Super-resolution stimulated Raman scattering imaging method and apparatus for drug distribution detection

Abstract

This invention discloses a super-resolution stimulated Raman scattering imaging method for drug distribution detection, comprising the following steps: The laser output from a laser is frequency-doubled and spatially shaped to generate three laser beams, one used as a Stokes beam, one as a pump beam, and one as a saturation beam; the Stokes beam and the pump beam are spatially collinear; the three beams are temporally coupled; the beams are connected to the scanning section of a microscope system; a digital acquisition card acquires signals, obtaining the modulation response of the pump beam to the Stokes beam, thereby monitoring drug distribution. This invention also discloses the application of this super-resolution stimulated Raman scattering imaging method for drug distribution detection in the cellular targeting and intracellular distribution of drugs in the nervous system. By controlling the spatial distribution of the laser, this invention enables a portion of molecules within the focal point to be in an electron absorption saturation state, which can suppress the pump detection signal and break the diffraction limit of non-fluorescent substances in far-field imaging.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical and optical imaging technology, and in particular relates to a label-free super-resolution stimulated Raman scattering (SRS) imaging method and device for detecting drug distribution. Background Technology

[0002] With the development of cell-targeting technologies, studying drug distribution at the subcellular level is crucial. Super-resolution stimulated Raman scattering (SRS) imaging, which breaks the diffraction limit, is a label-free, high-resolution imaging technique that can observe drug distribution in cells and tissues. This technique can detect and locate drugs at the molecular, cellular, and tissue levels by increasing the resolution of optical images to tens of nanometers. This technology has wide applications in the biomedical field, enabling the monitoring of receptors, enzymes, and other molecules on cell membranes, and the study of drug transport, metabolism, and distribution. The widespread application of super-resolution imaging technology will accelerate the development of new drug research and drug therapy. Using super-resolution imaging systems, we can not only study which tissues and organs drugs mainly accumulate in, but also refine the process down to the cellular level, observing which cells in the target organ take up the drug, and even delve into the substructures of cells to study the distribution and aggregation state of drugs within target cells.

[0003] Driven by disciplines such as computer science, molecular biology, and cell biology, drug distribution detection technology is developing towards greater precision and accuracy. Optical microscopy is becoming increasingly refined with the progress of biomedical research. However, the diffraction limit restricts the observation capabilities of optical microscopes, and the microscopic imaging techniques applicable to cells or tissues still face significant challenges. Fluorescent labeling-based microscopy can indirectly achieve specific super-resolution imaging of biomolecules by manipulating exogenous labeled molecules, but it is limited to fluorescently labeled cells and tissues. Whether based on endogenous gene alteration-based fluorescent protein labeling or exogenous staining, the fluorescent tags are often large molecules providing image contrast. These tags have a larger size or molecular weight than the proteins they modify or label, thus posing potential interference and altering the structure and functional expression of the target protein, potentially leading to inaccurate localization or loss of functional activity. Furthermore, fluorescent groups often exhibit photobleaching, showing instability and sometimes undergoing transient quenching, resulting in the loss of light signals. Therefore, far-field label-free technology has emerged, enabling high-resolution, precise localization analysis of active biochemical reactions, metabolic activities, small molecule metabolites, and drug distribution / metabolism within cells.

[0004] In recent years, label-free high-resolution microscopy has developed rapidly, enabling direct detection of intrinsic biomolecules and generating image contrast. Label-free molecular vibrational microscopy (e.g., infrared absorption microscopy, spontaneous Raman scattering microscopy) can utilize the intrinsic chemical bond vibrations of molecules for imaging. However, near-infrared excitation light limits the spatial resolution of infrared absorption microscopy, and the low imaging sensitivity of spontaneous Raman scattering microscopy limits its imaging speed. Although coherent anti-Stokes Raman scattering microscopy has high imaging sensitivity, it is susceptible to interference from strong non-resonant background signals.

[0005] SRS microscopy not only boasts high imaging sensitivity but also overcomes interference from non-resonant background signals, enabling chemically specific imaging without complex sample preparation or demanding imaging environments. This microscope is based on the resonance between photons and the vibrational energy levels of chemical bonds in sample molecules, achieving chemical bond imaging by detecting the modulation response of the pump light to Stokes light, without relying on fluorescence detection. However, the spatial resolution of SRS has been limited to around 300 nm, and super-resolution imaging remains a pressing area for development. Summary of the Invention

[0006] The purpose of this invention is to design a super-resolution hyperspectral SRS imaging method, device, and application based on drug distribution by combining the saturation effect.

[0007] To achieve the objective of this invention, the following technical solution is proposed:

[0008] A super-resolution stimulated Raman scattering imaging method for drug distribution, characterized by comprising the following steps:

[0009] Step S1: The laser outputs two laser beams. One beam, beam A, has a pulse width of 220 fs and a wavelength fixed at 1064 nm, and is used as a Stokes beam. The other beam, beam B, has a pulse width of 100 fs and a wavelength set to 903 nm, and is used as a pump beam.

[0010] S1.1, the A-beam laser passes through the first half-wave plate and the first polarization beam splitter, and then is sinusoidally intensity modulated at a frequency of 2.23MHz by an acousto-optic modulator to be used as Stokes light;

[0011] S1.2, the A-beam laser beam after passing through the acousto-optic modulator is connected to the first input terminal of the first dichroic mirror via the first time delay device;

[0012] S1.3, the B-beam laser is split into two beams, B1 and B2, by the second half-wave plate and the second polarization beam splitter; the B1 beam is connected to the second input terminal of the first dichroic mirror.

[0013] The B2 beam undergoes photon frequency doubling through a barium borate crystal to obtain a pulsed laser with a wavelength of 451.5 nm, which is used as saturation light.

[0014] S1.4, the 451.5nm B2 pulsed laser beam passes through a second time delay device, is shaped by pinhole filtering, and is finally connected to a vortex phase plate to generate vortex light;

[0015] Specifically, in step S1.4, the pinhole of the pinhole filter is selected with a diameter of 25μm for filtering, so that the saturated light presents a better Gaussian mode.

[0016] In step S2, the Stokes beam A output from the first input terminal and the pump beam B1 output from the second input terminal are spatially collinear after passing through a first dichroic mirror. Then, they pass through a dispersive medium with linear chirp coefficients to match the pump beam and the Stokes beam, and finally through a polarization-maintaining single-mode fiber before being incident on a second dichroic mirror. The first dichroic mirror ensures spatial collinearity between the Stokes beam after the first time-delay device and the pump beam after the second polarization beam splitter, guaranteeing signal strength, optimizing beam quality, and further improving stability. Furthermore, it avoids non-specific resonance signals while achieving high-spectral-resolution hyperspectral imaging.

[0017] Step S3: By adjusting the first delay device and the second delay device, the three beams of light, A beam Stokes beam, B1 beam pump beam and B2 beam pulsed laser, are coupled in time and collinear in space.

[0018] In step S4, the three collinear beams of light from step S3 are fed into the scanning section of the microscope system.

[0019] The microscope performs planar scanning using a biaxial two-dimensional scanning galvanometer, scanning three excitation beams that have been registered in the time and space domains. The sample is excited by two tightly focused laser beams, where the pump beam and the Stokes beam, which serves as the probe beam, have different wavelengths but are both located within the absorption band of the sample molecules. In this case, the pump beam will only excite one molecule and cause it to no longer remain in the ground state, so the photons from the probe beam cannot be absorbed.

[0020] Among them, three bandpass filters are installed in front of the photodiode to filter Stokes light with a wavelength of 1064nm as the detection beam;

[0021] The pump light is collected by an oil-immersed condenser lens, filtered out by a bandpass filter, and received by a photodiode.

[0022] Step S5, the rapid switching modulation of the strongly saturated pump light will cause the probe beam to have a modulation response at the same modulation frequency;

[0023] Step S6: A fast lock-in amplifier is used to realize heterodyne detection, and the imaging signal is acquired by a digital acquisition card and then analyzed and processed.

[0024] Step S7: When the pump light is applied to the sample, information including electronic energy levels and carrier dynamics can be obtained by measuring the changes in the transmittance and reflectance of the probe beam. This enables contactless and non-destructive detection of cells and tissues, thereby enabling the monitoring of drug distribution.

[0025] Preferably, in step S1.4, the saturated light B2 beam pulsed laser is collimated by a plano-convex lens and then generates vortex light by a phase-type diffraction element with a topological charge m=1 vortex phase plate.

[0026] Preferably, in step S1.4, the surface of the vortex phase plate has a spiral stepped structure. When the incident beam passes through, the surface structure of the vortex phase plate causes different changes in the optical path of the beam, thereby changing the phase change of the transmitted beam and generating vortex light. The optical path is simple and stable, and the output vortex beam has high precision and high energy efficiency (>90%). Introducing vortex light can suppress the SRS process around the focal spot, enabling the spatial resolution of near-infrared laser-based SRS microscopes to break through the diffraction limit.

[0027] Preferably, the dispersive medium in step S2 is a 0.25m long glass rod. This allows both beams of light to pass simultaneously through a sufficiently long dispersive medium, which is used to match the linear chirp coefficients of the pump light and the Stokes light.

[0028] Preferably, in step S2, the polarization-maintaining single-mode fiber has a length of 0.4m, achieving good Gaussian modes, absolute collinearity, and spatial stability for the two beams, which is also beneficial for super-resolution imaging. Simultaneously, these two beams are also used as excitation light for conventional SRS imaging. By matching the linear chirp coefficients of the pump light and the Stokes light, the spectral resolution is optimized.

[0029] Preferably, in step S3, both the first delay device and the second delay device are equipped with two devices arranged at 90 degrees. ° The system consists of an electrically driven two-dimensional displacement platform for the angled reflector. By moving the platform, the optical path can be changed, thereby achieving temporal coupling of three pulsed laser beams.

[0030] Preferably, in step S4, the scanning section is also equipped with two high-NA objectives.

[0031] This invention also provides an imaging apparatus for a super-resolution stimulated Raman scattering imaging method for drug distribution, comprising an excitation source module, an optical frequency doubling module, a laser scanning module, and a signal acquisition module, wherein...

[0032] The excitation source module includes a dual-output fly-laser system. The emitted laser is used as a laser source for stimulated Raman scattering imaging. It has two outputs, in which the A beam has a pulse width of 220 fs and a wavelength fixed at 1064 nm. After being sinusoidally intensity modulated at a frequency of 2.23 MHz by an acousto-optic modulator, it is used as Stokes light and passes through the first half-wave plate and the first polarization beam splitter.

[0033] Another beam, B, with a pulse width of 100 fs and a wavelength of 903 nm, is used as pump light. The pump light is split into two by a second half-wave plate and a polarizing beam splitter. The B2 beam undergoes photon frequency doubling via a barium borate crystal to generate a pulsed laser with a wavelength of 451.5 nm, which is used as saturation light. After being collimated by a plano-convex lens, the saturation light is used to generate vortex light by a vortex phase plate with a topological charge of m = 1.

[0034] Another B1 pump beam and the acousto-optic modulated A Stokes beam are combined through the first dichroic mirror to achieve spatial collinearity. Then, they are simultaneously coupled into a 0.4m long polarization-maintaining single-mode fiber. The collinear B1 pump beam and A Stokes beam are then spatially collinear with the B2 ring saturated beam passing through a quarter-wave plate via the second dichroic mirror, and then connected to the microscope system scanning module.

[0035] The sample carrier module includes a sample carrier platform and an objective lens. The sample carrier platform is used to place the sample. The signal acquisition module includes a photodetector and a lock-in amplifier, and then the data is collected through a data acquisition card.

[0036] Preferably, the saturated light after frequency doubling is shaped by passing it through a pinhole with a diameter of 25 μm;

[0037] Both the pump light and saturation light paths are equipped with time delay devices. These devices consist of an electrically driven two-dimensional displacement platform equipped with two mirrors at a 90° angle. The optical path can be changed by moving the platform, thus achieving time coupling of the three pulsed laser beams.

[0038] The objective lens is an Olympus super apochromatic immersion objective lens, used to compensate for chromatic aberration and spherical aberration from the ultraviolet to the near-infrared region, which can acquire clear images and maintain the good polarization characteristics of the laser.

[0039] The pump light is collected by an oil-immersion condenser lens, filtered out by a bandpass filter, and detected by a photodiode. In order to filter out the 1064nm Stokes light, three high-quality bandpass filters are installed in front of the photodiode, and a fast lock-in amplifier is used to achieve heterodyne detection.

[0040] The present invention also provides the application of super-resolution stimulated Raman scattering imaging method for drug distribution in the distribution of drugs in the nervous system, cellular targeting of drugs, and intracellular distribution.

[0041] Advantages and beneficial effects of the present invention:

[0042] 1. This invention proposes a super-resolution imaging method based on the saturated absorption effect and constructs a super-resolution SRS microscopy imaging device. This system has no special requirements on the conductivity of materials, surface cleanliness, or imaging environment, and can achieve super-resolution far-field imaging of samples, making the detection of drug distribution more precise and accurate.

[0043] 2. This invention, based on stimulated Raman scattering (SRS), introduces visible light into an SRS microscope. By introducing a saturated laser into the optical path system to induce electronic state transitions in ground-state sample molecules, the SRS process is effectively suppressed, and the SRS signal is switched reversibly by approximately 100%. This improves the spatial resolution of traditional SRS microscopes. By utilizing the ground-state loss process that occurs after electronic resonance to compensate for pump photons, and finally obtaining image contrast by directly detecting the Raman fingerprint spectrum, chemical imaging of fine structures in cells and tissues is demonstrated. This enables super-resolution chemical imaging of live cells and drug distribution.

[0044] 3. This invention controls the spatial distribution of the laser, so that a portion of the molecules within the focal point are in an electron absorption saturation state, which can suppress the pump detection signal and break the diffraction limit of non-fluorescent materials in far-field imaging.

[0045] 4. Based on the saturation effect, this invention designs a hollow laser beam to suppress electron transitions around the focal point of the excitation light by using a saturated electron beam, and introduces modulation only at the focal center. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 A schematic diagram of the pump detection principle provided for this invention;

[0048] Figure 2 The present invention provides energy level diagrams of the SRS process before and after saturation.

[0049] Figure 3 This is a schematic diagram of the scanning images of different spot patterns of the present invention and the fine features of the sample;

[0050] Figure 4 This is a schematic diagram of the super-resolution SRS system detection principle provided by the present invention;

[0051] Figure 5 This is a schematic diagram of the super-resolution SRS microscopic imaging system provided by the present invention;

[0052] Figure 6 This is a schematic diagram of the microscope structure in the microscopic imaging device provided by the present invention;

[0053] Figure 7 A schematic diagram of the overall structure of a super-resolution stimulated Raman scattering imaging device for drug distribution according to the present invention. Detailed Implementation

[0054] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0055] The super-resolution stimulated Raman scattering imaging method for drug distribution provided by this invention includes the following steps:

[0056] Step S1: The laser outputs two laser beams. One beam, beam A, has a pulse width of 220 fs and a wavelength fixed at 1064 nm, and is used as a Stokes beam. The other beam, beam B, has a pulse width of 100 fs and a wavelength set to 903 nm, and is used as a pump beam.

[0057] S1.1, the A-beam laser passes through the first half-wave plate and the first polarization beam splitter, and then is sinusoidally intensity modulated at a frequency of 2.23MHz by an acousto-optic modulator to be used as Stokes light;

[0058] S1.2, the A-beam laser beam after passing through the acousto-optic modulator is connected to the first input terminal of the first dichroic mirror via the first time delay device;

[0059] S1.3, the B-beam laser is split into two beams, B1 and B2, by the second half-wave plate and the second polarization beam splitter; the B1 beam is connected to the second input terminal of the first dichroic mirror.

[0060] The B2 beam undergoes photon frequency doubling through a barium borate crystal to obtain a pulsed laser with a wavelength of 451.5 nm, which is used as saturation light.

[0061] S1.4, the 451.5nm B2 pulsed laser beam passes through a second time delay device, is shaped by pinhole filtering, and is finally connected to a vortex phase plate to generate vortex light;

[0062] Preferably, in step S1.4, the saturated light B2 beam pulsed laser is collimated by a plano-convex lens and then generates vortex light by a phase-type diffraction element with a topological charge m=1 vortex phase plate.

[0063] Preferably, in step S1.4, the surface of the vortex phase plate has a spiral stepped structure. When the incident beam passes through, the surface structure of the vortex phase plate causes different changes in the optical path of the beam, thereby changing the phase change of the transmitted beam and generating vortex light. The optical path is simple and stable, and the output vortex beam has high precision and high energy efficiency (>90%). Introducing vortex light can suppress the SRS process around the focal spot, enabling the spatial resolution of near-infrared laser-based SRS microscopes to break through the diffraction limit.

[0064] In step S1.4, the pinhole of the pinhole filter is selected with a diameter of 25μm for filtering, so that the saturated light presents a better Gaussian mode.

[0065] In step S2, the Stokes beam A output from the first input terminal and the pump beam B1 output from the second input terminal are spatially collinear after passing through a first dichroic mirror. Then, they pass through a dispersive medium with linear chirp coefficients to match the pump beam and the Stokes beam, and finally through a polarization-maintaining single-mode fiber before being incident on a second dichroic mirror. The first dichroic mirror ensures spatial collinearity between the Stokes beam after the first time-delay device and the pump beam after the second polarization beam splitter, guaranteeing signal strength, optimizing beam quality, and further improving stability. Furthermore, it avoids non-specific resonance signals while achieving high-spectral-resolution hyperspectral imaging.

[0066] The dispersive medium in step S2 is a 0.25m long glass rod. Both beams of light pass simultaneously through a sufficiently long dispersive medium to match the linear chirp coefficients of the pump light and the Stokes light.

[0067] In step S2, the polarization-maintaining single-mode fiber has a length of 0.4m, achieving good Gaussian modes, absolute collinearity, and spatial stability for both beams, which is beneficial for super-resolution imaging. Simultaneously, these two beams are also used as excitation light for conventional SRS imaging. By matching the linear chirp coefficients of the pump light and the Stokes light, the spectral resolution is optimized.

[0068] Step S3: By adjusting the first delay device and the second delay device, the three beams of light, A beam Stokes beam, B1 beam pump beam and B2 beam pulsed laser, are coupled in time and collinear in space.

[0069] Preferably, in step S3, both the first delay device and the second delay device are equipped with two devices arranged at 90 degrees. ° The system consists of an electrically driven two-dimensional displacement platform for the angled reflector. By moving the platform, the optical path can be changed, thereby achieving temporal coupling of three pulsed laser beams.

[0070] In step S4, the three collinear beams of light from step S3 are fed into the scanning section of the microscope system.

[0071] In step S4, the scanning section is also equipped with two high-NA objectives.

[0072] The microscope performs planar scanning using a biaxial two-dimensional scanning galvanometer, scanning three excitation beams that have been registered in the time and space domains. The sample is excited by two tightly focused laser beams, where the Stokes beam and the pump beam, which serves as the probe beam, have different wavelengths but are both located within the absorption band of the sample molecules. In this case, the saturation light excites the sample molecules and puts them in an excited state, so photons from both the probe beam and the Stokes beam cannot be absorbed by the sample molecules.

[0073] Among them, three bandpass filters are installed in front of the photodiode to filter the pump light with a wavelength of 903nm as the detection beam;

[0074] The pump light is collected by an oil-immersed condenser lens, filtered out by a bandpass filter, and received by a photodiode.

[0075] Step S5, the rapid switching modulation of the strongly saturated pump light will cause the probe beam to have a modulation response at the same modulation frequency;

[0076] Step S6: A fast lock-in amplifier is used to realize heterodyne detection, and the imaging signal is acquired by a digital acquisition card and then analyzed and processed.

[0077] Step S7: When the pump light is applied to the sample, information including electronic energy levels and carrier dynamics can be obtained by measuring the changes in the transmittance and reflectance of the probe beam. This enables contactless and non-destructive detection of cells and tissues, thereby enabling the monitoring of drug distribution.

[0078] Combination Figure 1 , Figure 2 The principle of the super-resolution stimulated Raman scattering imaging device for drug distribution detection according to the present invention is explained as follows: The optical elements in the optical path exert a certain dispersion effect on the laser beam, causing the pulse width of pump light and Stokes light of different wavelengths to broaden to varying degrees over time after passing through the optical path. Due to the different refractive indices of different wavelengths of laser light in the dispersive medium, the resulting optical path difference ultimately causes photons of different energies to exhibit a linear distribution over time, ultimately leading to the broadening of the femtosecond pulse in both the time and frequency domains. The shorter the wavelength, the smaller the initial pulse width, and the greater the broadening. Figure 1The femtosecond excitation light introduces linear chirp, causing the frequency components of the excitation pulse to have a certain distribution along the time axis. Since the pump light and Stokes light have the same linear chirp parameter, the slopes of the linear distribution of the frequencies of the two laser beams over time are the same, and the instantaneous frequency difference between them is constant. Therefore, this method can be used to achieve high-spectral-resolution stimulated Raman scattering (SRS) imaging. When the pump light and Stokes light pass through the optical path, the pulse broadens in both the time and frequency domains. The overlapping portion of the two beams will produce the same frequency difference due to the same linear chirp coefficient, thus generating a high-spectral-resolution SRS imaging spectrum. The occurrence of SRS requires the excitation light to act on the ground-state molecules using photon energy differences to match the energy differences between the vibrational energy levels of the sample. The pump light pumps the ground-state molecules to a virtual state, and the Stokes light induces them to radiate back to a higher-order vibrational state. After a short relaxation time, these molecules cool back to the ground state. However, due to the additional saturated light excitation of the sample, the sample molecules are pumped to higher energy levels, resulting in the absence of ground-state molecules. This makes the sample transparent to the pump light and blocks the occurrence of the SRS effect.

[0079] Combination Figure 3 , Figure 4 The principle of a super-resolution stimulated Raman scattering (SRS) imaging device for drug distribution detection according to the present invention is explained as follows: In a microscopic imaging system based on point-scan imaging, the object size presented in the image is determined by the effective PSF of the excitation light on the sample plane, the PSF of the detection system, and the actual size of the object. The first two determine the lateral resolution of the imaging system. In a conventional SRS microscope, intensity-modulated or polarization-modulated Stokes light is used to induce a modulation response in the pump light, thereby enabling the detection of the SRS signal. The pump light, Stokes light, and saturation light, superimposed in the super-resolution space, are focused onto the sample through the objective lens. They are spatially collinear and simultaneously focused onto the sample. The annular saturation light draws molecules from the ground state to the excited state, while the pump light and Stokes light excite the sample to generate an SRS signal. Figure 3 Image 'a' is generated by scanning a cross-shaped sample using pump light and Stokes light. Figure 3 b is the image generated by scanning using a vortex light ring produced by a spiral phase plate. This image is generated by superimposing and scanning the pump light, Stokes light, and saturation light. Figure 3 The super-resolution SRS image of c. Before additional saturated light excitation of the sample, the SRS signal is obtained by detecting the modulation response of the pump light to the Stokes light at the focal point; after saturated light excitation of the sample, due to the loss of ground-state sample molecules, the sample molecules are transparent to near-infrared photons, and neither the pump light nor the Stokes light is absorbed, so the pump light no longer has a modulation response, and the signal cannot be detected.

[0080] Combination Figure 5 , Figure 6 , Figure 7The super-resolution stimulated Raman scattering imaging device for drug distribution according to the present invention is described below, comprising an excitation source module, a photon frequency doubling module, a laser scanning module, and a signal acquisition module. Specifically, it includes: an excitation source, a half-wave plate, a polarization beam splitter, a time delay device, an acousto-optic modulator, a β-phase barium borate crystal, a 25μm diameter pinhole, a dichroic mirror, a vortex phase plate, a polarization-maintaining single-mode fiber, a quarter-wave plate, a scanning module, a scanning lens, a barrel lens, a sample, an oil-immersed condenser lens, a bandpass filter, a photodiode, a lock-in amplifier, and a data acquisition module. The laser source outputs two laser beams. One beam has a fixed wavelength of 1064nm, which is sinusoidally intensity modulated at a frequency of 2.23MHz by the acousto-optic modulator and used as Stokes light. The other beam has a wavelength set to 903nm and is used as pump light. The input end of the first half-wave plate is connected to the first output end of the laser source, and the first half-wave plate is used to change the polarization direction of the linearly polarized light. The input of the second half-wave plate is connected to the second output of the laser source. The input of the first polarization beam splitter is connected to the output of the first half-wave plate, and the input of the second polarization beam splitter is connected to the output of the second half-wave plate. The first and second polarization beam splitters are used to filter out the vertical polarization component of the light. The input of the acousto-optic modulator is connected to the output of the first polarization beam splitter. The acousto-optic modulator is used to sinusoidally modulate the intensity of the first laser beam at a frequency of 2.23 MHz, and then use it as Stokes light. The output of the acousto-optic modulator is connected to the input of the first time-delay device, which consists of two beams angled at 90 degrees. °The system comprises an electrically driven two-dimensional displacement platform for the included-angle reflector. Moving the platform changes the optical path, enabling temporal coupling of the Stokes beam and the pump beam. The input of the first dichroic mirror is connected to the output of the first time-delay device and the output of the second polarization beam splitter. The first dichroic mirror is used to combine the pump beam and the acousto-optic modulated Stokes beam, achieving spatial collinearity. The input of the polarization-maintaining single-mode fiber is connected to the output of the first dichroic mirror. This fiber improves the beam pattern and stability of the two beams, optimizing the initially poor beam pattern into a better Gaussian mode. The output of the second polarization beam splitter is connected to the input of a barium borate crystal. This crystal performs photon frequency doubling on the pump beam, generating a 451.5nm pulsed laser, which is then used as the saturation beam. The input of the second time-delay device is connected to the output of the barium borate crystal. This device enables temporal coupling of the Stokes beam, the pump beam, and the saturation beam. The saturated light after passing through the second time-delay device is filtered through a 25μm diameter pinhole and then connected to the input of a vortex phase plate. The pinhole is used to shape the light spot to present a better Gaussian mode. The output of the vortex phase plate is connected to the input of a quarter-wave plate. The vortex phase plate is used to generate vortex light; specifically, it is a phase-type diffraction element with a "spiral step" structure on its surface. The saturated light generates vortex light through the vortex phase plate with a topological charge m=1. The optical path is simple and stable, and the output vortex light has high precision and high energy efficiency. The second input of the second dichroic mirror is connected to the output of the quarter-wave plate, which is used to convert circularly polarized light into linearly polarized light. The first input of the second dichroic mirror (bundler) is connected to the output of the polarization-maintaining single-mode fiber. The second dichroic mirror is used to achieve spatial collinearity between the already collinear pump light and Stokes light and the annular saturated light passing through the quarter-wave plate. The input end of the microscope is connected to the output end of the second dichroic mirror (beam combiner). The microscope is used to focus the combined laser beam onto the sample surface and acquire the signal of the focal plane by point scanning. The signal acquisition module is used to receive the light signal transmitted by the microscope, including a photodetector and a lock-in amplifier, and then collects it through a data acquisition card.

[0081] This invention provides a super-resolution SRS microscopic imaging device based on the saturation effect. By modulating the light field of saturated light and introducing high-photon-energy pulsed laser to consume sample molecules in the electronic ground state, the absorption of low-photon-energy near-infrared laser by the sample is blocked. This suppresses the SRS signal excited by both near-infrared pump light and Stokes light around the focal point in the traditional NIR-SRS system, reduces the volume of the effective focal point in the point scanning system, and provides a super-resolution SRS microscopic imaging device based on the saturation effect.

[0082] This invention utilizes a photonic frequency doubling system to split a 903nm pump light into two beams using a half-wave plate and a polarization beam splitter. One beam undergoes photonic frequency doubling via a barium borate crystal to generate a pulsed laser with a wavelength of 451.5nm, which is then used as the saturation light, thereby improving spatial resolution. The stability and collinearity of the pump light and Stokes light are ensured by introducing a single-mode polarization-maintaining fiber. Furthermore, by selecting a suitable dispersive medium (fiber) and matching the linear chirp coefficients of the pump light and Stokes light, the spectral resolution is optimized.

[0083] In traditional SRS microscopy, Stokes light is modulated by intensity or polarization to induce a modulation response in the pump light, thereby enabling the detection of the SRS signal. This invention proposes introducing a beam of saturated light in the visible light band into a super-resolution SRS imaging system. The superimposed pump light, Stokes light, and saturated light are focused onto the sample through the objective lens, achieving higher resolution.

[0084] This invention is based on the saturation effect. Because the SRS process in the annular region surrounding the focal spot is suppressed by the annular saturating light, and the sample is transparent to both the lower-energy pump light and Stokes light, the SRS process only occurs in the region near the center of the focused spot. At this point, the modulation response of the pump light to the Stokes light in the peripheral region disappears, and only the pump light at the center exhibits a modulation response to the Stokes light. Phase-locked detection can only demodulate and amplify the SRS signal in the central region of the focal spot. Therefore, the SRS signal detectable by the detector originates only from the center of the laser focused spot, and the effective volume of the laser focal spot is reduced to below the diffraction-limited size.

[0085] To generate an SRS signal, the pump light and Stokes light need to be coupled simultaneously in time and space. Since the pump light is detected by a photodiode (PD), its optical path is typically fixed to ensure stability. Because the pump light has higher photon energy than the Stokes light, a dichroic mirror, allowing short-wavelength lasers to pass through, is used for beam combining. The mirror and dichroic mirror shown in the figure are both mounted on a two-dimensional adjustable frame. By adjusting these two optical elements, the optical path of the Stokes light is altered, achieving spatial collinearity with the pump light.

[0086] The pump light and Stokes light are combined in free space by a dichroic mirror and then introduced into the same polarization-maintaining single-mode fiber (0.4m). The introduction of this fiber matches the chirp coefficient of the two laser beams, ensuring the spectral resolution of the hyperspectral imaging.

[0087] A more convenient pinhole filtering method was chosen for beam reshaping, which ensured signal strength while optimizing beam quality. The frequency-doubled saturated light, filtered through a 25μm diameter pinhole, exhibited a better Gaussian mode.

[0088] Both the pump light and saturation light paths are equipped with time delay devices, which consist of an electrically driven two-dimensional displacement platform with two mirrors at a 90° angle. The optical path can be changed by moving the platform to achieve time coupling of the three pulsed laser beams.

[0089] The objective lens is an Olympus super apochromatic immersion objective lens, which is used to compensate for chromatic aberration and spherical aberration from the ultraviolet to the near-infrared region. It can acquire clear images and maintain the good polarization characteristics of the laser.

[0090] The pump light is collected by an oil-immersion condenser lens, filtered out by a bandpass filter, and detected by a photodiode. In order to filter out the 1064nm Stokes light, three high-quality bandpass filters are installed in front of the photodiode, and a fast lock-in amplifier is used to achieve heterodyne detection.

Claims

1. A super-resolution stimulated Raman scattering imaging method for drug distribution detection, characterized in that, Includes the following steps: Step S1: The laser outputs two laser beams. One beam, beam A, has a pulse width of 220 fs and a wavelength fixed at 1064 nm, and is used as a Stokes beam. The other beam, beam B, has a pulse width of 100 fs and a wavelength set to 903 nm, and is used as a pump beam. S1.1, the A-beam laser passes through the first half-wave plate and the first polarization beam splitter, and then is sinusoidally intensity modulated at a frequency of 2.23 MHz by an acousto-optic modulator to be used as Stokes light; S1.2, the A-beam laser after passing through the acousto-optic modulator is connected to the first input terminal of the first dichroic mirror via the first delay device; S1.3, the B-beam laser is split into two beams, B1 and B2, by the second half-wave plate and the second polarization beam splitter; the B1 beam is connected to the second input terminal of the first dichroic mirror. The B2 beam undergoes photon frequency doubling through a β-phase barium borate crystal to obtain a pulsed laser with a wavelength of 451.5 nm, which is used as saturation light. S1.4, the 451.5 nm B2 pulsed laser beam is passed through a second delay device, and the beam spot is shaped by a pinhole filter. Finally, it is connected to a vortex phase plate to generate vortex light. Step S2: The A-beam Stokes beam output from the first input end and the B1-beam pump beam output from the second input end are spatially collinear after passing through the first dichroic mirror, then through the dispersive medium used to match the linear chirp coefficient of the pump beam and the Stokes beam, and then through the polarization-maintaining single-mode fiber before being incident on the second dichroic mirror. Step S3: By adjusting the first delay device and the second delay device, the A beam Stokes beam, the B1 pump beam and the B2 pulsed laser beam are coupled in time. In step S4, the three collinear beams of light from step S3 are fed into the scanning section of the microscope system. The microscope performs planar scanning using a biaxial two-dimensional scanning galvanometer, scanning three excitation beams that have been registered in the time and space domains. The sample is excited by two tightly focused laser beams, in which the Stokes beam and the pump beam, which is the probe beam, have different wavelengths, but both are located within the absorption band of the sample molecules. Among them, three bandpass filters are installed in front of the photodiode to filter Stokes light with a wavelength of 1064 nm as the detection beam; The pump light is collected by an oil-immersed condenser lens, filtered out by a bandpass filter, and received by a photodiode. Step S5, the rapid switching modulation of the Stokes light causes the probe beam to have a modulation response at the same modulation frequency; Step S6: A fast lock-in amplifier is used to realize heterodyne detection, and the imaging signal is acquired by a digital acquisition card and then analyzed and processed. Step S7: When the excitation light is applied to the sample, information including electronic energy levels and carrier dynamics can be obtained by measuring the changes in the transmittance and reflectance of the probe beam. This enables non-contact and non-damaging detection of cells and tissues, thereby achieving drug distribution monitoring.

2. The super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 1, characterized in that, In step S1.4, the saturated light B2 pulsed laser beam is collimated by a plano-convex lens and then generates vortex light by a phase-type diffraction element with a topological charge m=1 vortex phase plate. In step S1.4, the pinhole of the pinhole filter is selected with a diameter of 25μm for filtering, so that the saturated light presents a better Gaussian mode.

3. The super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 1, characterized in that, In step S1.4, the surface of the vortex phase plate has a spiral stepped structure. When the incident beam passes through, the surface structure of the vortex phase plate causes the optical path of the beam to change differently, thereby changing the phase change of the transmitted beam and generating vortex light.

4. The super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 1, characterized in that, The dispersive medium in step S2 is a 0.25 m long glass rod, which allows both beams of light to pass through a sufficiently long dispersive medium simultaneously, and is used to match the linear chirp coefficients of the pump light and the Stokes light.

5. The super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 1, characterized in that, In step S2, the polarization-maintaining single-mode fiber has a length of 0.4 m, which achieves good Gaussian mode for the two beams, absolute collinearity and spatial stability, and is also beneficial for super-resolution imaging. At the same time, these two beams are also used as excitation light for traditional SRS imaging. By matching the linear chirp coefficients of the pump light and the Stokes light, the spectral resolution is optimized.

6. The super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 1, characterized in that, In step S3, both the first delay device and the second delay device are equipped with two devices arranged at 90 degrees. ○ The system consists of an electrically driven two-dimensional displacement platform for the angled reflector. By moving the platform, the optical path can be changed, thereby achieving temporal coupling of three pulsed laser beams.

7. The super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 1, characterized in that, In step S4, the scanning section is also equipped with two high-NA objectives.

8. An imaging apparatus for a super-resolution stimulated Raman scattering imaging method for drug distribution detection as described in any one of claims 1-7, characterized in that, It includes an excitation light source module, an optical frequency doubling module, a laser scanning module, and a signal acquisition module, among which, The excitation source module includes a dual-output fly-laser system. The emitted laser is used as a laser source for stimulated Raman scattering imaging. It has two outputs, in which the A beam has a pulse width of 220 fs and a wavelength fixed at 1064 nm. After being sinusoidally intensity modulated at a frequency of 2.23 MHz by an acousto-optic modulator, it is used as Stokes light and passes through the first half-wave plate and the first polarization beam splitter. Another beam, B, with a pulse width of 100 fs and a wavelength of 903 nm, is used as pump light. The pump light is split into two by a second half-wave plate and a polarizing beam splitter. The B2 beam undergoes photon frequency doubling via a barium borate crystal to generate a pulsed laser with a wavelength of 451.5 nm, which is used as saturation light. After being collimated by a plano-convex lens, the saturation light is used to generate vortex light by a vortex phase plate with a topological charge of m=1. Another B1 pump beam and the acousto-optic modulated A Stokes beam are combined through the first dichroic mirror to achieve spatial collinearity. Then, they are simultaneously coupled into a 0.4 m long polarization-maintaining single-mode fiber. The collinear B1 pump beam and A Stokes beam are then spatially collinear with the B2 ring saturated beam passing through a quarter-wave plate via the second dichroic mirror, and then connected to the microscope system scanning module. The sample carrier module includes a sample carrier platform and an objective lens. The sample carrier platform is used to place the sample. The signal acquisition module includes a photodetector and a lock-in amplifier, and then the data is collected through a data acquisition card.

9. The imaging device for a super-resolution stimulated Raman scattering imaging method for drug distribution detection according to claim 8, characterized in that, The frequency-doubled saturated light is shaped by passing it through a pinhole with a diameter of 25 μm; Both the pump light and saturation light paths are equipped with time delay devices. These devices consist of an electrically driven two-dimensional displacement platform equipped with two mirrors at a 90° angle. The optical path can be changed by moving the platform, thus achieving time coupling of the three pulsed laser beams. The objective lens is an Olympus super apochromatic immersion objective lens, used to compensate for chromatic aberration and spherical aberration from the ultraviolet to the near-infrared region, which can acquire clear images and maintain the good polarization characteristics of the laser. The pump light is collected by an oil-immersion condenser lens, filtered out by a bandpass filter, and detected by a photodiode. In order to filter out the 1064 nm wavelength Stokes light, three high-quality bandpass filters are installed in front of the photodiode, and a fast lock-in amplifier is used to achieve heterodyne detection.

10. The application of the super-resolution stimulated Raman scattering imaging method for drug distribution detection as described in any one of claims 1-7 in the distribution of drugs in the nervous system, the cellular targeting of drugs, and intracellular distribution.

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