parameters controlled by the light field control module include at least one of wavelength, polarization, wave vector, and phase; wherein super-diffraction limit modulation includes: Based on the point spread function modification of the additional pump light, the light source module also generates an additional light beam as the additional pump light. The point spread function modification process based on the additional pump light is as follows: a ring-shaped additional pump light is introduced to deplete the phonons or energy around the detection light spot to reduce the range of the point spread function, specifically, the frequencies of the pump light, the detection light, and the ring-shaped additional pump light meet f P - f S = f S - f L = f B ,in, f P , f S , f L are the frequencies of pump light, probe light, and annular additional pump light, f B The Brillouin frequency shift of the sample at the focus point is based on the stimulated Brillouin scattering mechanism. The energy of the pump spot is transferred to the detection spot, and the energy around the detection spot is further transferred to the annular additional pump spot, thereby reducing the effective signal area of ​​the detection spot, realizing the point spread function transformation, and obtaining a detection spot beyond the diffraction limit. Or, based on the point spread function modification of the additional detection light, the light source module also generates an additional light beam as the additional detection light, wherein the point spread function modification process based on the additional detection light is: introducing a beam of annular additional detection light to induce phonon depletion around the detection light spot, or forming interference destructive phase, thereby reducing the range of the point spread function, specifically, the frequency of the annular additional detection light is consistent with that of the detection light, but the phase is opposite, so as to achieve interference destructive phase and reduce the range of the point spread function; or specifically designing the annular additional detection light and the pump light to meet the Brillouin scattering condition to deplete the phonons around the Airy disk, while ensuring that the Gaussian detection light and the pump light meet the Brillouin scattering condition to maintain the phonon effect in the central area of ​​the Airy disk, so that there is a slight difference in the wave vector between the annular additional detection light and the Gaussian detection light to form an energy competition mode, and to facilitate the separation of optical signals, and finally reduce the range of the point spread function to obtain a detection light spot beyond the diffraction limit; Or, based on polarization-controlled structured light illumination, specifically by adjusting the polarization parameters to change the spatial polarization distribution of the pump spot and the detection spot, the structured light is focused onto the sample through the microscope module, so that the Brillouin gain of the high polarization matching area in the focus point is enhanced, and the Brillouin gain of the low polarization matching area is suppressed, so as to achieve spatial selective enhancement of stimulated Brillouin scattering in the detection spot and obtain detection spot information beyond the diffraction limit; Or, structured light modulation based on phase modulation, specifically, by adjusting the spatial frequency, phase parameters and direction parameters to generate periodic structured light of different phases and directions, focusing the structured light onto the sample through a microscope module, obtaining the high spatial spectrum Brillouin scattering signal within the focus point, encoding the high spatial spectrum information that originally exceeds the resolution limit of the optical system into an observable low-frequency range, and obtaining the detection spot information beyond the diffraction limit; Or, high-order Brillouin modulation, specifically adjusting the frequency and spot distribution of the probe light and pump light to match the excitation conditions of the high-order Brillouin scattering process, focusing the modulated light beam onto the sample through a microscopic module, inducing the generation of high-order acoustic modes within the focus point, obtaining high-order Brillouin scattering signals containing sample microstructure information, and obtaining detection spot information beyond the diffraction limit; A microscopy module, which is used to focus the detection light and pump light after light field modulation on the sample, so that stimulated Brillouin scattering occurs on the surface or inside of the sample, generate a super-resolution Brillouin spatial spot, capture the detection light and transmit it to the signal acquisition and processing module, wherein the microscopy module includes a first reflector, a first microscope objective lens, a second reflector, a spectroscope, a second microscope objective lens, and a three-dimensional electric translation stage, the pump light after light field modulation sequentially passes through the first reflector, the spectroscope, and the first microscope objective lens to focus on the sample on the three-dimensional electric translation stage, and the detection light after light field modulation sequentially passes through the second reflector and the second microscope objective lens to focus on the sample on the three-dimensional electric translation stage, so as to stimulate stimulated Brillouin scattering inside or on the surface of the sample; A signal acquisition and processing module, which is used to filter, collect and process the detection light to obtain single-point Brillouin scattering spectrum information beyond the diffraction limit; The scanning and imaging module is used to control the operation of the light source module, the light field control module and the microscopy module, and to perform point scanning. It is also used to realize multi-dimensional Brillouin scattering microscopy imaging beyond the diffraction limit based on single-point Brillouin scattering spectrum information.

2. A Brillouin scattering microscopy method beyond the diffraction limit, characterized in that: Using the Brillouin scattering microscopy imaging system of claim 1, the method comprises the following steps: A light source module is used to generate two excitation light beams as detection light and pump light and output them; The optical field control module is used to control the optical parameters of the excitation beam to achieve optical field control, change the spatial distribution of the Brillouin gain in the detection spot, and achieve super-diffraction limit modulation; The microscopy module is used to focus the probe light and pump light after light field control on the sample, so as to cause stimulated Brillouin scattering on the surface or inside of the sample, generate a super-resolution Brillouin spatial spot, capture the probe light and transmit it to the signal acquisition and processing module; The signal acquisition and processing module is used to filter, collect and process the detection light to obtain single-point Brillouin scattering spectrum information beyond the diffraction limit; The scanning and imaging module is used to control the operation of the light source module, the light field control module and the microscopy module, and to perform point scanning, so as to realize multi-dimensional Brillouin scattering microscopy imaging beyond the diffraction limit based on the single-point Brillouin scattering spectrum information.

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