CN109164084B - Super-resolution Raman spectrum imaging system and method - Google Patents
Super-resolution Raman spectrum imaging system and method Download PDFInfo
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- CN109164084B CN109164084B CN201810916840.0A CN201810916840A CN109164084B CN 109164084 B CN109164084 B CN 109164084B CN 201810916840 A CN201810916840 A CN 201810916840A CN 109164084 B CN109164084 B CN 109164084B
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Abstract
The invention provides a super-resolution Raman spectrum imaging system and a method, wherein the system comprises: an excitation light source module for generating an excitation light source; the polarization modulation module is used for modulating the polarization direction of the excitation light source; the galvanometer scanning module is used for scanning and finishing the focusing of the exciting light source at different positions in the sample imaging area; the microscope system module focuses an excitation light source through a sample imaging area and excites the excitation light source to generate a Raman signal, wherein the sample imaging area comprises a surface enhanced Raman spectrum substrate and a detection sample positioned on the surface enhanced Raman spectrum substrate; the super-resolution imaging module is used for generating a super-resolution image of the detection sample according to the Raman signal; and the Raman spectrum analysis module is used for generating a Raman spectrum according to the Raman signal and analyzing the detection sample. The Raman super-resolution imaging method is used for unmarked super-resolution imaging of biological samples and chemical samples and super-resolution imaging of Raman spectra, and effectively solves the problems of small application range, long imaging time, uncontrollable SERS scintillation behavior and the like in Raman super-resolution imaging.
Description
Technical Field
The invention relates to the technical fields of surface plasmon polariton Raman enhancement (SERS), super-resolution optical imaging, laser modulation and the like, in particular to a super-resolution Raman spectrum imaging system and method.
Background
The search for microscopic cells or organelles has long been of interest. In recent years, the research of brain science and brain-like intelligence is more and more attracting attention of all countries in the world, and dynamic imaging and research on the interaction of brain cells and complex neural circuits need to develop the leading advanced imaging, tracing and marking technology from micro to mesoscopic to macro. Therefore, the newly developed optical field regulation and control and nanometer optical imaging subversive technology breaking through the diffraction optical limit provides a powerful method for solving the key scientific technology.
The single molecule positioning super-resolution technology utilizes the spontaneous scintillation effect of fluorescent molecules of marked cells, and obtains a super-resolution image by positioning the position of a fluorescent light source and acquiring multi-frame image reconstruction. The technology has the advantages of small excitation intensity, high wide-field resolution capability, multicolor and 3D imaging capability and the like, but the imaging speed is lower due to the fact that multi-frame reconstruction imaging is needed.
The Surface Enhanced Raman Spectroscopy (SERS) technology is a novel biomarker means, and has the characteristics of difficult bleaching of optical signals, wide range of applicable labeled biological samples, small damage to the samples and the like. Meanwhile, the Raman signal scattered by the original sample is greatly enhanced, so that the method has extremely high sensitivity to the spectrum detection based on the SERS effect. The SERS technology can also acquire chemical component information in cells and biological tissues by raman spectroscopy, and has the natural advantage of label-free imaging, and is widely used in the biomedical field.
In 2010, Willets et al firstly combine the super-resolution microscopy technology with the SERS technology, study single-molecule SERS super-resolution reconstruction imaging, respectively locate the centroids of single molecules and nanoparticles through two-dimensional Gaussian fitting, obtain the spatial position information of the single molecules and the nanoparticles, and the resolution can reach 10 nm. With the combination of single-molecule positioning super-resolution technology and SERS technology, researchers have also begun to use super-resolution technology to obtain SERS signals of subcellular or tissue, and to realize label-free raman super-resolution imaging. However, the period of the scintillation action of SERS is usually tens of milliseconds, generally 100ms is required for acquiring a frame of image, and in combination with a multi-frame acquisition and reconstruction method, it usually takes tens of minutes to obtain a super-resolution image, and if it is used for imaging multiple molecules or tissue cells, it takes several hours, which has a great influence on the study of some living cell tissues. And the illumination mode adopts the static illumination of plasma excimer to produce static hot spot, and it is very successful to realize super-resolution Raman imaging to some single molecules, and can not utilize the scintillation action of molecule to the formation of image of multimolecular and cell tissue to lead to the formation of image result to leave a blank, have problems such as background SERS signal interference.
Disclosure of Invention
To solve the above and other potential technical problems, an embodiment of the present invention provides a super-resolution raman spectroscopy imaging system, including: the excitation light source module is used for generating an excitation light source; the polarization modulation module is used for modulating the polarization direction of the excitation light source; the galvanometer scanning module is used for scanning and finishing different positions of the excitation light source in the sample imaging area to focus; the microscope system module is used for focusing the excitation light source through a sample imaging area and exciting to generate a Raman signal, wherein the sample imaging area comprises a surface enhanced Raman spectroscopy substrate and a detection sample positioned on the surface enhanced Raman spectroscopy substrate; the super-resolution imaging module is used for generating a super-resolution image of the detection sample according to the Raman signal; and the Raman spectrum analysis module is used for generating a Raman spectrum according to the Raman signal and analyzing the detection sample.
In an embodiment of the invention, the excitation light source module includes: a laser generating the excitation light source; and the optical fiber collimator is connected with the laser through an optical fiber and is used for collimating an excitation light source output by the laser and outputting the collimated excitation light source to the polarization modulation module.
In an embodiment of the invention, the polarization modulation module includes: the polaroid receives an excitation light source output by the optical fiber collimator; and a polarization controller controlling a rotation angle and a rotation speed of the polarization of the laser in the polarizing plate.
In an embodiment of the present invention, the galvanometer scanning module includes: the laser galvanometer receives an excitation light source subjected to polarization modulation by the polaroid and scans to finish focusing of the excitation light source at different positions in the sample imaging area; and the galvanometer controller is used for controlling the scanning angle range and the angle change rate of the laser galvanometer.
In an embodiment of the present invention, one way of controlling the polarizer and the laser galvanometer is: keeping the angle of the laser galvanometer unchanged, continuously changing the polarization angle of the polaroid, changing the angle of the laser galvanometer after the polaroid rotates for one circle in a vibrating manner, rotating the polaroid for one circle again, and repeating the processes until the scanning of the imaging area of the sample is completed; another way to control the polarizer and the laser galvanometer is: keeping the angle of the polaroid unchanged, controlling the laser galvanometer to complete scanning of the imaging area of the sample, rotating the polaroid after the scanning is completed, controlling the laser galvanometer to scan the imaging area of the sample again, and repeating the processes until the polaroid rotates for a circle.
In an embodiment of the invention, the super-resolution raman spectroscopy imaging system further includes: the excitation light coupling module is positioned between the laser galvanometer and the microscope system module and couples an excitation light source output by the laser galvanometer to the microscope system module; the microscope system module includes: the input excitation light source is focused on the sample imaging area through the objective lens to excite and generate a Raman signal; and the light splitting module is used for splitting the Raman signal into two paths, wherein one path enters the super-resolution imaging module, and the other path enters the Raman spectrum analysis module.
In an embodiment of the invention, the excitation light coupling module includes one or more combinations of a reflecting mirror, a dichroic mirror, a beam splitter mirror, or a turning mirror.
In an embodiment of the present invention, the super-resolution imaging module includes: the Raman signal filter, the band-pass filter, the imaging lens and the array detector are sequentially arranged; the array detector records the luminous state of the detection sample in real time, performs multi-frame acquisition on the same position of the detection sample, and performs Raman hotspot positioning reconstruction on an image by using a scintillation effect generated by a Raman signal to generate a super-resolution image.
In an embodiment of the 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: the Raman spectrum super-resolution imaging system comprises a Raman signal filter plate, an optical fiber coupler and a spectrometer for generating the Raman spectrum super-resolution image.
In an embodiment of the invention, the surface-enhanced raman spectroscopy substrate is composed of one or more of nanoparticle dimers, nanowires and nanoparticle systems with polarization-dependent properties, nanoparticle array-nanowire systems, nanocubes or mixed systems with nanocubes core-shell structure systems.
The embodiment of the invention also provides a super-resolution Raman spectrum imaging method, which comprises the following steps: generating an excitation light source and modulating the polarization direction of the excitation light source; focusing the excitation light source on a sample imaging area through a laser galvanometer, and exciting to generate a Raman signal through the sample imaging area; wherein the sample imaging area comprises a surface enhanced Raman spectroscopy substrate and a detection sample positioned on the surface enhanced Raman spectroscopy substrate; continuously adjusting the polarization direction of the laser excitation light source or the scanning direction of the laser galvanometer, and controlling different excitation positions of the sample imaging area to generate Raman signals; and respectively generating a super-resolution image and a Raman spectrum super-resolution image of the detection sample according to the Raman signal.
In an embodiment of the present invention, a process of acquiring the super-resolution image includes: keeping the angle of the laser galvanometer unchanged, continuously adjusting the polarization direction of the laser excitation light source, respectively acquiring hot spot position information images of Raman signals generated in the sample imaging area under each polarization direction, and overlapping the hot spot position information images acquired under each polarization direction to form a super-resolution image of the detected sample; and adjusting 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 repeating the process to obtain the super-resolution images of the detection samples at different positions on the sample imaging area.
In an embodiment of the invention, the hotspot position information in the hotspot position information image is obtained by a fitting positioning method; the positioning method is a Gaussian distribution fitting reconstruction method, a multi-hot-point super Gaussian fitting reconstruction method or a 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 focusing position of the laser galvanometer, and acquiring hotspot position information of the excited position and Raman spectrum information corresponding to the hotspot position information when the focusing position of the laser galvanometer is adjusted each time; constructing a Raman spectrum imaging graph through the acquired hotspot position information and Raman spectrum information corresponding to the hotspot position information; changing the polarization direction of the excitation light source, repeating the process until the laser polarization rotation is finished after one circle, and acquiring a plurality of Raman spectrum imaging graphs in different polarization directions; and reconstructing the obtained Raman spectrum imaging graph to obtain the Raman spectrum super-resolution image.
In an embodiment of the present invention, one way of forming the raman spectrum image is: extracting the intensity of a Raman peak with the same wavelength and overlapping the information of the position of a hot spot on the detection sample of the Raman signal excited by the excitation light source to obtain a Raman spectrum imaging graph of the excited position under the wavelength; the Raman spectrum imaging graph is position information of a Raman signal excited by an excitation light source on a detection sample in the dimension of the direction X, Y, and the gray value of the image is intensity information of a Raman peak at the wavelength.
In an embodiment of the present invention, one way to obtain the raman spectrum super-resolution image is as follows: reconstructing the Raman spectrum imaging images with the same wavelength and all polarization directions to obtain the Raman spectrum super-resolution image; and acquiring position information of Raman signals of the Raman spectrum super-resolution image in dimensions of x and y directions, wherein the gray scale of the image represents the intensity integral of Raman peaks under different polarization and certain wavelength conditions.
In one embodiment of the present invention, the polarization direction of the excitation light source is modulated by a liquid crystal polarization rotator or an 1/2 wave plate polarization rotator.
As described above, the super-resolution raman spectroscopy imaging system and method of the present invention have the following beneficial effects:
the invention adjusts the scintillation characteristic of SERS by actively modulating the polarization of exciting light based on polarization modulation and Raman hotspot positioning super-resolution technology, controls the SERS scintillation rate and combines with high-efficiency and quick reconstruction technology, not only can expand the application range of a super-resolution imaging sample, but also can efficiently obtain unmarked super-resolution imaging and Raman spectrum super-resolution imaging for biological samples and chemical samples, greatly increases the information content of the Raman spectrum of the sample, effectively solves the problems of small application range, long imaging time, uncontrollable SERS scintillation behavior and the like in the prior art in Raman super-resolution imaging, effectively improves the analysis capability of sample components, and plays an important role in researching the influence of polarization on the Raman spectrum of the sample.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 is a schematic block diagram of a super-resolution raman spectroscopy imaging system of the present invention.
Fig. 2 is a schematic structural diagram showing a specific principle of the super-resolution raman spectroscopy imaging system of the present invention.
Fig. 3 is a schematic diagram showing a polarization modulation surface enhanced raman spectroscopy super-resolution imaging process in the super-resolution raman spectroscopy imaging system of the present invention.
Fig. 4 is a schematic diagram showing a raman spectroscopy super-resolution imaging process in the super-resolution raman spectroscopy imaging system of the present invention.
Fig. 5 is a schematic overall flow chart of the super-resolution raman spectroscopy imaging method of the present invention.
Description of the element reference numerals
100 super-resolution Raman spectrum imaging system
110 excitation light source module
111 Raman excitation laser
112 optical fiber collimator
120 polarization modulation module
121 polarizer
122 polarization controller
130 galvanometer scanning module
131 laser galvanometer
132 galvanometer controller
140 microscope system module
141 surface enhanced Raman spectroscopy substrate
142 test sample
143 objective lens
144 light splitting module
150 super-resolution imaging module
151 Raman signal filter
152 band-pass filter
153 imaging lens
154 array detector
160 Raman spectrum analysis module
161 spectrometer
162 Raman signal filter
163 optical fiber coupler
170 excitation light coupling module
171 reflecting mirror
172 dichroic mirror
173 beam splitter
S110 to S140
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be noted that the features in the following embodiments and examples may be combined with each other without conflict.
Please refer to fig. 1 to 5. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Aiming at the problems of small application range, long imaging time, uncontrollable SERS (surface enhanced Raman scattering) scintillation behavior and the like when a single molecule positioning technology is used for Raman super-resolution imaging, the present embodiment aims to provide a super-resolution Raman spectrum imaging system 100 and a method, which are used for label-free super-resolution imaging of biological samples and chemical samples and super-resolution imaging of Raman spectra, and effectively solve the problems of small application range, long imaging time, uncontrollable SERS scintillation behavior and the like in Raman super-resolution imaging in the prior art.
The embodiment is a surface plasmon polariton raman enhanced super-resolution raman spectroscopy imaging system 100 and method based on polarization modulation, and is a label-free super-resolution imaging system utilizing a surface plasmon polariton raman enhancement effect and a raman spectroscopy imaging method based on the system. The super-resolution raman spectroscopy imaging system 100 of the present 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. The imaging method of the super-resolution raman spectroscopy imaging system 100 based on this embodiment is to modulate the polarization of the excitation light, so as to have a surface raman enhanced hot spot with random excitation polarization dependence, so as to generate a polarization-dependent scintillation effect on a sample raman signal, and perform random optical reconstruction super-resolution imaging and raman spectroscopy super-resolution imaging by using the collected scintillation raman signal.
The principles and embodiments of the super-resolution raman spectroscopy imaging system 100 and the method of the present 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 the method of the present embodiment without creative efforts.
An embodiment of the present invention provides a super-resolution raman spectroscopy imaging system 100, where the super-resolution raman spectroscopy imaging system 100 includes: the system comprises an excitation light source module 110, a polarization modulation module 120, a galvanometer scanning module 130, a microscope system module 140, an excitation light coupling module 170, a super-resolution imaging module 150 and a raman spectrum analysis module 160.
The super-resolution raman spectroscopy imaging system 100 of the present embodiment is described in detail below.
In this embodiment, the excitation light source module 110 is used for generating an excitation light source.
Specifically, in the present embodiment, as shown in fig. 2, the excitation light source module 110 includes: a raman excitation laser 111 that generates the excitation light source; and the optical fiber collimator 112 is connected to the raman excitation laser 111 through an optical fiber, and collimates the excitation light source output by the raman excitation laser 111 and outputs the collimated excitation light source to the polarization modulation module 120.
The laser wavelength output by the raman excitation laser 111 includes, but is not limited to, commonly used raman excitation wavelengths: 405nm, 488nm, 532nm, 632nm, 785nm and the like, the output aperture of the optical fiber meets the requirements of confocal scanning microimaging, so that the system has confocal microimaging capability, and the Raman excitation laser 111 outputs laser through the 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, i.e. a polarizer, is used for modulating the polarization direction of the excitation light source.
Specifically, the polarization modulation module 120 generates a polarization rotation of zero to 2 π for the incident linearly polarized laser light.
Specifically, in the present embodiment, as shown in fig. 2, the polarization modulation module 120 includes: a polarizer 121 for receiving the excitation light source output by the fiber collimator 112; and a polarization controller 122 for controlling the rotation angle and rotation speed of the polarization of the laser light in the polarizing plate 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. The means for providing polarization include, but are not limited to, liquid crystal polarization rotators and 1/2 wave plate polarization rotators, among others.
In this embodiment, the galvanometer scanning module 130 is configured to scan different positions of the excitation light from the sample imaging area.
Specifically, in the present embodiment, as shown in fig. 2, the galvanometer scanning module 130 includes: the laser galvanometer 131 receives the excitation light source subjected to polarization modulation by the polarizer 121, and scans to finish focusing of the excitation light source at different positions in the sample imaging area; and a galvanometer controller 132 for controlling the scanning angle range and the angle change rate of the laser galvanometer 131.
The laser galvanometer 131 is used for realizing the rapid two-dimensional scanning of the sample imaging area in the confocal microscope system module 140, and the scanning angle range and the angle change rate of the laser galvanometer 131 are simultaneously controlled by the galvanometer controller 132 and a time sequence control program in the galvanometer controller 132.
Specifically, in the present embodiment, one way of controlling the polarizer 121 and the laser galvanometer 131 is as follows: keeping the angle of the laser galvanometer 131 unchanged, continuously changing the polarization angle of the polarizer 121, changing the angle of the laser galvanometer 131 after the polarizer 121 rotates for one circle in a vibrating manner, rotating the polarizer 121 for one circle again, and repeating the above processes until the scanning of the sample imaging area is completed.
Another way to control the polarizer 121 and the laser galvanometer 131 is: keeping the angle of the polarizer 121 unchanged, controlling the laser galvanometer 131 to complete scanning of the sample imaging area, rotating the polarizer 121 after the scanning is completed, controlling the laser galvanometer 131 to scan the sample imaging area again, and repeating the above processes until the polarizer 121 rotates for one circle.
In this embodiment, the excitation light coupling module 170 is disposed between the laser galvanometer 131 and the microscope system module 140, and outputs an excitation light source output by the laser galvanometer 131 to the microscope system module 140.
Specifically, in the present embodiment, as shown in fig. 2, the excitation light coupling module 170 includes one or more combinations of a reflecting 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 excite raman signal generation through a sample imaging area, wherein the sample imaging area includes a surface-enhanced raman spectroscopy substrate 141 and a detection sample 142 disposed on the surface-enhanced raman spectroscopy substrate 141, as shown in fig. 2.
Specifically, in the present embodiment, the surface-enhanced raman spectroscopy substrate 141 is composed of one or more of nanoparticle dimers, nanowires and nanoparticle systems with polarization-dependent properties, nanoparticle array-nanowire systems, nanocubes, or mixed systems with nanocubes core-shell structure systems.
The excitation light source after polarization modulation enters the objective lens 143 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) in a suspension, adsorption, or other form, and inelastic raman scattering occurs between the focused excitation light source and the detection sample 142 to generate a raman signal.
Because the excitation light source has polarization in a certain direction, the hot spot in the SERS substrate can achieve the maximum Raman enhancement effect only in the polarized plasma mode which is consistent with the polarization direction of the excitation light source, so that the Raman signal on the SERS substrate can be regulated and controlled by the polarization of the excitation light. The raman signal generated by the sample molecules near the excited hot spot is enhanced while the raman signal near the unexcited hot spot is not enhanced. Fig. 3 illustrates an example of a silver nanoparticle dimer: the polarization direction of the dimer is related to the long axis direction of the dimer, and is in a random arrangement on the substrate. When the excitation light is polarized as in the form of long arrows in fig. 3. It can be seen that only hot spots with long axis directions coinciding with the polarization direction of the excitation light are excited. Thereby generating a surface raman scattering enhancement effect to enhance the raman signal in the sample signal. While unexcited dimers do not enhance the raman signal in the sample.
Specifically, as shown in fig. 2, the microscope system module 140 includes: an objective lens 143, through which an input excitation light source is focused on the sample imaging area to excite and generate a raman signal; the optical splitting module 144 is configured to split the raman signal into two paths, where one path enters the super-resolution imaging module 150 and the other path enters the raman spectrum analysis module 160. The light splitting module 144 is an electric rotating mirror or a beam splitter.
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 the present embodiment, as shown in fig. 2, the super-resolution imaging module 150 includes: a Raman signal filter 151, a band-pass 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, performs multi-frame collection on the same position of the detection sample 142, and performs raman hotspot positioning reconstruction on an image by using a scintillation effect generated by a raman signal to generate a super-resolution image.
Specifically, in the present 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 to be imaged. Since light is an electromagnetic wave with diffraction characteristics, the raman signal collected in the CCD/EMCCD/CMOS array detector 154 is a diffraction spot modulated by a point spread function determined by an imaging system. The light intensity of the diffraction light spot has the characteristic of Gaussian distribution approximately, and the nano-precision hot spot position information of the Raman hot spot can be found through a fitting positioning method. The positioning method adopted in the system comprises but is not limited to a Gaussian distribution fitting reconstruction method, a multi-hot-point super Gaussian fitting reconstruction method and a compressed sensing data reconstruction method.
The polarization modulator is changed in real time while the vibrating mirror is kept unchanged, and the polarization direction of the exciting light is continuously changed, so that hot spots with randomly distributed polarization directions can be randomly excited along with the change of the polarization direction of the laser. Every time the polarization direction of the exciting light is changed, the CCD/EMCCD/CMOS array detector 154 collects and positions each frame of image to obtain an image of the hot spot position information. All the images obtained in the process are superposed to realize super-resolution imaging. FIG. 3 illustrates the process in detail, taking the substrate of nanoparticle dimers as an example: the polarization direction angle of the excitation light in fig. 3A is 0 °, only a part of the nanoparticle dimers (black dimer particles in fig. 3A) shown in fig. 3A have the strongest raman enhancement effect, and the diffraction spot image is obtained after the enhanced raman signal is collected by the array detector 154. By the positioning method, the position information of the Raman signal is further obtained. The polarization direction angle of the excitation light is continuously changed, (fig. 3B is 30 °, fig. 3C is 45 °, and fig. 3D is 90 °) to acquire raman signal information at different positions, and all the acquired images are reconstructed to obtain a super-resolution image of the sample (fig. 3E). The laser galvanometer 131 is continuously adjusted to change the position of the exciting light focused on the sample, and a plurality of super-resolution images of the detection sample at other positions are obtained.
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 the present 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 requirement of confocal scanning microscopic imaging on the collected optical signal, and is matched with the aperture of the output optical fiber in the excitation light source module 110, so that the system has the confocal scanning microscopic imaging capability.
The raman signal enters the spectrometer 161 through fiber coupling, and the raman spectrum information at a certain polarization angle and the position information of the excited hot spot are acquired. Raman spectra at different wavelengths (lambda)1，λ2，λ3，λ4… …) will show a raman peak. Tong (Chinese character of 'tong')The same wavelength (lambda) of different sites is extracted1) The Raman peak intensity and the Raman signal position information are reconstructed to obtain the Raman signal with the wavelength (lambda)1) And (3) Raman spectrum imaging of the excited site. Obtaining other wavelengths (lambda) by the same principle2，λ3，λ4… …) in the sample.
The raman spectrum imaging graph acquisition process is described in detail by taking fig. 4 as an example.
When the polarization angle of the excitation light is 0 ° (black double arrow direction in fig. 3A), the positional information (fig. 4A) and raman spectrum (fig. 4A) focused by the excitation light at this time are acquired by the spectrometer 1615). Raman spectra at these positions are respectively at λ1，λ2，λ3，λ4Has Raman peaks with different intensities, will be lambda1The raman peak intensity at wavelength and the source position of the raman signal reconstruct a raman spectrum image as shown in fig. 4a 1. The figure 4A1Position information of Raman signal in x and y directions, gray value of image representing polarization angle of 0 degree and lambda1Intensity information of Raman peak under the wavelength condition of (1) (FIG. 4A)1Black dot in (c) indicates a raman peak at this position). In the same way, λ can be obtained2，λ3，λ4wavelength-down-Raman spectral imaging (FIG. 4A)2FIG. 4A3FIG. 4A4). The polarizing plate 121 is rotated to continuously change the polarization direction of the exciting light, and the above process is repeated to obtain raman spectrum imaging images under different polarization angles. As shown in fig. 4B, when the polarization direction changes by θ, the raman spectrum changes. The reconstructed Raman spectrum imaging chart is shown in FIG. 4B1FIG. 4B2FIG. 4B3FIG. 4B4As shown.
And reconstructing the obtained Raman spectrum imaging graph to obtain a Raman spectrum super-resolution image. The reconstruction method comprises the following steps: and superposing the Raman spectrum imaging graphs of the same wavelength and different polarization angles. FIG. 4C is λ1Conditional raman spectral super-resolution images consisting of different polarization angles (theta)1，θ2，θ3...) of the same wavelength (λ)1) Raman spectrum distribution of (FIG. 4A)1FIG. 4B1… …) obtained by reconstruction. FIG. 4 shows the position information of the Raman signal in the x and y dimensions, and the gray scale of the image represents different polarization, λ1(iv) the intensity of the raman peak under the wavelength condition of (iv) (different raman peak intensities are represented by different filling patterns in fig. 4C and 4D).
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, and the laser light is collimated by the fiber collimator 112 to form parallel light, and the parallel light enters the continuously rotating polarizing plate 121 controlled by software to generate polarized light with continuously changing polarization direction. Then, the polarized light enters the objective lens 143 after passing through the laser vibrating mirror 131 and the reflecting mirror 171 and is focused on the SERS substrate, and the detection sample is excited to emit a Raman signal. The raman signal is collected by the objective lens 143 and then divided into two paths by the electric rotating mirror or beam splitter 173: the first path enters a CCD/EMCCD/CMOS array detector 154 for imaging, and the adopted super-resolution imaging method is a Raman hotspot positioning reconstruction super-resolution imaging method. The other path enters the spectrometer 161 for Raman spectrum super-resolution imaging.
As shown in fig. 5, this embodiment further provides a super-resolution raman spectroscopy imaging method, where the super-resolution raman spectroscopy imaging method includes:
step S110, generating an excitation light source and modulating the polarization direction of the excitation light source;
step S120, focusing the excitation light source on a sample imaging area through a laser galvanometer 131, and generating a Raman signal through excitation of the sample imaging area; wherein the sample imaging area comprises a surface enhanced raman spectroscopy substrate 141 and a detection sample 142 positioned on the surface enhanced 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 different excitation positions of the sample imaging area to generate Raman signals;
and 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 acquiring 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, respectively obtaining the hot spot position information images of the raman signal generated in the sample imaging area under each polarization direction, and overlapping the hot spot position information images obtained under each polarization direction to form a super-resolution image of the detection sample 142; the angle of the laser galvanometer 131 is adjusted to change the position of the raman signal excited by the excitation light source on the detection sample 142, 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.
I.e. the polarization characteristics of the excitation light source are changed by adjusting the polarization rotator. And exciting a hot spot in the SERS substrate plasmon enhancement mode, wherein the hot spot is consistent with the polarization direction of exciting light, and generating the maximum Raman enhancement effect. The raman signal is collected by the array detector 154 to obtain random hotspot location information. The polarization direction of the exciting light is continuously changed, the acquisition rate of the CCD/EMCCD/CMOS array detector 154 is controlled, and a series of random hot spot position information is obtained. And carrying out Raman hot spot positioning and reconstruction on the acquired image to obtain the super-resolution image of the detection sample 142.
Specifically, in the present embodiment, the complete process of generating the super-resolution image of the detection sample 142 is as follows:
1) the imaged target was transferred to a Surface Enhanced Raman Spectroscopy (SERS) substrate with strong polarization dependence. The polarization-dependent SERS substrate can be formed by one or a mixture of several of metal dimer, polymer, cube, core-shell structure and nanoparticle-nanowire structure which have a polarization plasma mode.
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 the rotation speed of the polarizer 121 may be adjusted by a controller. Polarization rotation means include, but are not limited to, liquid crystal polarization rotators and 1/2 wave plate polarization rotators. Keeping the laser galvanometer 131 still, rotating the polarizer, modulating the polarization direction of the excitation light source, and controlling the polarization included angle θ of the excitation light to excite the plasma mode of polarization on the SERS substrate consistent with the polarization direction of the excitation light source (defined as shown in fig. 3).
3) The polarization-modulated excitation light enters the objective lens 143 through the laser galvanometer 131, the reflecting mirror 171 and the dichroic mirror 172, and is focused on the sample on the SERS substrate. The detection sample 142 may be immobilized on the SERS substrate by suspension, adsorption, or the like. Inelastic raman scattering occurs between the focused excitation light and the sample analyte to generate a raman signal. Because the SERS substrate has strong polarization dependence, the maximum Raman enhancement effect can be achieved only in a polarized plasma mode consistent with the polarization direction of the excitation light source. The detection targets at these sites emit detectable raman signals, while the remaining polarization-polarized plasmon mode sites have no enhancement effect. Namely, the effect of bright and dark flicker of SERS hot spots at different sites is realized.
Because the exciting light has polarization in a certain direction, the hot spot in the SERS substrate can achieve the maximum Raman enhancement effect only in the polarized plasma mode which is consistent with the polarization direction of the exciting light source, so that the Raman signal on the SERS substrate can be regulated and controlled by the polarization of the exciting light. The raman signal generated by the sample molecules near the excited hot spot is enhanced while the raman signal near the unexcited hot spot is not enhanced. Fig. 3 illustrates an example of a silver nanoparticle dimer: the polarization direction of the dimer is related to the long axis direction of the dimer, and is in a random arrangement on the substrate. When the excitation light is polarized as in the form of long arrows in fig. 3. It can be seen that only hot spots with long axis directions coinciding with the polarization direction of the excitation light are excited. Thereby generating a surface raman scattering enhancement effect to enhance the raman signal in the sample signal. While unexcited dimers do not enhance the raman signal in the sample.
4) The polarization direction of the laser is continuously changed by adjusting the rotary polarizer, so that Raman luminescence of hot spots at different positions and different polarization directions is randomly modulated, and the scintillation effect of the SERS detection target is generated.
In this embodiment, the polarization direction of the excitation light source is modulated by a liquid crystal polarization rotator or an 1/2 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 luminous state of the sample in real time, and multi-frame collection is carried out on the same position of the sample. And carrying out Raman hotspot positioning reconstruction on the image by utilizing the generated flicker effect to generate a super-resolution image.
Specifically, the enhanced raman signal enters the CCD/EMCCD/CMOS array detector 154 all the way to be imaged. Since light is an electromagnetic wave with diffraction characteristics, the raman signal collected in the CCD/EMCCD/CMOS array detector 154 is a diffraction spot modulated by a point spread function determined by an imaging system. The light intensity of the diffraction light spot has the characteristic of Gaussian distribution approximately, and the nanometer precision position information of the Raman hot spot can be found through a fitting positioning method. The positioning method adopted in the system comprises but is not limited to a Gaussian distribution fitting reconstruction method, a multi-hot-point super Gaussian fitting reconstruction method and a compressed sensing data reconstruction method.
The polarization modulator is changed in real time while the vibrating mirror is kept unchanged, and the polarization direction of the exciting light is continuously changed, so that hot spots with randomly distributed polarization directions can be randomly excited along with the change of the polarization direction of the laser. Every time the polarization direction of the exciting light is changed, the CCD/EMCCD/CMOS array detector 154 collects and positions each frame of image to obtain an image of the hot spot position information. All the images obtained in the process are superposed to realize super-resolution imaging. FIG. 3 illustrates the process in detail, taking the substrate of nanoparticle dimers as an example: the polarization direction angle θ of the excitation light in fig. 3A is 0 °, only a part of the nanoparticle dimers (black dimer particles in fig. 3A) shown in fig. 3 have the strongest raman enhancement effect, and the enhanced raman signal is collected by the CCD/EMCCD/CMOS array detector 154 to obtain a diffraction spot image. And further acquiring the position information of the Raman signal by the positioning method in the step 5). Continuously varying the polarization direction angle theta of the excitation light (FIG. 3B theta)130 ° fig. 3C θ245 ° fig. 3D θ3The raman signal information at different positions is acquired at 90 °, and all the acquired images are reconstructed to obtain a super-resolution image of the sample (fig. 3E).
6) And (3) adjusting the laser galvanometer 131, namely adjusting the laser galvanometer 131 to change the position of the exciting light focused on the sample, changing the focusing position of the exciting light, and repeating the steps 2) to 5) to obtain a super-resolution image of another position.
In this embodiment, the hotspot location information in the hotspot location information image is obtained by a fitting and positioning method; the positioning method is a Gaussian distribution fitting reconstruction method, a multi-hot-point super Gaussian fitting reconstruction method or a compressed sensing data reconstruction 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 focusing position of the laser galvanometer 131, and acquiring hotspot position information of the excited position and Raman spectrum information corresponding to the hotspot position information when adjusting the focusing position of the laser galvanometer 131 each time; constructing a Raman spectrum imaging graph through the acquired hotspot position information and Raman spectrum information corresponding to the hotspot position information; changing the polarization direction of the excitation light source, repeating the process until the laser polarization rotation is finished after one circle, and acquiring a plurality of Raman spectrum imaging graphs in different polarization directions; and reconstructing the obtained Raman spectrum imaging graph to obtain the Raman spectrum super-resolution image.
I.e. the polarization characteristics of the excitation light source are changed by adjusting the polarization rotator. The polarized light excites a hot spot in the SERS substrate plasmon polariton enhancement mode, wherein the hot spot is consistent with the polarization direction of the exciting light, and the maximum Raman enhancement effect is generated. 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 micro-raman spectrum. And completing two-dimensional confocal quick scanning of the sample through a galvanometer. During the scanning process, the spectrometer 161 collects a raman spectrum each time the galvanometer changes the scanning position. And constructing a Raman spectrum imaging graph through the scanned position information and the Raman spectrum of the position. And changing the polarization direction of the exciting light, and performing confocal scanning Raman spectrum imaging in the same region to obtain a Raman imaging image. This process is repeated until the laser polarization is rotated one revolution. And reconstructing the Raman spectrum distribution diagram with the same wavelength to obtain a Raman spectrum super-resolution diagram.
In this embodiment, one way to construct the raman spectrum imaging graph is as follows: extracting the intensity of the Raman peak with the same wavelength and overlapping the information of the position of the hot spot of the Raman signal excited by the excitation light source on the detection sample 142 to obtain a Raman spectrum imaging graph of the excited position under the wavelength; the raman spectrum imaging graph represents the position information of the raman signal excited by the excitation light source on the detection sample 142 in the dimensions of x and y directions, and the gray value of the image is the intensity information of the wavelength raman peak.
Because the Raman spectrogram is at different wavelengths (lambda)1，λ2，λ3，λ4… …) will show a raman peak. The Raman peak intensity and the position information focused on the sample by the laser in the same wavelength band can be extracted for reconstruction, namely, the Raman peak intensity and the position information focused on the sample in the wavelength band (lambda)1) And (3) Raman spectrum imaging of the excited site. The Raman spectrum imaging image is position information of laser focusing on a sample in x and y directions, and the gray scale of the image is intensity information of Raman peaks in wavelength ranges.
In this embodiment, one way to obtain the raman spectrum super-resolution image is as follows: reconstructing the Raman spectrum imaging images with the same wavelength and all polarization directions to obtain the Raman spectrum super-resolution image; and acquiring the position information of the Raman signal of the Raman spectrum super-resolution image in the dimensions of the x direction and the y direction, wherein the gray scale of the image is the intensity integral of the Raman peak under the conditions of different polarizations and a certain wavelength.
Specifically, the process of acquiring the raman spectrum super-resolution image is as follows:
1) transferring the imaged target to a SERS substrate having polarization dependence, the SERS substrate structure having polarization dependence including but not limited to: nanoparticle dimers, nanowires and nanoparticle systems, nanoparticle array-nanowire systems, nanocubes, or systems with nanocubes core-shell structures.
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) Polarized light is focused on the SERS substrate after passing through the laser galvanometer 131, the reflector 171, the dichroic mirror 172 and the objective lens 143, and the controller controls the laser galvanometer 131 to scan in two dimensions and scan a sample confocal microscopic imaging area rapidly.
4) Because the exciting light has polarization in a certain direction, the hot spot in the SERS substrate can achieve the maximum Raman enhancement effect only in a polarization-related plasma mode consistent with the polarization direction of the exciting light source, the Raman signal generated by the sample near the excited hot spot is enhanced, and the Raman signal near the unexcited hot spot is not enhanced.
5) The raman signal enters the spectrometer 161 through fiber coupling, and the raman spectrum information at a certain polarization angle and the position information of the excited hot spot are acquired. Raman spectra at different wavelengths (lambda)1，λ2，λ3，λ4… …) will show a raman peak. By extracting the same wavelength (lambda) from different sites1) The Raman peak intensity and the Raman signal position information are reconstructed to obtain the Raman signal with the wavelength (lambda)1) And (3) Raman spectrum imaging of the excited site. Obtaining other wavelengths (lambda) by the same principle2，λ3，λ4… …) in the sample. The raman spectrum imaging graph acquisition method is described in detail by taking fig. 4 as an example. When the polarization angle of the excitation light is 0 ° (black double arrow direction in fig. 3A), the positional information (fig. 4A) and raman spectrum (fig. 4A) focused by the excitation light at this time are acquired by the spectrometer 1615). Raman spectra at these positions are respectively at λ1，λ2，λ3，λ4Has Raman peaks with different intensities, will be lambda1The raman peak intensity at wavelength and the source position of the raman signal reconstruct a raman spectrum image as shown in fig. 4a 1. The image has position information of Raman signal in x and y directions, and the gray scale of the image is 0 degree and lambda1Intensity information of Raman peak under the wavelength condition of (1) (FIG. 4A)1Black dot in (c) indicates a raman peak at this position). In the same way, λ can be obtained2，λ3，λ4wavelength-down-Raman spectral imaging (FIG. 4A)2FIG. 4A3FIG. 4A4)。
6) Rotating the polarizer 121 to continuously change the polarization direction of the excitation lightAnd repeating the step 5) to obtain Raman spectrum imaging images under different polarization angles. As shown in fig. 4B, when the polarization direction changes by θ, the raman spectrum changes. The reconstructed Raman spectrum imaging chart is shown in FIG. 4B1FIG. 4B2FIG. 4B3FIG. 4B4As shown.
7) And reconstructing the obtained Raman spectrum imaging graph to obtain a Raman spectrum super-resolution image. The reconstruction method comprises the following steps: and superposing the Raman spectrum imaging graphs of the same wavelength and different polarization angles. FIG. 4C is λ1Conditional raman spectral super-resolution images consisting of different polarization angles (theta)1，θ2，θ3...) of the same wavelength (λ)1) Raman spectrum distribution of (FIG. 4A)1FIG. 4B1… …) obtained by reconstruction. FIG. 4 shows the position information of the Raman signal in the x and y dimensions, and the gray scale representation of the image is different polarization, λ1The intensity of the raman peak under the wavelength condition (different raman peak intensities are shown by different filling patterns in fig. 4C and 4D).
In summary, the invention adjusts the scintillation characteristic of SERS by actively modulating the polarization of the excitation light based on the polarization modulation and raman hotspot positioning super-resolution technology, controls the SERS scintillation rate, and combines with the high-efficiency and fast reconstruction technology, not only can expand the use range of the super-resolution imaging sample, but also can efficiently obtain the unmarked super-resolution imaging and the super-resolution imaging of raman spectrum for biological samples and chemical samples, greatly increase the information content of the raman spectrum of the sample, effectively solve the problems of small application range, long imaging time, uncontrollable SERS scintillation behavior and the like in the raman super-resolution imaging in the prior art, effectively improve the analysis capability of sample components, and play an important role in researching the influence of polarization on the raman spectrum of the sample. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention shall be covered by the claims of the present invention.
Claims (16)
1. A super-resolution raman spectroscopy imaging system, comprising:
the excitation light source module is used for generating an excitation light source;
the polarization modulation module is used for modulating the polarization direction of the excitation light source;
the galvanometer scanning module is used for scanning and finishing different positions of the excitation light source in the sample imaging area to focus;
the microscope system module is used for focusing the excitation light source through a sample imaging area and exciting to generate a Raman signal, wherein the sample imaging area comprises a surface enhanced Raman spectroscopy substrate and a detection sample positioned on the surface enhanced Raman spectroscopy substrate;
the super-resolution imaging module is used for generating a super-resolution image of the detection sample according to the Raman signal;
the Raman spectrum analysis module is used for generating a Raman spectrum according to the Raman signal and analyzing the detection sample;
the super-resolution imaging module includes: the Raman signal filter, the band-pass filter, the imaging lens and the array detector are sequentially arranged; the array detector records the luminous state of the detection sample in real time, performs multi-frame acquisition on the same position of the detection sample, and performs Raman hotspot positioning reconstruction on an image by using a scintillation effect generated by a Raman signal 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 generating the excitation light source;
and the optical fiber collimator is connected with the laser through an optical fiber and is used for collimating an excitation light source output by the laser and outputting the collimated excitation light source to the polarization modulation module.
3. The super-resolution raman spectral imaging system of claim 2, wherein said polarization modulation module comprises:
the polaroid receives an excitation light source output by the optical fiber collimator;
and a polarization controller controlling a rotation angle and a rotation speed of the polarization of the laser in the polarizing plate.
4. The super-resolution raman spectroscopy imaging system of claim 3, wherein said galvanometer scanning module comprises:
the laser galvanometer receives an excitation light source subjected to polarization modulation by the polaroid and scans to finish focusing of the excitation light source at different positions in the sample imaging area;
and the galvanometer controller is used for controlling the scanning angle range and the angle change rate of the laser galvanometer.
5. The super-resolution raman spectral imaging system of claim 4, wherein:
one way of controlling the polarizer and the laser galvanometer is: keeping the angle of the laser galvanometer unchanged, continuously changing the polarization angle of the polaroid, changing the angle of the laser galvanometer after the polaroid rotates for a circle, rotating the polaroid for a circle again, and repeating the processes until the scanning of the imaging area of the sample is completed;
another way to control the polarizer and the laser galvanometer is: keeping the angle of the polaroid unchanged, controlling the laser galvanometer to complete scanning of the imaging area of the sample, rotating the polaroid after the scanning is completed, controlling the laser galvanometer to scan the imaging area of the sample again, and repeating the processes until the polaroid rotates for a circle.
6. The super-resolution raman spectral imaging system of claim 4, further comprising: the exciting light coupling module is positioned between the laser galvanometer and the microscope system module and is used for coupling exciting light output by the laser galvanometer to the microscope system module;
the microscope system module includes:
the input excitation light source is focused on the sample imaging area through the objective lens to excite and generate a Raman signal;
and the light splitting module is used for splitting the Raman signal into two paths, wherein 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 of claim 6, wherein the excitation light coupling module comprises one or more combinations of mirrors, dichroic mirrors, beam splitters, or turning mirrors.
8. The super-resolution raman spectroscopy imaging system of claim 1, wherein the array detector is a CCD array detector, an EMCCD array detector, or a CMOS array detector.
9. The super-resolution raman spectroscopy imaging system of claim 1, wherein the raman spectroscopy analysis module comprises: the Raman spectrum super-resolution imaging system comprises a Raman signal filter plate, an optical fiber coupler and a spectrometer for generating the Raman spectrum super-resolution image.
10. The super-resolution raman spectroscopy imaging system according to claim 1, wherein said surface enhanced raman spectroscopy substrate is comprised of one or more mixed systems of nanoparticle dimers, nanowires and nanoparticle systems, nanoparticle array-nanowire systems, nanocubes, or core-shell structures systems with nanocubes with polarization dependent properties.
11. A super-resolution Raman spectrum imaging method is characterized by comprising the following steps:
generating an excitation light source and modulating the polarization direction of the excitation light source;
focusing the excitation light source on a sample imaging area through a laser galvanometer, and exciting to generate a Raman signal through the sample imaging area; wherein the sample imaging area comprises a surface enhanced Raman spectroscopy substrate and a detection sample positioned on the surface enhanced Raman spectroscopy substrate;
continuously adjusting the polarization direction of the laser excitation light source or the scanning direction of the laser galvanometer, and controlling 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 a detection sample according to the Raman signals;
one process of acquiring the super-resolution image includes:
keeping the angle of the laser galvanometer unchanged, continuously adjusting the polarization direction of the laser excitation light source, respectively acquiring hot spot position information images of Raman signals generated in the sample imaging area under each polarization direction, and overlapping the hot spot position information images acquired under each polarization direction to form a super-resolution image of the detected sample;
and adjusting 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 repeating the process to obtain the super-resolution images of the 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 location information in the hotspot location information image is obtained by a fitting and positioning method; the positioning method is a Gaussian distribution fitting reconstruction method, a multi-hot-point super Gaussian fitting reconstruction method or a compressed sensing data reconstruction method.
13. The method of claim 11, wherein acquiring the raman spectroscopy super-resolved image comprises:
keeping the polarization direction of the excitation light source unchanged, continuously adjusting the focusing position of the laser galvanometer, and acquiring hotspot position information of the excited position and Raman spectrum information corresponding to the hotspot position information when the focusing position of the laser galvanometer is adjusted each time;
constructing a Raman spectrum imaging graph through the acquired hotspot position information and Raman spectrum information corresponding to the hotspot position information;
changing the polarization direction of the excitation light source, repeating the process until the laser polarization rotation is finished after one circle, and acquiring a plurality of Raman spectrum imaging graphs in different polarization directions;
and reconstructing the obtained Raman spectrum imaging graph to obtain the Raman spectrum super-resolution image.
14. The method of claim 13, wherein the raman spectroscopy image is generated by: extracting the intensity of a Raman peak with the same wavelength and overlapping the information of the position of a hot spot on the detection sample of the Raman signal excited by the excitation light source to obtain a Raman spectrum imaging graph of the excited position under the wavelength; the Raman spectrum imaging graph is position information of a Raman signal excited by an excitation light source on a detection sample in the dimension of the direction X, Y, and the gray value of the image is intensity information of a Raman peak at the wavelength.
15. The method of claim 13, wherein the raman spectroscopy super-resolved image is obtained by: reconstructing the Raman spectrum under the same wavelength and all polarization directions to obtain a Raman spectrum super-resolution image; and acquiring position information of Raman signals of the Raman spectrum super-resolution image in dimensions of x and y directions, wherein the gray scale of the image represents the intensity integral of Raman peaks under different polarization and certain wavelength conditions.
16. The method according to claim 11, wherein the polarization direction of the excitation light source is modulated by a liquid crystal polarization rotator or an 1/2 wave plate polarization rotator.
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