Super-diffraction limit Brillouin scattering microscopic imaging method and system

Abstract

The invention discloses a super-diffraction limit Brillouin scattering microscopic imaging method and system, stimulated Brillouin scattering on the surface or in a sample is stimulated in a pumping-detection mode, and based on a stimulated Brillouin scattering mechanism, spatial distribution of Brillouin gain in a detection light spot is regulated and changed by using a light field, so that the Brillouin scattering microscopic imaging is realized. Unification of spatial super-diffraction imaging and Brillouin scattering spectral measurement is realized through point spread function transformation, structured light illumination or high-order Brillouin scattering, and finally non-contact super-diffraction limit Brillouin scattering microscopic imaging of a sample is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of Brillouin spectrum measurement and imaging, and in particular relates to a super-diffraction-limited Brillouin scattering microscopic imaging method and system. Background Art

[0002] Brillouin scattering microscopy is a technology based on the interaction between light and sound waves in a medium. It has the following notable features: it is label-free and does not require chemical dyes or fluorescent labels, making it suitable for imaging living cells and tissues and avoiding interference that may be introduced by the labeling process; it is specific and can directly measure the mechanical properties of samples (such as elastic modulus and viscosity), providing information that cannot be obtained using traditional optical microscopy techniques; it is non-contact and uses optical methods for measurement without causing physical contact or damage to the sample, making it 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, existing technologies still have limitations in terms of resolution. The resolution of traditional Brillouin microscopy is usually at the sub-micron level, which is significantly lower than other super-resolution imaging technologies. This technical bottleneck limits the widespread application of Brillouin scattering microscopy in fine structure research. Summary of the invention

[0004] In view of the above, the purpose of the present invention is to provide a super-diffraction-limited Brillouin scattering microscopy method and system, which utilizes a pump-probe method to excite stimulated Brillouin scattering on the surface or inside of a sample, and based on the stimulated Brillouin scattering mechanism, utilizes light field control to change the spatial distribution of Brillouin gain in 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, ultimately realizing non-contact, super-diffraction-limited Brillouin scattering microscopy of the sample.

[0005] To achieve the above-mentioned object of the invention, an embodiment provides a super-diffraction-limited Brillouin scattering microscopic imaging system, comprising:

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

[0007] The 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;

[0008] The microscopy module is used to focus the probe light and pump light after light field regulation 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;

[0009] 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;

[0010] 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.

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

[0012] Preferably, the super-diffraction limit modulation includes: based on the point spread function modification of the additional pump light, at this time the light source module also generates an additional light beam as the additional pump light, wherein the point spread function modification process based on the additional pump light is:

[0013] 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 , where f P , f S , f L are the frequencies of pump light, probe light, and annular additional pump light, respectively, and f B The energy of the pump spot is transferred to the detection spot based on the stimulated Brillouin scattering mechanism due to the Brillouin frequency shift of the sample at the focusing point, 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.

[0014] Preferably, the super-diffraction limit modulation includes: based on the point spread function modification of the additional detection light, at this time 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:

[0015] A beam of annular additional probe light is introduced to induce phonon depletion around the detection spot, or to form interference destructiveness, thereby reducing the range of the point spread function. Specifically, the frequency of the annular additional probe light is consistent with that of the detection light, but the phase is opposite, so as to achieve interference destructiveness and reduce the range of the point spread function; or the annular additional probe light and the pump light are specifically designed to meet the Brillouin scattering condition to deplete the phonons around the Airy disk, while ensuring that the Gaussian probe 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 probe light and the Gaussian probe light to form an energy competition mode, and to facilitate optical signal separation, ultimately reducing the range of the point spread function and obtaining a detection spot beyond the diffraction limit.

[0016] Preferably, the super-diffraction limit modulation includes: structured light illumination based on polarization regulation, specifically changing the spatial polarization distribution of the pump spot and the detection spot by adjusting the polarization parameters, focusing the structured light onto the sample through the microscope module, so that the Brillouin gain of the high polarization matching area in the focusing point is enhanced, and the Brillouin gain of the low polarization matching area is suppressed, thereby achieving spatial selective enhancement of stimulated Brillouin scattering in the detection spot and obtaining super-diffraction limit detection spot information.

[0017] Preferably, the super-diffraction limit modulation includes: structured light modulation based on phase modulation, specifically generating periodic structured light of different phases and directions by adjusting the spatial frequency, phase parameters and direction parameters, 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 super-diffraction limit.

[0018] Preferably, the super-diffraction limited modulation includes: high-order Brillouin modulation, specifically adjusting the frequency and spot distribution of the detection light and the pump light to match the excitation conditions of the high-order Brillouin scattering process, focusing the modulated light beam onto the sample through a microscope module, inducing high-order acoustic modes in the focal point, obtaining high-order Brillouin scattering signals containing the microstructure information of the sample, and obtaining detection spot information beyond the diffraction limit.

[0019] Preferably, the microscope module includes a first reflector, a first microscope objective lens, a second reflector, a beam splitter, a second microscope objective lens, and a three-dimensional electric translation stage. The pump light after light field modulation passes through the first reflector, the beam splitter, and the first microscope objective lens in sequence and is focused into the sample on the three-dimensional electric translation stage. The detection light after light field modulation passes through the second reflector and the second microscope objective lens in sequence and is focused into the sample on the three-dimensional electric translation stage to stimulate stimulated Brillouin scattering inside or on the surface of the sample.

[0020] To achieve the above-mentioned object of the invention, an embodiment of the present invention provides a super-diffraction-limited Brillouin scattering microscopic imaging method, using the above-mentioned Brillouin scattering microscopic imaging system, and the method comprises the following steps:

[0021] A light source module is used to generate two excitation light beams as detection light and pump light and output them;

[0022] 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;

[0023] 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;

[0024] 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;

[0025] 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.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

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

[0028] 2. There are no special requirements for the type of sample. It can be either a labeled sample or an unlabeled sample, either a high Brillouin gain sample or an ordinary sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 A schematic diagram of the structure of a super-diffraction-limited Brillouin scattering microscopic imaging system provided by the present invention;

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

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

[0033] Figure 4 is a schematic diagram of a polarization pattern in Example 3;

[0034] Figure 5 is a schematic diagram of a structured light mask in Example 4;

[0035] Figure 6 is a high-order Brillouin scattering spectrum diagram in Example 5;

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

[0037] To make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0038] like Figure 1As shown, the super-diffraction-limited Brillouin scattering microscopic imaging system provided in the embodiment includes a light source module 1, a light field control 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 line width. The laser light beam includes a detection light and a pump light, etc. The line width of the light beam is less than 100kHz to ensure high coherence and high frequency resolution; the light field control module 2 is used to control the wavelength (frequency), polarization, wave vector, phase and other optical parameters of the excitation light beam, thereby changing the spatial distribution of the Brillouin gain in the detection light spot and realizing super-diffraction-limited modulation; the microscopy module 3 is used to focus the light beam after light field control on the sample, so that stimulated Brillouin scattering occurs on the surface or inside of the sample, generate a super-resolution Brillouin spatial light spot, capture the detection light signal and transmit it to the signal acquisition and processing module 5, and the key components in the microscopy module 3 include a first reflector 301, a spectroscope 302, a first microscope objective 303, a three-dimensional electric translation stage 304, and a second microscope 305. The objective lens 305 and the second reflector 306, the pump light after light field modulation passes through the first reflector 301, the beam splitter 302, and the first microscope objective lens 303 in sequence and is focused into the sample 4 on the three-dimensional electric translation stage 304, and the detection light after light field modulation passes through the second reflector 306 and the second microscope objective lens 305 in sequence and is focused into the sample 4 on the three-dimensional electric translation 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 detection light to obtain a single-point Brillouin scattering image beyond the diffraction limit; the scanning and imaging module 6 is used to control the light source module 1, the light field control module 2, and the three-dimensional electric translation stage 304 to perform point scanning, and realize multi-dimensional Brillouin scattering microscopic imaging beyond the diffraction limit based on the single-point Brillouin scattering spectrum information.

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

[0040] (1) Beam generation: using a light source module to generate at least two excitation beams as pump light and probe light based on a pump-probe method, and optionally generating a third additional beam to modify the point spread function, wherein the line width of each beam is less than 100 kHz to ensure high coherence and high frequency resolution;

[0041] (2) Super-diffraction-limited modulation: The optical field control module is used to control the optical parameters of the excitation light beam generated by the light source module. By changing the spatial distribution of the Brillouin gain in the detection spot, the gain in some areas remains unchanged or enhanced, while the gain in other areas is lost / suppressed, thus achieving super-diffraction-limited modulation.

[0042] (3) Stimulated Brillouin excitation: The light beam modulated by the light field control module is focused on the sample through the microscope module. The pump light and the probe light meet the stimulated Brillouin scattering conditions 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, achieving specific energy conversion and acoustic wave excitation.

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

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

[0045] The occurrence of stimulated Brillouin scattering in the sample requires satisfying the stimulated Brillouin scattering conditions, namely the momentum matching and energy matching formulas between the pump light, the probe light and the generated acoustic wave:

[0046]

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

[0048] The super-diffraction limit modulation method of the light field control module in the embodiment includes point spread function modification, structured light modulation, and high-order Brillouin scattering, and each method is described in detail below.

[0049] Example 1

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

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

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

[0053] like Figure 2 As shown in (b), when the pump light, the probe light, and the annular additional pump light exist at the same time, the pump light and the probe light meet the stimulated Brillouin scattering condition, so the pump light energy is transferred to the probe light, and the probe light and the annular additional pump light also meet the stimulated Brillouin scattering condition, so the probe light energy is transferred to the annular additional pump light, resulting in the frequency domain intensity diagram of the probe light at the frequency f S A dip appears at , generating a Brillouin gain-loss spectrum.

[0054] As shown in Figure 2(c), since the pump light and the probe light are circular spots, and the additional pump light is an annular spot, only the pump light and the probe light interact in the center of the probe spot, and the frequency domain intensity diagram of the central area of ​​the probe light is the Brillouin gain spectrum; the annular area of ​​the probe spot interacts with the pump light and the additional pump light at the same time, and the frequency domain intensity diagram of the probe light here is the Brillouin gain-loss spectrum. Therefore, the frequency is f S The spatial size of the detection light signal is the size of the circular detection light spot minus the size of the annular additional pump light spot. Since the detection light spot reaches the diffraction limit under the focus of the microscope module, after subtracting the annular additional pump light spot, the remaining central light spot is the spatial light spot that breaks through the optical diffraction limit.

[0055] Example 2

[0056] The point spread function modification based on additional probe light is used as the super-diffraction limit modulation method in this embodiment. The core is to introduce a beam of annular additional probe light to induce phonon depletion around the detection spot, or to form interference destructiveness by setting opposite phase conditions, thereby reducing the range of the point spread function.

[0057] Specifically, the key to achieving phonon depletion is to control the wave vector mismatch Δk (Δk = k P -k s -q), Brillouin gain g B The theoretical relationship with the wave vector mismatch is:

[0058]

[0059] Where a is the damping constant related to the phonon properties of the medium, reflecting the mechanical and optical properties of the sample. When Δk is 0, the Brillouin gain reaches its 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 adjusting the wave vectors of the two probe lights to change the wave vector mismatch, energy competition can be formed between the Gaussian probe light and the annular additional probe light, thereby transforming the point spread function and achieving a super-diffraction limit effect.

[0060] Specifically, Figure 3 As shown, the wave vector of the annular additional probe light is adjusted to make it mismatch with the wave vector of the pump light by Δk I = 0, that is, the Brillouin scattering condition is satisfied, thereby maximally exhausting the phonons around the Airy disk; at the same time, the wave vector of the Gaussian probe light is adjusted to make it mismatch with the wave vector of the pump light by Δk s ≈0, that is, the Brillouin scattering condition is met, 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 area of ​​the Airy disk will interact with the annular additional probe light and be depleted, and the phonons in the central area will interact with the Gaussian probe light, ultimately reducing the range of the point spread function and generating a spatial light spot that breaks the optical diffraction limit.

[0061] Example 3

[0062] Structured light illumination based on polarization control is used as the super-diffraction limit modulation method in this embodiment. This embodiment achieves super-diffraction limit Brillouin scattering microscopy imaging by controlling the polarization state of the pump light and the probe light and using the influence of polarization matching on the Brillouin gain. By optimizing the polarization distribution of light, the intensity and resolution of the signal can be controlled. The specific implementation plan is as follows:

[0063] (1) Polarized illumination: Use a spatial light modulator to control the polarization states of the pump light and the detection light at different spatial positions respectively. To this end, a preset polarization pattern is designed to cover the entire imaging area. In the high polarization matching area, the polarization directions of the pump light and the detection light are kept the same or similar as much as possible to maximize the Brillouin gain, thereby enhancing the signal strength; in the low polarization matching area, the polarization states of the pump light and the detection light are set to be orthogonal or have significant differences to reduce the Brillouin gain and suppress the signal strength; a gradient polarization distribution is set between the high and low polarization matching areas to ensure a smooth transition of the light field and avoid edge effects or artifacts. The spatial polarization distribution of the pump light spot and the detection light spot is dynamically adjusted by controlling to achieve gain enhancement and high-resolution imaging of specific areas. Such as Figure 4 Shown is a schematic diagram of the polarization patterns of a detection light spot and a pump light spot.

[0064] (2) Signal acquisition: The structured light with different patterns of polarization modulation is focused onto the sample through the microscope module. Under each polarization pattern condition, the single-point stimulated Brillouin scattering spectrum signal generated by the sample beyond the diffraction limit is collected to form a series of image stacks. These images reflect the intensity distribution of the scattering spectrum signal under different polarization matching conditions. Specifically, a high-sensitivity detector can be used in the signal acquisition and processing module to ensure that the changes in the scattering spectrum signal under various polarization conditions can be accurately captured.

[0065] (3) Scanning imaging: The obtained image stack is processed to extract and synthesize super-resolution information, and the super-resolution information under various polarization matching conditions is fused to finally generate a multi-dimensional Brillouin scattering microscopy image that exceeds the diffraction limit.

[0066] Example 4

[0067] The phase-modulated structured light illumination modulation is used as the super-diffraction limit modulation method in this embodiment. This embodiment uses structured light illumination technology to improve the spatial resolution of Brillouin scattering imaging through the moiré fringe effect. Specifically, by adjusting the phase parameters and direction parameters to generate periodic structured light of different phases and directions, structured light illumination can encode high-frequency information that exceeds the resolution limit of the optical system into an observable low-frequency range, thereby achieving super-resolution in subsequent image processing. The specific implementation process is as follows:

[0068] (1) Structured light illumination: Use digital micromirror devices or spatial light modulators to generate a variety of structured light patterns, such as Figure 5 As shown. Depending on the experimental requirements, these structured light patterns can be stripes, lattices, or other periodic structures. The structured light pattern is projected onto the sample through the microscopy module, and a controllable moiré effect is produced by changing the spatial frequency, phase, and direction of the light. In this process, precise adjustment of the light pattern can optimize the illumination of the sample to achieve the best signal encoding effect.

[0069] (2) Frequency encoding: Structured light patterns of different spatial frequencies are superimposed on the sample to generate moiré fringes. These moiré fringes can transfer the high-frequency information present in the sample to the low-frequency range detectable by the optical system. Through structured light modulation, the high-frequency characteristics of the sample can be effectively encoded, so that the information can be decoded and recovered in subsequent signal processing;

[0070] (3) Data acquisition and reconstruction: Illuminate the sample with structured light of different phases and directions, collect the single-point stimulated Brillouin scattering spectral signals beyond the diffraction limit generated by the sample, and form multiple sets of moiré fringe images; the classic three-step phase shift method or five-step phase shift method can be used to obtain image data of 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, and finally reconstruct a Brillouin scattering image with spatial super-resolution.

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

[0072] Example 5

[0073] High-order Brillouin modulation is used as the super-diffraction limit modulation method in this embodiment. Multi-order Stokes signals are generated through high-order nonlinear optical processes, thereby achieving super-resolution imaging. Figure 6 As shown, in the stimulated Brillouin scattering process, in addition to the fundamental frequency Stokes signal, a series of high-order Stokes signals with frequencies higher than the fundamental frequency are generated. The frequency interval between these signals is equal to an integer multiple of the Brillouin frequency shift. The high-order Stokes signal provides additional spectral information, which helps to distinguish more subtle structural features in the sample. In addition, the high-order Brillouin scattering process produces 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 realizing a super-resolution Brillouin space spot. This embodiment specifically includes the following steps:

[0074] (1) High-order Brillouin excitation: The frequency, phase and spatial distribution of the pump and probe lights are adjusted by combining the wavelength tuning and spatial modulation techniques using the light field control module to match the excitation conditions of the high-order Brillouin scattering process. The modulated pump and probe lights are focused into the sample through a high numerical aperture microscope objective to induce high-order acoustic modes. These modes are excited through multiphoton 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 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 use of a phase-locked amplifier or coherent detection technology can further improve the detection sensitivity and accuracy of the spectral information.

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

[0077] The specific implementation methods described above provide a detailed description of 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 intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in 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; The 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; The microscopy module is used to focus the probe light and pump light after light field regulation 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; 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. The super-diffraction-limited Brillouin scattering microscopy system according to claim 1, characterized in that: The line width of the excitation light beam generated by the light source module is lower than 100 kHz, and the optical parameters regulated by the light field regulation module include at least one of wavelength (frequency), polarization, wave vector, and phase.

3. The super-diffraction-limited Brillouin scattering microscopy imaging system according to claim 1, characterized in that: The super-diffraction limit modulation includes: a point spread function modification based on additional pump light, in which case the light source module also generates an additional light beam as additional pump light, wherein the point spread function modification process based on the additional pump light is: 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 , where f P , f S , f L are the frequencies of pump light, probe light, and annular additional pump light, respectively, and f B The energy of the pump spot is transferred to the detection spot based on the stimulated Brillouin scattering mechanism due to the Brillouin frequency shift of the sample at the focusing point, 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.

4. The super-diffraction-limited Brillouin scattering microscopy imaging system according to claim 1, characterized in that: The super-diffraction limit modulation includes: a point spread function modification based on the additional detection light, in which case 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: A beam of annular additional probe light is introduced to induce phonon depletion around the detection spot, or to form interference destructiveness, thereby reducing the range of the point spread function. Specifically, the frequency of the annular additional probe light is consistent with that of the detection light, but the phase is opposite, so as to achieve interference destructiveness and reduce the range of the point spread function; or the annular additional probe light and the pump light are specifically designed to meet the Brillouin scattering condition to deplete the phonons around the Airy disk, while ensuring that the Gaussian probe 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 probe light and the Gaussian probe light to form an energy competition mode, and to facilitate optical signal separation, ultimately reducing the range of the point spread function and obtaining a detection spot beyond the diffraction limit.

5. The super-diffraction-limited Brillouin scattering microscopy imaging system according to claim 1, characterized in that: Super-diffraction-limited modulation includes: structured light illumination based on polarization regulation, specifically changing the spatial polarization distribution of the pump spot and the detection spot by adjusting the polarization parameters, focusing the structured light 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, thereby achieving spatial selective enhancement of stimulated Brillouin scattering in the detection spot and obtaining detection spot information beyond the diffraction limit.

6. The super-diffraction-limited Brillouin scattering microscopy imaging system according to claim 1, characterized in that: Super-diffraction-limited modulation includes: structured light modulation based on phase modulation, specifically, generating periodic structured light of different phases and directions by adjusting the spatial frequency, phase parameters and direction parameters, 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 exceeded the resolution limit of the optical system into an observable low-frequency range, and obtaining the detection spot information beyond the diffraction limit.

7. The super-diffraction-limited Brillouin scattering microscopy imaging system according to claim 1, characterized in that: Super-diffraction-limited modulation includes: high-order Brillouin modulation, which specifically adjusts the frequency and spot distribution of the probe light and pump light to match the excitation conditions of the high-order Brillouin scattering process, focuses the modulated light beam onto the sample through a microscope module, induces high-order acoustic modes within the focal point, obtains high-order Brillouin scattering signals containing the sample's microscopic structure information, and obtains super-diffraction-limited detection spot information.

8. The super-diffraction-limited Brillouin scattering microscopy imaging system according to claim 1, characterized in that: The microscope module includes a first reflector, a first microscope objective lens, a second reflector, a beam splitter, a second microscope objective lens, and a three-dimensional electric translation stage. The pump light after light field modulation passes through the first reflector, the beam splitter, and the first microscope objective lens in sequence and is focused into the sample on the three-dimensional electric translation stage. The detection light after light field modulation passes through the second reflector and the second microscope objective lens in sequence and is focused into the sample on the three-dimensional electric translation stage to stimulate stimulated Brillouin scattering inside or on the surface of the sample.

9. A Brillouin scattering microscopy method beyond the diffraction limit, characterized in that: Using the Brillouin scattering microscopy imaging system according to any one of claims 1 to 8, 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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