CN109164084B - Super-resolution Raman spectroscopy imaging system and method - Google Patents

Abstract

The present invention provides a super-resolution Raman spectroscopy imaging system and method. The system includes: an excitation light source module for generating an excitation light source; a polarization modulation module for modulating the polarization direction of the excitation light source; a galvanometer scanning module for scanning the excitation light source Focus at different positions in the sample imaging area; the microscope system module focuses the excitation light source and generates Raman signals through the sample imaging area, wherein the sample imaging area includes a surface-enhanced Raman spectroscopy substrate and is located on the surface-enhanced Raman spectroscopy substrate. The super-resolution imaging module generates a super-resolution image of the detection sample according to the Raman signal; the Raman spectrum analysis module is used to generate the Raman spectrum according to the Raman signal and analyze the detection sample. The invention is used for label-free super-resolution imaging of biological samples and chemical samples and super-resolution imaging of Raman spectroscopy, and effectively solves the problems of small applicable range, long imaging time and uncontrollable SERS scintillation behavior in Raman super-resolution imaging.

Description

Super-resolution Raman spectroscopy imaging system and method

technical field

The invention relates to the technical fields of surface plasmon Raman enhancement (SERS), super-resolution optical imaging, laser modulation and the like, in particular to a super-resolution Raman spectroscopy imaging system and method.

Background technique

The exploration of microscopic cells or organelles has long been of interest. In recent years, the research on brain science and brain-like intelligence has attracted more and more attention from all over the world. To conduct dynamic imaging and research on the interaction of brain cells and complex neural circuits, it is necessary to develop the most cutting-edge microscopic to mesoscopic to macroscopic Advanced imaging, tracing and marking technologies. In this regard, the newly developed subversive technology of light field regulation and nano-optical imaging that breaks through the limit of diffractive optics provides a powerful method for solving this key science and technology.

Single-molecule localization super-resolution technology utilizes the spontaneous flickering effect of fluorescent molecules that label cells, and obtains super-resolution images by locating the position of the fluorescent light source and acquiring multiple frames of image reconstruction. This technology has the advantages of low excitation intensity, high wide-field resolution, multi-color and 3D imaging capabilities, etc., but the imaging speed is slow due to the need for multi-frame reconstruction imaging.

Surface-enhanced Raman spectroscopy (SERS) technology is a new type of biological labeling method, which has the characteristics of not easy to bleach the light signal, suitable for a wide range of biological samples, and small damage to the samples. At the same time, the Raman signal scattered by the original sample is greatly enhanced, so that the spectral detection based on the SERS effect has extremely high sensitivity. SERS technology can also obtain chemical composition information in cells and biological tissues through Raman spectroscopy, and has the natural advantage of label-free imaging, which is widely used in the field of biomedicine.

In 2010, Willets et al. first combined super-resolution microscopy and SERS technology to study single-molecule SERS super-resolution reconstruction imaging, and locate the centroids of single molecules and nanoparticles through two-dimensional Gaussian fitting to obtain single molecules and nanoparticles. The spatial position information of the particles, the resolution can reach 10nm. With the combination of single-molecule localization super-resolution technology and SERS technology, researchers have also begun to use super-resolution technology to obtain subcellular or tissue SERS signals to achieve label-free Raman super-resolution imaging. However, since the blinking behavior of SERS usually has a period of tens of milliseconds, it generally takes 100ms to acquire a frame of image. Combined with the method of multi-frame acquisition and reconstruction, it usually takes tens of minutes to obtain a super-resolution image. Imaging, which takes several hours, can have a big impact on studying some living cell tissues. And the illumination method uses plasmon static illumination to generate static hot spots. It is very successful to achieve super-resolution Raman imaging for some single molecules. For the imaging of multiple molecules and cellular tissues, the blinking behavior of molecules cannot be used, resulting in residual imaging results. There are also problems such as background SERS signal interference.

SUMMARY OF THE INVENTION

In order to solve the above and other potential technical problems, an embodiment of the present invention provides a super-resolution Raman spectroscopy imaging system, the super-resolution Raman spectroscopy imaging system includes: an excitation light source module for generating an excitation light source; polarization; The modulation module is used to modulate the polarization direction of the excitation light source; the galvanometer scanning module is used to scan and focus the excitation light source at different positions in the sample imaging area; the microscope system module is used to focus the excitation light source through the sample imaging area Exciting a light source and exciting to generate a Raman signal, wherein the sample imaging area includes a surface-enhanced Raman spectroscopy substrate and a detection sample located on the surface-enhanced Raman spectroscopy substrate; a super-resolution imaging module, used for according to the Raman signal generating a super-resolution image of the detection sample; a Raman spectrum analysis module for generating a Raman spectrum according to the Raman signal and analyzing the detection sample.

In an embodiment of the present invention, the excitation light source module includes: a laser, which generates the excitation light source; a fiber collimator, which is connected to the laser through an optical fiber, collimates the excitation light source output by the laser, and outputs it to the laser source. The polarization modulation module described above.

In an embodiment of the present invention, the polarization modulation module includes: a polarizer, which receives the excitation light source output by the fiber collimator; and a polarization controller, which controls the rotation angle and rotation speed of the laser polarization in the polarizer.

In an embodiment of the present invention, the galvanometer scanning module includes: a laser galvanometer, which receives the excitation light source after polarization modulation by the polarizer, and scans and completes the focusing of the excitation light source at different positions in the sample imaging area; The galvanometer controller controls the scanning angle range and angle change rate of the laser galvanometer.

In an embodiment of the present invention, one way to control the polarizer and the laser galvanometer is to keep the angle of the laser galvanometer unchanged, and constantly change the polarization angle of the polarizer. The angle of the laser galvanometer is changed after the polarizer is vibrated for one rotation, the polarizer is rotated once again, and the above process is repeated until the scanning of the sample imaging area is completed; the control of the polarizer and the laser galvanometer Another way is: keep the angle of the polarizer unchanged, control the laser galvanometer to scan the sample imaging area, rotate the polarizer after scanning, and control the laser galvanometer to scan the sample imaging area again , repeat the above process until the polarizer rotates for one cycle.

In an embodiment of the present invention, the super-resolution Raman spectroscopy imaging system further includes: an excitation light coupling module, which is located between the laser galvanometer and the microscope system module, and outputs the output of the laser galvanometer. The excitation light source is coupled to the microscope system module; the microscope system module includes: an objective lens, the input excitation light source is focused on the sample imaging area through the objective lens, so as to generate a Raman signal; The Raman signal is divided into two paths, one path enters the super-resolution imaging module, and the other path enters the Raman spectrum analysis module.

In an embodiment of the present invention, the excitation light coupling module includes one or more combinations of a reflection mirror, a dichroic mirror, a beam splitter or a rotating mirror.

In an embodiment of the present invention, the super-resolution imaging module includes: a Raman signal filter, a bandpass filter, an imaging lens and an array detector arranged in sequence; wherein, the array detector records the detection in real time. In the light-emitting state of the sample, multi-frame acquisition is performed on the same position of the detection sample, and the image is reconstructed by Raman hot spot location and reconstruction by using the flickering effect generated by the Raman signal, so as to generate a super-resolution image.

In an embodiment of the present invention, the array detector is a CCD array detector, an EMCCD array detector or a CMOS array detector.

In an embodiment of the present invention, the Raman spectrum analysis module includes: a Raman signal filter, an optical fiber coupler, and a spectrometer for generating the Raman spectrum super-resolution image.

In one embodiment of the present invention, the surface-enhanced Raman spectroscopy substrate is composed of nanoparticle dimers, nanowires and nanoparticle systems, nanoparticle array-nanowire systems, nanocubes, or nanoparticle systems with polarization-dependent properties. One or more hybrid systems of cubic core-shell structural systems.

An embodiment of the present invention also provides a super-resolution Raman spectral imaging method, the super-resolution Raman spectral imaging method includes: generating an excitation light source and modulating the polarization direction of the excitation light source; Focus on the sample imaging area, and generate a Raman signal through the excitation of the sample imaging area; wherein, the sample imaging area includes a surface-enhanced Raman spectroscopy substrate and a detection sample on the surface-enhanced Raman spectroscopy substrate; continuously adjust the laser Exciting the polarization direction of the light source or adjusting the scanning direction of the laser galvanometer, and controlling the different excitation positions of the sample imaging area to generate Raman signals; respectively generating a super-resolution image and a Raman spectrum of the detected sample according to the Raman signals super-resolved images.

In an embodiment of the present invention, a process for obtaining the super-resolution image includes: keeping the angle of the laser galvanometer unchanged, continuously adjusting the polarization direction of the laser excitation light source, and obtaining the polarization direction of each polarization direction. The hot spot position information image of the Raman signal generated in the sample imaging area, and the hot spot position information images obtained in each polarization direction are superimposed to form a super-resolution image of the detected sample; the angle of the laser galvanometer is adjusted to change the excitation light source The position of the Raman signal on the detection sample is excited, and the above process is repeated to obtain the super-resolution images of multiple detection samples at different positions on the sample imaging area.

In an embodiment of the present invention, the hotspot position information in the hotspot position information image is obtained by a fitting positioning method; wherein, the positioning method is a Gaussian distribution fitting and reconstruction method, a multi-hotspot Gaussian fitting and reconstruction method or Compressed sensing data reconstruction method.

In an embodiment of the present invention, a process of acquiring the Raman spectrum super-resolution image includes: keeping the polarization direction of the excitation light source unchanged, continuously adjusting the focus position of the laser galvanometer, and performing the same operation every time. When adjusting the focus position of the laser galvanometer, collect the hot spot position information of the excited position and the Raman spectrum information corresponding to the hot spot position information; obtain the hot spot position information and the Raman spectrum corresponding to the hot spot position information information to construct a Raman spectral imaging map; change the polarization direction of the excitation light source, repeat the above process until the laser polarization rotates for one week, and obtain a plurality of the Raman spectral imaging maps under different polarization directions; image the acquired Raman spectrum Image reconstruction to obtain the Raman spectral super-resolution image.

In an embodiment of the present invention, one method of the Raman spectral imaging image is to extract the Raman peak intensity of the same wavelength and superimpose the hot spot position information of the Raman signal excited by the excitation light source on the detection sample, and obtain The Raman spectrum imaging image of the excited position at this wavelength; wherein, the Raman spectrum imaging image in the X and Y directions is the position information of the Raman signal excited by the excitation light source on the detection sample, and the gray value of the image is Intensity information of the Raman peak at this wavelength.

In an embodiment of the present invention, one way to obtain the Raman spectral super-resolution image is to reconstruct the Raman spectral imaging image at the same wavelength and under all polarization directions to obtain the Raman spectral super-resolution image. Image; obtaining the Raman spectrum super-resolution image is the position information of the Raman signal in the x and y directions, and the grayscale of the image represents the intensity integral of the Raman peaks under different polarizations and a certain wavelength.

In an embodiment of the present invention, the polarization direction of the excitation light source is modulated by a liquid crystal polarization rotator or a half-wave plate polarization rotator.

As mentioned above, the super-resolution Raman spectroscopy imaging system and method of the present invention has the following beneficial effects:

Based on polarization modulation and Raman hot spot localization super-resolution technology, the invention adjusts the scintillation characteristics of SERS by actively modulating the polarization of excitation light, controls the scintillation rate of SERS and combines with efficient and fast reconstruction technology, which not only can expand the scintillation characteristics of super-resolution imaging samples The scope of application can also efficiently obtain label-free super-resolution imaging and Raman spectroscopy super-resolution imaging for biological samples and chemical samples, greatly increasing the amount of information in the Raman spectrum of the sample, and effectively solving the Raman spectrum in the existing technology. The problems of small application range, long imaging time, and uncontrollable SERS scintillation behavior in super-resolution imaging can effectively improve the resolution of sample components and play an important role in studying the influence of polarization on Raman spectra of samples.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

FIG. 1 is a schematic block diagram of the super-resolution Raman spectroscopy imaging system of the present invention.

FIG. 2 is a schematic diagram showing the specific principle structure of the super-resolution Raman spectroscopy imaging system of the present invention.

FIG. 3 is a schematic diagram showing the super-resolution imaging process of polarization-modulated surface-enhanced Raman spectroscopy in the super-resolution Raman spectroscopy imaging system of the present invention.

FIG. 4 is a schematic diagram of the Raman spectrum super-resolution imaging process in the super-resolution Raman spectrum imaging system of the present invention.

FIG. 5 is a schematic diagram showing the overall flow of the super-resolution Raman spectroscopy imaging method of the present invention.

Component label description

100 Super-resolution Raman Spectroscopy Imaging System

110 Excitation light source module

111 Raman excitation laser

112 Fiber Collimator

120 Polarization Modulation Module

121 Polarizer

122 Polarization Controller

130 Galvo Scanning Module

131 Laser Galvo

132 Galvo Controller

140 Microsystem Modules

141 Surface Enhanced Raman Spectroscopy Substrate

142 Test samples

143 Objectives

144 splitting module

150 super-resolution imaging module

151 Raman Signal Filter

152 bandpass filter

153 Imaging Lenses

154 Array Detectors

160 Raman Spectroscopy Module

161 Spectrometer

162 Raman Signal Filter

163 Fiber Optic Couplers

170 Excitation Light Coupling Module

171 Mirrors

172 Dichroic mirror

173 Beamsplitters

Steps S110～S140

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other under the condition of no conflict.

See Figures 1 to 5. It should be noted that the structures, proportions, sizes, etc. shown in the drawings in this specification are only used to cooperate with the contents disclosed in the specification, so as to be understood and read by those who are familiar with the technology, and are not used to limit the implementation of the present invention. Restricted conditions, it does not have technical substantive significance, any structural modification, proportional relationship change or size adjustment, without affecting the effect that the present invention can produce and the purpose that can be achieved, should still fall within the present invention. The disclosed technical content must be within the scope of coverage. At the same time, the terms such as "up", "down", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description and clarity, and are not used to limit this specification. The implementable scope of the invention, and the change or adjustment of the relative relationship thereof, shall also be regarded as the implementable scope of the present invention without substantially changing the technical content.

Aiming at the problems of small application range, long imaging time, and uncontrollable SERS scintillation behavior in Raman super-resolution imaging by single-molecule localization technology, the purpose of this embodiment is to provide a super-resolution Raman spectroscopy imaging system 100 and a method, using It is used for label-free super-resolution imaging of biological samples and chemical samples and super-resolution imaging of Raman spectroscopy, effectively solving the problems of small application range, long imaging time, and uncontrollable SERS scintillation behavior in Raman super-resolution imaging in the prior art.

This embodiment is a polarization modulation-based surface plasmon Raman-enhanced super-resolution Raman spectroscopy imaging system 100 and a method, which is a label-free super-resolution imaging system using the surface plasmon Raman enhancement effect and a super-resolution imaging system based on Raman spectroscopy imaging method of this system. The super-resolution Raman spectroscopy imaging system 100 of this embodiment includes an excitation light source module 110 , a polarization modulation module 120 , a galvanometer scanning module 130 , a microscope system module 140 , a super-resolution imaging module 150 and a Raman spectroscopy analysis module 160 . Based on the imaging method of the super-resolution Raman spectroscopy imaging system 100 of this embodiment, the polarization of the excitation light is modulated, so that the surface Raman-enhanced hot spot with random excitation polarization dependence can make the sample Raman signal produce polarization-dependent scintillation effect, The stochastic optical reconstruction super-resolution imaging and Raman spectroscopy super-resolution imaging are performed using the collected scintillation Raman signals.

The principles and implementations of the super-resolution Raman spectroscopy imaging system 100 and method of this embodiment will be described in detail below, so that those skilled in the art can understand the super-resolution Raman spectroscopy imaging system 100 and method of this embodiment without creative work. .

An embodiment of the present invention provides a super-resolution Raman spectroscopy imaging system 100, the super-resolution Raman spectroscopy imaging system 100 includes: an excitation light source module 110, a polarization modulation module 120, a galvanometer scanning module 130, and a microscope system module 140 , the excitation light coupling module 170 , the super-resolution imaging module 150 and the Raman spectrum analysis module 160 .

The super-resolution Raman spectroscopy imaging system 100 of this embodiment will be described in detail below.

In this embodiment, the excitation light source module 110 is used to generate an excitation light source.

Specifically, in this embodiment, as shown in FIG. 2 , the excitation light source module 110 includes: a Raman excitation laser 111 , which generates the excitation light source; a fiber collimator 112 , which communicates with the Raman excitation laser through an optical fiber. 111 is connected, and the excitation light source output by the Raman excitation laser 111 is collimated and then output to the polarization modulation module 120 .

The laser wavelengths output by the Raman excitation laser 111 include but are not limited to commonly used Raman excitation wavelengths: 405nm, 488nm, 532nm, 632nm, 785nm, etc. The output aperture of the optical fiber meets the requirements of confocal scanning microscopy imaging, making the system capable of confocal microscopy imaging The Raman excitation laser 111 outputs laser light through an optical fiber, and enters the polarization modulation module 120 after being collimated by the optical fiber collimator 112 .

In this embodiment, the polarization modulation module 120 is a polarizer, which is used to modulate the polarization direction of the excitation light source.

Specifically, the polarization modulation module 120 generates a polarization rotation of zero to 2π for the incident linearly polarized laser light.

Specifically, in this embodiment, as shown in FIG. 2 , the polarization modulation module 120 includes: a polarizer 121 , which receives the excitation light source output by the fiber collimator 112 ; and a polarization controller 122 , which controls the polarizer Rotation angle and rotation speed of laser polarization in 121.

The rotation angle and rotation speed of the laser polarization are controlled by the polarization controller 122 and a specific timing control program within the polarization controller 122 . Wherein, the devices for providing polarization include but are not limited to liquid crystal polarization rotators and half-wave plate polarization rotators.

In this embodiment, the galvanometer scanning module 130 is used to scan and focus the excitation light source at different positions of the sample imaging area.

Specifically, in this embodiment, as shown in FIG. 2 , the galvanometer scanning module 130 includes: a laser galvanometer 131 , which receives the excitation light source subjected to polarization modulation by the polarizer 121 , and completes the scanning of the excitation light source. Focus on different positions of the sample imaging area; the galvanometer controller 132 controls the scanning angle range and angle change rate of the laser galvanometer 131 .

The fast two-dimensional scanning of the sample imaging area in the confocal microscope system module 140 is realized by the laser galvanometer 131 , and the scanning angle range and angle change rate of the laser galvanometer 131 are simultaneously controlled by the galvanometer controller 132 and the timing sequence in the galvanometer controller 132 Control program control.

Specifically, in this embodiment, one way to control the polarizer 121 and the laser galvanometer 131 is to keep the angle of the laser galvanometer 131 unchanged, and constantly change the polarization of the polarizer 121 When the polarizer 121 rotates for one round, the angle of the laser galvanometer 131 is changed, the polarizer 121 is rotated again for one round, and the above process is repeated until the scanning of the sample imaging area is completed.

Another way to control the polarizer 121 and the laser galvanometer 131 is to keep the angle of the polarizer 121 unchanged, and control the laser galvanometer 131 to scan the imaging area of the sample. Rotate the polarizer 121 , control the laser galvanometer 131 to scan the imaging area of the sample again, and repeat the above process until the polarizer 121 rotates once.

In this embodiment, the excitation light coupling module 170 is installed between the laser galvanometer 131 and the microscope system module 140, and outputs the excitation light source output by the laser galvanometer 131 to the microscope. System module 140 .

Specifically, in this embodiment, as shown in FIG. 2 , the excitation light coupling module 170 includes one or more combinations of a reflection mirror 171 , a dichroic mirror 172 , a beam splitter 173 or a turning mirror.

In this embodiment, the microscope system module 140 is configured to focus the excitation light source and generate a Raman signal through a sample imaging area, wherein, as shown in FIG. 2 , the sample imaging area includes surface-enhanced Raman spectroscopy. The substrate 141 and the detection sample 142 on the surface-enhanced Raman spectroscopy substrate 141.

Specifically, in this embodiment, the surface-enhanced Raman spectroscopy substrate 141 is composed of a nanoparticle dimer, a nanowire and nanoparticle system, a nanoparticle array-nanowire system, a nanocube or a One or more hybrid systems of nanocube core-shell structural systems.

The polarization-modulated excitation light source enters the objective lens 143 after passing through the laser galvanometer 131, the reflecting mirror 171 and the dichroic mirror 172, and is focused on the detection sample 142 on the SERS substrate. The detection sample 142 can be fixed on the surface-enhanced Raman spectroscopy substrate 141 (SERS substrate) by means of suspension, adsorption, etc., and inelastic Raman scattering occurs between the focused excitation light source and the detection sample 142 to generate Raman Signal.

Since the excitation light source has a certain direction of polarization, the hot spot in the SERS substrate can only achieve the maximum Raman enhancement effect in the polarization polarization plasma mode consistent with the polarization direction of the excitation light source, so that the Raman signal on the SERS substrate can be excited. Regulation of the polarization of light. Raman signals from sample molecules near excited hotspots are enhanced, while Raman signals near unexcited hotspots are not. Figure 3 illustrates by taking silver nanoparticle dimer as an example: the polarization direction of the dimer is related to the direction of the long axis of the dimer, and it presents a random arrangement on the substrate. When the excitation light is polarized in the form of a long arrow in Figure 3. It can be seen that only the hot spots whose long axis direction coincides with the polarization direction of the excitation light are excited. In turn, a surface Raman scattering enhancement effect is generated to enhance the Raman signal in the sample signal. The unexcited dimer cannot enhance the Raman signal in the sample.

Specifically, as shown in FIG. 2 , the microscope system module 140 includes: an objective lens 143, the input excitation light source is focused on the sample imaging area through the objective lens 143, so as to generate a Raman signal; a spectroscopic module 144, which uses The Raman signal is divided into two paths, one path enters the super-resolution imaging module 150 , and the other path enters the Raman spectrum analysis module 160 . , wherein, the light splitting module 144 is an electric rotating mirror or a beam splitting mirror.

In this embodiment, the super-resolution imaging module 150 is configured to generate a super-resolution image of the detection sample 142 according to the Raman signal.

Specifically, in this embodiment, as shown in FIG. 2 , the super-resolution imaging module 150 includes: a Raman signal filter 151 , a bandpass filter 152 , an imaging lens 153 and an array detector 154 , which are arranged in sequence; , the array detector 154 records the light-emitting state of the detection sample 142 in real time, collects multiple frames at the same position of the detection sample 142, and uses the flickering effect generated by the Raman signal to perform Raman hot spot positioning and reconstruction on the image to generate super-resolved images.

Specifically, in this embodiment, the array detector 154 is a CCD array detector, an EMCCD array detector or a CMOS array detector.

The enhanced Raman signal enters the CCD/EMCCD/CMOS array detector 154 all the way for imaging. Since light is an electromagnetic wave with diffraction characteristics, the Raman signal we collect in the CCD/EMCCD/CMOS array detector 154 is the diffracted light spot modulated by the point spread function determined by the imaging system. The light intensity of the diffracted spot has the characteristics of a Gaussian distribution, and the nano-precision hotspot position information of the Raman hotspot can be found by the method of fitting and localization. The positioning methods adopted in this system include but are not limited to Gaussian distribution fitting and reconstruction methods, multi-hot spot Gaussian fitting and reconstruction methods, and compressed sensing data reconstruction methods.

Keep the galvanometer unchanged, change the polarization modulator in real time, and constantly change the polarization direction of the excitation light, so that the hot spots with randomly distributed polarization directions can be randomly excited with the change of the polarization direction of the laser. Every time the polarization direction of the excitation light is changed, the CCD/EMCCD/CMOS array detector 154 collects and locates images of each frame to obtain the hot spot position information. Super-resolution imaging is achieved by superimposing all images obtained in this process. Fig. 3 takes the substrate of the nanoparticle dimer as an example to illustrate the process in detail: the polarization direction angle of the excitation light in Fig. 3A is 0°, and only a part of the nanoparticle dimer is shown in Fig. 3A (Fig. 3A). The Raman enhancement effect of black dimer particles) reaches the strongest, and after the enhanced Raman signal is collected by the array detector 154, a diffraction spot image is obtained. Through the above positioning method, the position information of the Raman signal is further obtained. Continuously change the polarization direction angle of the excitation light, (Figure 3B=30°, Figure 3C=45°, Figure 3D=90°) to obtain Raman signal information at different positions, and reconstruct all the obtained images to obtain the sample’s Super-resolved images (Figure 3E). The laser galvanometer 131 is continuously adjusted to change the position where the excitation light is focused on the sample to obtain super-resolution images of the detected samples at multiple other positions.

In this embodiment, the Raman spectrum analysis module 160 is configured to generate a Raman spectrum according to the Raman signal and analyze the detection sample.

Specifically, in this embodiment, as shown in FIG. 2 , the Raman spectrum analysis module 160 includes: a Raman signal filter 162 , a fiber coupler 163 and a spectrometer 161 for generating the Raman spectrum super-resolution image. The aperture of the coupling optical fiber meets the requirements of confocal scanning microscopy imaging for collecting optical signals, and cooperates with the aperture of the output optical fiber in the excitation light source module 110 to enable the system to have the capability of confocal scanning microscopy imaging.

The Raman signal is coupled into the spectrometer 161 through the optical fiber, and the Raman spectral information under a certain polarization angle and the position information of the excited hot spot are obtained. The Raman spectrum will show Raman peaks at different wavelengths (λ ₁ , λ ₂ , λ ₃ , λ ₄ . . . ). By extracting the Raman peak intensity and Raman signal position information of the same wavelength (λ ₁ ) at different sites for reconstruction, the Raman spectral imaging image of the site excited at this wavelength (λ ₁ ) is obtained. In the same way, obtain Raman spectral imaging images at other wavelengths (λ ₂ , λ ₃ , λ ₄ . . . ).

Taking FIG. 4 as an example, the process of acquiring the Raman spectrum imaging map will be described in detail.

When the polarization angle of the excitation light is 0° (the direction of the black double arrow in FIG. 3A ), the position information ( FIG. 4A ) and the Raman spectrum ( FIG. 4A ₅ ) of the focus of the excitation light are obtained by the spectrometer 161 . The Raman spectra at these positions have Raman peaks with different intensities at λ ₁ , λ ₂ , λ ₃ , and λ ₄ respectively. The intensity of the Raman peak at the wavelength of λ ₁ and the light source position of the Raman signal are reconstructed as follows Figure 4A1 Raman spectroscopic imaging map. This _Fig . 4A1 shows the position information of the Raman signal in the x and y direction dimensions, and the gray value of the image represents the intensity information of the Raman peak under the wavelength condition of the polarization angle of 0° and λ ₁ (the black point in Fig. 4A1 ₎ . bit indicates that there is a Raman peak at this position). In the same way, the Raman spectral imaging images of λ ₂ , λ ₃ and λ ₄ wavelengths can be obtained ( FIG. 4A ₂ , FIG. 4A ₃ , and FIG. 4A ₄ ). The polarizer 121 is rotated to continuously change the polarization direction of the excitation light, and the above process is repeated to obtain Raman spectral imaging images under different polarization angles. As shown in Figure 4B, the Raman spectrum changes when the direction of polarized light changes by θ. The reconstructed Raman spectral imaging images are shown in Fig. 4B ₁ , Fig. 4B ₂ , Fig. 4B ₃ , and Fig. 4B ₄ .

The acquired Raman spectral imaging image is reconstructed to obtain a Raman spectral super-resolution image. Reconstruction method: Superimpose the Raman spectral imaging images of the same wavelength and different polarization angles. Fig. 4C is a super-resolution image of Raman spectrum under the condition of λ ₁ , which is a Raman spectrum distribution diagram of different polarization angles (θ ₁ , θ ₂ , θ ₃ . . . ) and the same wavelength (λ ₁ ) (Fig. 4A ). _1. Figure 4B ₁ ...) obtained by reconstruction. Figure 4 is the position information of the Raman signal in the x and y directions, and the grayscale of the image represents the Raman peak intensities of different polarizations and wavelengths of λ ₁ (in Figure 4C and Figure 4D, different filling patterns are used to represent different Raman peak intensity).

The working process of the super-resolution Raman spectroscopy imaging system 100 in this embodiment is as follows:

The Raman excitation laser 111 outputs laser light from the fiber, which is collimated by the fiber collimator 112 to form parallel light and enters the continuously rotating polarizer 121 controlled by software to generate polarized light whose polarization direction changes continuously. Then, the polarized light passes through the laser galvanometer 131 and the mirror 171 and then enters the objective lens 143 to be focused on the SERS substrate, and the detection sample is excited to emit a Raman signal. After the Raman signal is collected by the objective lens 143, it is divided into two paths by the motorized rotating mirror or the beam splitter 173: the first path enters the CCD/EMCCD/CMOS array detector 154 for imaging. Constructed super-resolution imaging method. The other way enters the spectrometer 161 for Raman spectral super-resolution imaging.

As shown in FIG. 5 , this embodiment also provides a super-resolution Raman spectral imaging method, and the super-resolution Raman spectral imaging method includes:

Step S110, generating an excitation light source and modulating the polarization direction of the excitation light source;

In step S120, the excitation light source is focused on the sample imaging area by the laser galvanometer 131, and a Raman signal is generated by excitation in the sample imaging area; wherein, the sample imaging area includes a surface-enhanced Raman spectroscopy substrate 141 and a surface-enhanced Raman spectroscopy substrate 141. The detection sample 142 on the Raman spectroscopy substrate 141;

Step S130, continuously adjusting the polarization direction of the laser excitation light source or adjusting the scanning direction of the laser galvanometer 131, and controlling and completing different excitation positions in the sample imaging area to generate Raman signals;

Step S140, respectively generating a super-resolution image and a Raman spectrum super-resolution image of the detection sample 142 according to the Raman signal.

In this embodiment, a process of obtaining the super-resolution image includes: keeping the angle of the laser galvanometer 131 unchanged, continuously adjusting the polarization direction of the laser excitation light source, and obtaining images of the sample under each polarization direction respectively. The hot spot position information image of the Raman signal generated in the region is obtained, and the hot spot position information images obtained in each polarization direction are superimposed to form a super-resolution image of the detection sample 142; the angle of the laser galvanometer 131 is adjusted to change the excitation light source The position of the Raman signal on the detection sample 142 is excited, and the above process is repeated to obtain the super-resolution images of the plurality of detection samples 142 at different positions on the sample imaging area.

That is, the polarization characteristics of the excitation light source are changed by adjusting the polarization rotator. The hot spot in the plasmon enhancement mode of the SERS substrate is excited in the same direction as the polarization direction of the excitation light, resulting in the maximum Raman enhancement effect. The Raman signal is collected by the array detector 154 to obtain random hot spot location information. The polarization direction of the excitation light is continuously changed, and the acquisition rate of the CCD/EMCCD/CMOS array detector 154 is controlled to obtain a series of random hot spot position information. The super-resolution image of the detection sample 142 is obtained by performing Raman hot spot localization and reconstruction on the collected image.

Specifically, in this embodiment, the complete process of generating the super-resolution image of the detection sample 142 is as follows:

1) Transfer the imaging target to a surface-enhanced Raman spectroscopy (SERS) substrate with strong polarization dependence. The polarization-dependent SERS substrate can be composed of one or several mixed random distributions of metal dimers, polymers, cubes, core-shell structures, and nanoparticle-nanowire structures with polarization-polarized plasmonic modes.

2) The excitation light generated by the excitation light source module 110 passes through the optical fiber and the optical fiber collimator 112 to generate parallel light and then enters the polarization modulation module 120 . In the polarization modulation module 120, the rotation direction and rotation speed of the polarizer 121 can be adjusted by the controller. The polarization rotation methods include but are not limited to liquid crystal polarization rotators and half-wave plate polarization rotators. Keep the laser galvanometer 131 still, rotate the polarizer, modulate the polarization direction of the excitation light source, and control the polarization angle θ of the excitation light to excite the polarized plasma mode on the SERS substrate that is consistent with the polarization direction of the excitation light source (see Fig. 3 shown).

3) The polarization-modulated excitation light enters the objective lens 143 through the laser galvanometer 131, the reflection mirror 171 and the dichroic mirror 172, and is focused on the sample on the SERS substrate. The detection sample 142 can be immobilized on the SERS substrate by means of suspension, adsorption, or the like. Inelastic Raman scattering occurs between the focused excitation light and the sample analyte, resulting in a Raman signal. Due to the strong polarization dependence of the SERS substrate, only the polarization-polarized plasmon mode consistent with the polarization direction of the excitation light source can achieve the maximum Raman enhancement effect. The detection targets at these sites emit detectable Raman signals, while the remaining polarization-polarized plasmonic mode sites have no enhancement effect. That is to realize the bright and dark flickering effect when the SERS hotspots are at different sites.

Since the excitation light has a polarization in a certain direction, the hot spot in the SERS substrate can only achieve the maximum Raman enhancement effect in the polarization polarization plasmon mode that is consistent with the polarization direction of the excitation light source, so that the Raman signal on the SERS substrate can be excited. Regulation of the polarization of light. Raman signals from sample molecules near excited hotspots are enhanced, while Raman signals near unexcited hotspots are not. Figure 3 illustrates by taking silver nanoparticle dimer as an example: the polarization direction of the dimer is related to the direction of the long axis of the dimer, and it presents a random arrangement on the substrate. When the excitation light is polarized in the form of a long arrow in Figure 3. It can be seen that only the hot spots whose long axis direction coincides with the polarization direction of the excitation light are excited. In turn, a surface Raman scattering enhancement effect is generated to enhance the Raman signal in the sample signal. The unexcited dimer cannot enhance the Raman signal in the sample.

4) Adjusting the rotating polarizer to continuously change the polarization direction of the laser light, thereby randomly modulating the Raman luminescence of hot spots with different polarization directions at different positions, resulting in the flickering effect of the SERS detection target.

In this embodiment, the polarization direction of the excitation light source is modulated by a liquid crystal polarization rotator or a half-wave plate polarization rotator.

5) The SERS signal of the imaging target enters the objective lens 143 and then enters the CCD/EMCCD/CMOS array detector 154. The CCD/EMCCD/CMOS array detector 154 records the luminescence state of the sample in real time, and collects multiple frames at the same position of the sample. Using the generated flickering effect, the image is reconstructed by Raman hot spot location, and a super-resolution image is generated.

Specifically, the enhanced Raman signal enters the CCD/EMCCD/CMOS array detector 154 all the way for imaging. Since light is an electromagnetic wave with diffraction characteristics, the Raman signal we collect in the CCD/EMCCD/CMOS array detector 154 is the diffracted light spot modulated by the point spread function determined by the imaging system. The light intensity of the diffracted spot has the characteristics of Gaussian distribution, and the nanometer-precision position information of the Raman hot spot can be found by the method of fitting and positioning. The positioning methods adopted in this system include but are not limited to Gaussian distribution fitting and reconstruction methods, multi-hot spot Gaussian fitting and reconstruction methods, and compressed sensing data reconstruction methods.

Keep the galvanometer unchanged, change the polarization modulator in real time, and constantly change the polarization direction of the excitation light, so that the hot spots with randomly distributed polarization directions can be randomly excited with the change of the polarization direction of the laser. Every time the polarization direction of the excitation light is changed, the CCD/EMCCD/CMOS array detector 154 collects and locates images of each frame to obtain the hot spot position information. Super-resolution imaging is achieved by superimposing all images obtained in this process. Fig. 3 takes the substrate of the nanoparticle dimer as an example to illustrate the process in detail: the polarization direction angle θ of the excitation light in Fig. 3A is 0°, and only a part of the nanoparticle dimer is shown in Fig. 3 (Fig. 3A The Raman enhancement effect of the medium-black dimer particles) reaches the strongest, and the enhanced Raman signal is collected by the CCD/EMCCD/CMOS array detector 154 to obtain a diffraction spot image. Through the positioning method described in step 5), the position information of the Raman signal is obtained. Continuously change the polarization direction angle θ of the excitation light, (Fig. 3B θ ₁ =30°, Fig. 3C θ ₂ =45°, Fig. 3D θ ₃ =90°) to obtain Raman signal information at different positions, and reconstruct all the obtained images, namely A super-resolved image of the sample can be obtained (Figure 3E).

6) Adjust the laser galvanometer 131, that is, adjust the laser galvanometer 131 to change the position where the excitation light is focused on the sample, change the focus position of the excitation light, and repeat steps 2) to 5) to obtain a super-resolution image of another position.

In this embodiment, the hotspot position information in the hotspot position information image is obtained by a fitting positioning method; wherein, the positioning method is a Gaussian distribution fitting and reconstruction method, a multi-hotspot Gaussian fitting and reconstruction method, or compressed sensing data Refactoring method.

In this embodiment, a process of acquiring the Raman spectrum super-resolution image includes: keeping the polarization direction of the excitation light source unchanged, continuously adjusting the focus position of the laser galvanometer 131, and adjusting all When the focus position of the laser galvanometer 131 is described, the hot spot position information of the excited position and the Raman spectral information corresponding to the hot spot position information are collected; the obtained hot spot position information and the Raman spectral information corresponding to the hot spot position information Constructing a Raman spectral imaging map; changing the polarization direction of the excitation light source, repeating the above process until the laser polarization rotates for one week, and obtaining a plurality of the Raman spectral imaging maps under different polarization directions; Reconstruction acquires the Raman spectral super-resolution image.

That is, the polarization characteristics of the excitation light source are changed by adjusting the polarization rotator. The polarized light excites the hot spot in the plasmon enhancement mode of the SERS substrate, which is aligned with the polarization direction of the excitation light, resulting in the maximum Raman enhancement effect. The Raman signal is coupled to the spectrometer 161 through an optical fiber, and the Raman signal is collected by the spectrometer 161 to obtain a confocal microscopic Raman spectrum. The two-dimensional confocal fast scanning of the sample is completed by the galvanometer. During the scanning process, each time the scanning position of the galvanometer changes, the spectrometer 161 collects a Raman spectrum. A Raman spectral imaging map is constructed from the scanned location information and the Raman spectrum of the location. Change the polarization direction of the excitation light, perform confocal scanning Raman spectroscopy imaging in the same area, and obtain a Raman image. This process is repeated until the laser polarization is rotated one revolution. The Raman spectral super-resolution map is obtained by reconstructing the Raman spectral distribution map of the same wavelength.

In this embodiment, one way of constructing the Raman spectral imaging map is: extracting the Raman peak intensity of the same wavelength and superimposing the hot spot position information of the Raman signal excited by the excitation light source on the detection sample 142 to obtain the Raman peak intensity. The Raman spectral imaging diagram of the excited position at the wavelength; wherein, the Raman spectral imaging diagram in the x and y directions is the position information of the Raman signal excited by the excitation light source on the detection sample 142, and the gray value of the image is Intensity information of wavelength Raman peaks.

Since the Raman spectrum will show Raman peaks at different wavelengths (λ ₁ , λ ₂ , λ ₃ , λ ₄ . . . ). The Raman peak intensity in the same wavelength band and the position information of the laser focused on the sample can be extracted and reconstructed, that is, the Raman spectral imaging map of the excited site in this wavelength band (λ ₁ ). The Raman spectrum imaging image is the position information of the laser focused on the sample in the x and y directions, and the grayscale of the image is the intensity information of the Raman peak in the wavelength band.

In this embodiment, one way to obtain the Raman spectral super-resolution image is as follows: reconstructing the Raman spectral imaging image at the same wavelength and under all polarization directions to obtain the Raman spectral super-resolution image; obtaining the Raman spectral super-resolution image; The Raman spectrum super-resolution image is the position information of the Raman signal in the x and y directions, and the grayscale of the image is the intensity integral of the Raman peaks under different polarizations and a certain wavelength.

Specifically, the process of acquiring the Raman spectral super-resolution image is as follows:

1) Transfer the imaging target to a polarization-dependent SERS substrate. The polarization-dependent SERS substrate structure includes but is not limited to: nanoparticle dimer, nanowire and nanoparticle system, nanoparticle array-nanowire system , nanocubes, or a core-shell structure system with nanocubes.

2) The excitation light passes through the optical fiber and the optical fiber collimator 112 to generate parallel light and enters the polarization modulation module 120 to generate polarized light.

3) The polarized light is focused on the SERS substrate after passing through the laser galvanometer 131, the mirror 171, the dichroic mirror 172 and the objective lens 143. The controller controls the laser galvanometer 131 to scan two-dimensionally to quickly scan the sample confocal microscopy imaging area.

4) Since the excitation light has a polarization in a certain direction, the hot spot in the SERS substrate can only achieve the maximum Raman enhancement effect in the polarization-dependent plasmon mode that is consistent with the polarization direction of the excitation light source. The Mann signal is enhanced, while the Raman signal near the unexcited hotspot is not.

5) The Raman signal is coupled into the spectrometer 161 through the optical fiber, and the Raman spectrum information under a certain polarization angle and the position information of the excited hot spot are obtained. The Raman spectrum will show Raman peaks at different wavelengths (λ ₁ , λ ₂ , λ ₃ , λ ₄ . . . ). By extracting the Raman peak intensity and Raman signal position information of the same wavelength (λ ₁ ) at different sites for reconstruction, the Raman spectral imaging image of the site excited at this wavelength (λ ₁ ) is obtained. In the same way, obtain Raman spectral imaging images at other wavelengths (λ ₂ , λ ₃ , λ ₄ . . . ). Taking FIG. 4 as an example, the method for acquiring the Raman spectrum imaging image will be described in detail. When the polarization angle of the excitation light is 0° (the direction of the black double arrow in FIG. 3A ), the position information ( FIG. 4A ) and the Raman spectrum ( FIG. 4A ₅ ) of the focus of the excitation light are obtained by the spectrometer 161 . The Raman spectra at these positions have Raman peaks with different intensities at λ ₁ , λ ₂ , λ ₃ , and λ ₄ respectively. The Raman peak intensity at the wavelength of λ ₁ and the light source position of the Raman signal are reconstructed as follows Figure 4A1 Raman spectroscopic imaging map. In this figure, the position information of the Raman signal in the x and y direction dimensions, the grayscale of the image is the intensity information of the Raman peak under the wavelength condition of the polarization angle of 0° and λ ₁ (the black points in Fig. 4A ₁ are shown here position has a Raman peak). In the same way, the Raman spectral imaging images of λ ₂ , λ ₃ and λ ₄ wavelengths can be obtained ( FIG. 4A ₂ , FIG. 4A ₃ , and FIG. 4A ₄ ).

6) Rotate the polarizer 121 to continuously change the polarization direction of the excitation light, and repeat step 5) to obtain Raman spectral imaging images under different polarization angles. As shown in Figure 4B, the Raman spectrum changes when the direction of polarized light changes by θ. The reconstructed Raman spectral imaging images are shown in Fig. 4B ₁ , Fig. 4B ₂ , Fig. 4B ₃ , and Fig. 4B ₄ .

7) Reconstructing the acquired Raman spectrum imaging image to obtain a Raman spectrum super-resolution image. Reconstruction method: Superimpose the Raman spectral imaging images of the same wavelength and different polarization angles. Fig. 4C is a super-resolution image of Raman spectrum under the condition of λ ₁ , which is a Raman spectrum distribution diagram of different polarization angles (θ ₁ , θ ₂ , θ ₃ . . . ) and the same wavelength (λ ₁ ) (Fig. 4A ). _1. Figure 4B ₁ ...) obtained by reconstruction. This figure 4 is the position information of the Raman signal in the x and y directions, and the grayscale representation of the image is the intensity integral of the Raman peak under the wavelength conditions of different polarizations and λ ₁ (different filling patterns are used in Fig. 4C and Fig. 4D ). represent different Raman peak intensities).

To sum up, the present invention adjusts the scintillation characteristics of SERS by actively modulating the polarization of excitation light based on polarization modulation and Raman hot spot localization super-resolution technology, controls the scintillation rate of SERS and combines it with efficient and fast reconstruction technology, not only in the ability to expand The application range of super-resolution imaging samples can also efficiently obtain label-free super-resolution imaging and super-resolution imaging of Raman spectroscopy for biological samples and chemical samples, which greatly increases the amount of information of sample Raman spectroscopy and effectively solves current problems. Raman super-resolution imaging in the existing technology has problems such as small application range, long imaging time, and uncontrollable SERS scintillation behavior. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (16)

1. a super-resolution Raman spectral imaging system, is characterized in that, described super-resolution Raman spectral imaging system comprises: The excitation light source module is used to generate the excitation light source; a polarization modulation module for modulating the polarization direction of the excitation light source; a galvanometer scanning module, used to scan and complete the focusing of the excitation light source at different positions in the sample imaging area; A microscope system module, configured to focus the excitation light source and generate a Raman signal through a sample imaging area, wherein the sample imaging area includes a surface-enhanced Raman spectroscopy substrate and a detection sample located on the surface-enhanced Raman spectroscopy substrate ; a super-resolution imaging module, configured to generate a super-resolution image of the detected sample according to the Raman signal; a Raman spectrum analysis module, used for generating a Raman spectrum according to the Raman signal and analyzing the detection sample; The super-resolution imaging module includes: a Raman signal filter, a bandpass filter, an imaging lens and an array detector arranged in sequence; wherein, the array detector records the luminescence state of the detection sample in real time, and the detection Collect multiple frames at the same position of the sample and use the flickering effect generated by the Raman signal to locate and reconstruct the Raman hotspot to generate a super-resolution image. 2. The super-resolution Raman spectroscopy imaging system according to claim 1, wherein the excitation light source module comprises: a laser that generates the excitation light source; The optical fiber collimator is connected to the laser through an optical fiber, and the excitation light source output by the laser is collimated and then output to the polarization modulation module. 3. The super-resolution Raman spectral imaging system according to claim 2, wherein the polarization modulation module comprises: a polarizer, receiving the excitation light source output by the fiber collimator; The polarization controller controls the rotation angle and rotation speed of the laser polarization in the polarizer. 4. super-resolution Raman spectroscopy imaging system according to claim 3, is characterized in that, described galvanometer scanning module comprises: a laser galvanometer, receiving the excitation light source after polarization modulation by the polarizer, and scanning and focusing the excitation light source at different positions of the sample imaging area; The galvanometer controller controls the scanning angle range and angle change rate of the laser galvanometer. 5. super-resolution Raman spectroscopy imaging system according to claim 4, is characterized in that: One way to control the polarizer and the laser galvanometer is to keep the angle of the laser galvanometer unchanged, constantly change the polarization angle of the polarizer, and change the polarizer after one rotation of the polarizer. The angle of the laser galvanometer, rotate the polarizer for one turn again, and repeat the above process until the scanning of the imaging area of the sample is completed; Another way to control the polarizer and the laser galvanometer is to keep the angle of the polarizer unchanged, control the laser galvanometer to scan the imaging area of the sample, and rotate the polarization after scanning is completed. The scanning of the imaging area of the sample by the laser galvanometer is controlled again, and the above process is repeated until the polarizer rotates for one revolution. 6. The super-resolution Raman spectroscopic imaging system according to claim 4, wherein the super-resolution Raman spectroscopic imaging system further comprises: an excitation light coupling module located at the laser galvanometer and the microscope system Between modules, the excitation light output by the laser galvanometer is coupled to the microscope system module; The microscope system module includes: an objective lens, the input excitation light source is focused on the sample imaging area through the objective lens, so as to generate a Raman signal by excitation; The spectroscopic module is used for dividing the Raman signal into two paths, one path enters the super-resolution imaging module, and the other path enters the Raman spectrum analysis module. 7 . The super-resolution Raman spectroscopy imaging system according to claim 6 , wherein the excitation light coupling module comprises one or more combinations of a reflection mirror, a dichroic mirror, a beam splitter or a turning mirror. 8 . 8 . The super-resolution Raman spectroscopy imaging system according to claim 1 , wherein the array detector is a CCD array detector, an EMCCD array detector or a CMOS array detector. 9 . 9. The super-resolution Raman spectral imaging system according to claim 1, wherein the Raman spectral analysis module comprises: a Raman signal filter, a fiber coupler, and a Raman spectral super-resolution image generator. spectrometer. 10. The super-resolution Raman spectroscopy imaging system according to claim 1, wherein the surface-enhanced Raman spectroscopy substrate is composed of nanoparticle dimers, nanowires and nanoparticle systems, nanoparticle systems with polarization-dependent properties. Particle arrays - nanowire systems, nanocubes, or one or more hybrid systems with nanocube core-shell structural systems. 11. A super-resolution Raman spectroscopy imaging method, wherein the super-resolution Raman spectroscopy imaging method comprises: generating an excitation light source and modulating the polarization direction of the excitation light source; The excitation light source is focused on the sample imaging area by a laser galvanometer, and a Raman signal is generated by excitation in the sample imaging area; wherein, the sample imaging area includes a surface-enhanced Raman spectroscopy substrate and a surface-enhanced Raman spectroscopy substrate located on the surface-enhanced Raman spectroscopy substrate test samples; Continuously adjust the polarization direction of the laser excitation light source or adjust the scanning direction of the laser galvanometer, and control and complete the different excitation positions of the sample imaging area to generate Raman signals; respectively generating a super-resolution image and a Raman spectrum super-resolution image of the detection sample according to the Raman signal; A process for obtaining the super-resolved image includes: Keep the angle of the laser galvanometer unchanged, continuously adjust the polarization direction of the laser excitation light source, obtain the hot spot position information image of the Raman signal generated by the sample imaging area under each polarization direction, and obtain the image under each polarization direction. The hotspot location information images of the samples are superimposed to form a super-resolution image of the detection sample; Adjust the angle of the laser galvanometer to change the position of the Raman signal excited by the excitation light source on the detection sample, and repeat the above process to obtain the super-resolution images of multiple detection samples at different positions on the sample imaging area. 12 . The super-resolution Raman spectroscopy imaging method according to claim 11 , wherein the hotspot position information in the hotspot position information image is obtained by a fitting positioning method; wherein, the positioning method is Gaussian distribution fitting reconstruction. 13 . method, multi-hot spot Gaussian fitting reconstruction method or compressed sensing data reconstruction method. 13. The super-resolution Raman spectral imaging method according to claim 11, wherein a process of acquiring the Raman spectral super-resolution image comprises: Keep the polarization direction of the excitation light source unchanged, continuously adjust the focus position of the laser galvanometer, and collect the hot spot position information of the excited position and the hot spot position information each time the focus position of the laser galvanometer is adjusted. Corresponding Raman spectral information; Constructing a Raman spectral imaging map by using the acquired hotspot location information and Raman spectral information corresponding to the hotspot location information; Changing the polarization direction of the excitation light source, repeating the above process until the laser polarization rotates for one week, and obtaining a plurality of the Raman spectral imaging images under different polarization directions; Reconstructing the acquired Raman spectrum imaging image to obtain the Raman spectrum super-resolution image. 14 . The super-resolution Raman spectral imaging method according to claim 13 , wherein a method of the Raman spectral imaging map is: extracting the Raman peak intensity of the same wavelength and the excitation Raman signal of the excitation light source. 15 . The location information of the hot spot on the detection sample is superimposed, and the Raman spectral imaging map of the excited position at the wavelength is obtained; wherein, the Raman spectral imaging map in the X and Y directions is the excitation light source excitation Raman signal in the detection The position information on the sample, the gray value of the image is the intensity information of the Raman peak at this wavelength. 15 . The super-resolution Raman spectroscopy imaging method according to claim 13 , wherein a method for obtaining the Raman spectroscopy super-resolved image is: using the Raman spectroscopy at the same wavelength and in all polarization directions. 16 . Reconstructing and obtaining the Raman spectral super-resolution image; obtaining the Raman spectral super-resolution image is the position information of the Raman signal in the x and y directions, and the grayscale of the image represents Raman under different polarization and a certain wavelength condition Intensity integration of peaks. 16 . The super-resolution Raman spectroscopy imaging method according to claim 11 , wherein the polarization direction of the excitation light source is modulated by a liquid crystal polarization rotator or a half-wave plate polarization rotator. 17 .

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