CN109164084A - Super-resolution Raman spectrum imaging system and method - Google Patents
Super-resolution Raman spectrum imaging system and method Download PDFInfo
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Abstract
The present invention provides a kind of super-resolution Raman spectrum imaging system and method, the system comprises: excitation light source module generates excitation light source；Polarization Modulation module, for modulating the polarization direction of excitation light source；Vibration mirror scanning module, scanning are completed excitation light source and are focused in the different location of sample imaging area；Microscopic system module by sample imaging area focused activating light source and excites generation Raman signal, wherein sample imaging area includes Surface enhanced Raman spectroscopy substrate and the test sample on Surface enhanced Raman spectroscopy substrate；Super-resolution imaging module generates the super resolution image of test sample according to Raman signal；Raman spectrum analysis module, for generating Raman spectrum and analysis detection sample according to Raman signal.Super-resolution imaging of the present invention for the unmarked super-resolution imaging and Raman spectrum of biological sample and chemical sample, effectively solves the problems such as scope of application is small, imaging time is long, SERS flicker behavior is uncontrollable in Raman super-resolution imaging.
Description
Technical field
The present invention relates to the technologies such as surface plasmons Raman enhancing (SERS), super-resolution optical imaging, Laser Modulation
Field, more particularly to a kind of super-resolution Raman spectrum imaging system and method.
Background technique
The exploration of micro-cell or organelle is aroused people's interest for a long time.Brain science and class brain intelligence in recent years
The research of energy increasingly causes the attention of countries in the world, and to move to brain cell and complicated neural circuit interaction
The imaging and research of state need to develop the microcosmic to advanced imaging, tracer and the labelling technique seen to macroscopic view that be situated between of forefront.It is right
This, subversiveness technology is imaged in the light field regulation of the breakthrough diffraction optics limit of latest development and nanocomposite optical, to solve this key
Science and technology provide strong method.
The scintillation effect for marking the fluorescent molecule of cell spontaneous is utilized in unimolecule positioning super resolution technology, glimmering by positioning
Radiant position obtains multiple image reconstruct acquisition super resolution image.The technology small, wide field resolution capability with excitation intensity
Height has many advantages, such as polychrome and 3D imaging capability, but due to needing multiframe reconstruct imaging, image taking speed is slower.
Surface enhanced Raman spectroscopy (SRES) technology is a kind of novel biomarker means, and there is optical signal to be not easy to float
Biological sample that is white, being applicable in label damages the features such as small extensively, for sample.Have simultaneously to the Raman signal of script sample scattering
Greatly enhancing, so that there is high sensitivity for the spectral detection based on SERS effect.SERS technology can be with
The chemical component information in cell and biological tissue, and the inherent advantage with unmarked imaging are obtained by Raman spectrum,
It is used widely in field of biomedicine.
Willets in 2010 et al. first combines super-resolution microtechnic with SERS technology, studies unimolecule SERS
Super-resolution reconstruct imaging, positions the mass center of unimolecule and nano particle by two-dimensional Gauss curve fitting respectively, obtain unimolecule and
The spatial positional information of nano particle, resolution ratio can reach 10nm.With unimolecule positioning super resolution technology and SERS technology
In conjunction with researcher starts with super resolution technology also to obtain the SERS signal of subcellular or tissue, realizes that unmarked Raman is super
Resolution imaging.But since the flicker behavior of the SERS usual period is a few tens of milliseconds, the general frame image that acquires needs 100ms, ties
The method for closing multiple frame acquisitions reconstruct, obtains a super-resolution picture and usually requires dozens of minutes, if for polymolecular or tissue
The imaging of cell then needs several hours, this can have a great impact to some active somatic cell tissues are studied.And lighting method
Static hot spot is generated using plasmon static illumination, and super-resolution Raman image ten, which is divided into, to be realized to some unimolecules
Function can stay the imaging of polymolecular and cell tissue due to that can not utilize the flicker behavior of molecule so as to cause imaging results
It is white, also have powerful connections SERS signal interference the problems such as.
Summary of the invention
In order to solve above-mentioned and other potential technical problems, the embodiment provides a kind of drawings of super-resolution
Graceful spectrum imaging system, the super-resolution Raman spectrum imaging system includes: excitation light source module, for generating excitation light source；
Polarization Modulation module, for modulating the polarization direction of the excitation light source；Vibration mirror scanning module completes the excitation for scanning
Different location of the light source in sample imaging area focuses；Microscopic system module, for focusing the exciting light by sample imaging area
Source simultaneously excites generation Raman signal, wherein the sample imaging area includes Surface enhanced Raman spectroscopy substrate and increases positioned at surface
Test sample on strong Raman spectrum substrate；Super-resolution imaging module, for generating test sample according to the Raman signal
Super resolution image；Raman spectrum analysis module, for generating Raman spectrum and analysis detection sample according to the Raman signal.
In one embodiment of the invention, the excitation light source module includes: laser, generates the excitation light source；Light
Fine collimator is connected by optical fiber with laser, is output to after collimating to the excitation light source of laser output described
Polarization Modulation module.
In one embodiment of the invention, the Polarization Modulation module includes: polarizing film, and it is defeated to receive the optical fiber collimator
Excitation light source out；Polarization Controller controls the rotation angle and rotation speed of laser polarization in the polarizing film.
In one embodiment of the invention, the vibration mirror scanning module includes: laser galvanometer, receive through the polarizing film into
Excitation light source after row Polarization Modulation, scanning are completed the excitation light source and are focused in the different location of sample imaging area；Galvanometer control
Device processed controls the scanning angle range and angulation change rate of the laser galvanometer.
In one embodiment of the invention, to a kind of mode of the control of the polarizing film and the laser galvanometer are as follows: protect
It is constant to hold the laser galvanometer angle, constantly changes the polarization angle of the polarizing film, after polarizing film vibration rotates a circle
Change the laser galvanometer angle, rotate the polarizing film again one week, repeat the above process until complete to the sample at
As the scanning in region；To the another way of the control of the polarizing film and the laser galvanometer are as follows: keep the polarizing film angle
It spends constant, controls the scanning of the complete paired samples imaging region of the laser galvanometer, scanning completes back spin and turns the polarizing film, again
Scanning of the laser galvanometer to sample imaging region is controlled, repeats the above process and is tied after the polarizing film rotates a circle
Beam.
In one embodiment of the invention, the super-resolution Raman spectrum imaging system further include: excitation optical coupler module,
Between the laser galvanometer and the microscopic system module, couple the excitation light source that the laser galvanometer exports to described
Microscopic system module；The microscopic system module includes: object lens, and the excitation light source of input focuses on the sample through the object lens
Imaging area, to excite generation Raman signal；Spectral module enters described super all the way for the Raman signal to be divided into two-way
Resolution imaging module, another way enter the Raman spectrum analysis module.
In one embodiment of the invention, the excitation optical coupler module includes reflecting mirror, dichroscope, beam splitter or turns
One or more combinations in mirror.
In one embodiment of the invention, the super-resolution imaging module include: the Raman signal filter plate set gradually,
Band pass filter, imaging len and detector array；Wherein, the detector array records what the test sample shone in real time
State is carried out multiple frame acquisitions to the test sample same position and is carried out using the flicker effect that Raman signal generates to image
The positioning reconstruct of Raman hot spot, generates super resolution image.
In one embodiment of the invention, the detector array be ccd array detector, EMCCD detector array or
CMOS array detector.
In one embodiment of the invention, the Raman spectrum analysis module includes: Raman signal filter plate, fiber coupling
Device and the spectrometer for generating the Raman spectrum super resolution image.
In one embodiment of the invention, the Surface enhanced Raman spectroscopy substrate is by the nanometer with polarization independent characteristic
Particle dimer, nano wire and nanoparticle system, nano-grain array-nano wire system, nanocube or have receive
One or more mixed systems of rice cube core-shell structure system.
The embodiment of the present invention also provides a kind of super-resolution Raman spectrum imaging method, the super-resolution Raman spectrum imaging
Method includes: the polarization direction for generating excitation light source and modulating the excitation light source；By laser galvanometer by the excitation light source
Sample imaging area is focused on, and Raman signal is generated by the excitation of sample imaging area；Wherein, the sample imaging area includes surface
Enhance Raman spectrum substrate and the test sample on Surface enhanced Raman spectroscopy substrate；Constantly adjust the laser excitation light
The different excitation positions of the sample imaging area are completed in the polarization direction in source or the scanning direction of the adjustment laser galvanometer, control
Generate Raman signal；The super resolution image and Raman spectrum super-resolution figure of test sample are generated according to the Raman signal respectively
Picture.
In one embodiment of the invention, a kind of process for obtaining the super resolution image includes: to keep described to swash
Light galvanometer angle is constant, constantly adjusts the polarization direction of the laser excitation light source, obtains sample under each polarization direction respectively
The hotspot location information image for the Raman signal that imaging area generates, and the hotspot location information figure that will be obtained under each polarization direction
As being overlapped, the super resolution image of test sample is formed；The laser galvanometer angle is adjusted to change the excitation light source and swash
Position of the Raman signal in test sample is sent out, and is repeated the above process, the more of different location on the sample imaging area are obtained
The super resolution image of a test sample.
In one embodiment of the invention, the hotspot location in hotspot location information image is obtained by fitting localization method
Information；Wherein, the localization method is Gauss Distribution Fitting reconstructing method, more hot spot super-Gaussian fitting reconfiguration methods or compression sense
Primary data reconstructing method.
In one embodiment of the invention, a kind of process for obtaining the Raman spectrum super resolution image includes: to keep institute
The polarization direction for stating excitation light source is constant, constantly adjusts the focal position of the laser galvanometer, and adjusting the laser every time
When the focal position of galvanometer acquisition be excited position hotspot location information and Raman spectrum corresponding with the hotspot location information
Information；Raman light is constructed by the hotspot location information of acquisition and Raman spectral information corresponding with the hotspot location information
Compose image；The polarization direction for changing the excitation light source, repeating the above process terminates after laser polarization rotates a circle, and obtains
Take multiple Raman spectrum imaging figures under different polarization direction；The Raman spectrum imaging figure reconstruct that will acquire obtains the Raman
Spectrum super resolution image.
In one embodiment of the invention, a kind of mode of the Raman spectrum imaging figure are as follows: extract Same Wavelength Raman
Peak intensity is overlapped with hotspot location information of the excitation light source Raman emission in test sample, obtains the wavelength
Under be excited the Raman spectrum imaging figure of position；Wherein, the Raman spectrum imaging figure is excitation light source in X, Y-direction dimension
Location information of the Raman emission in test sample, the gray value of image are the strength information of the Raman peaks under the wavelength.
In one embodiment of the invention, a kind of mode of the Raman spectrum super resolution image is obtained are as follows: by same wave
Raman spectrum imaging figure reconstruct under long, all polarization directions obtains the Raman spectrum super resolution image；Described in acquisition
Raman spectrum super resolution image is the location information of Raman signal in the dimension of the direction x, y, the gray scale of image indicate different polarization,
The intensity integral of Raman peaks under a certain wavelength condition.
In one embodiment of the invention, modulated by liquid crystal polarization rotator or 1/2 wave plate polarization rotator described sharp
The polarization direction of light emitting source.
As described above, super-resolution Raman spectrum imaging system and method for the invention has the advantages that
The present invention is based on Polarization Modulations and Raman hot spot positioning super resolution technology to modulate excitation light polarization by active to adjust
The blinking characteristic of SERS is saved, control SERS flashing rate is simultaneously combined with efficient quickly reconfiguration technique, can not only extended
The use scope of super-resolution imaging sample, additionally it is possible to effective acquisition for biological sample and chemical sample unmarked super-resolution at
The super-resolution imaging of picture and Raman spectrum largely increases the information content of sample Raman spectrum, effectively the solution prior art
The problems such as scope of application is small in middle Raman super-resolution imaging, imaging time is long, SERS flicker behavior is uncontrollable, effectively improve for
The analytic ability of sample composition, the influence for research polarization for sample Raman spectrum play an important role.
Detailed description of the invention
To describe the technical solutions in the embodiments of the present invention more clearly, make required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings other
Attached drawing.
Fig. 1 is shown as the functional block diagram of super-resolution Raman spectrum imaging system of the invention.
Fig. 2 is shown as the concrete principle structural schematic diagram of super-resolution Raman spectrum imaging system of the invention.
Fig. 3 is shown as Polarization Modulation Surface enhanced Raman spectroscopy oversubscription in super-resolution Raman spectrum imaging system of the invention
Distinguish imaging process schematic diagram.
Fig. 4 is shown as Raman spectrum super-resolution imaging process signal in super-resolution Raman spectrum imaging system of the invention
Figure.
Fig. 5 is shown as the overall flow schematic diagram of super-resolution Raman spectrum imaging method of the invention.
Component label instructions
100 super-resolution Raman spectrum imaging systems
110 excitation light source modules
111 Raman excitation lasers
112 optical fiber collimators
120 Polarization Modulation modules
121 polarizing films
122 Polarization Controllers
130 vibration mirror scanning modules
131 laser galvanometers
132 galvanometer controllers
140 microscopic system modules
141 Surface enhanced Raman spectroscopy substrates
142 test samples
143 object lens
144 spectral modules
150 super-resolution imaging modules
151 Raman signal filter plates
152 band pass filters
153 imaging lens
154 detector arrays
160 Raman spectrum analysis modules
161 spectrometers
162 Raman signal filter plates
163 fiber couplers
170 excitation optical coupler modules
171 reflecting mirrors
172 dichroscopes
173 beam splitters
S110~S140 step
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.It should be noted that in the absence of conflict, following embodiment and implementation
Feature in example can be combined with each other.
Fig. 1 is please referred to Fig. 5.It should be clear that this specification structure depicted in this specification institute accompanying drawings, ratio, size etc., only to
Cooperate the revealed content of specification, so that those skilled in the art understands and reads, being not intended to limit the invention can be real
The qualifications applied, therefore do not have technical essential meaning, the tune of the modification of any structure, the change of proportionate relationship or size
It is whole, in the case where not influencing the effect of present invention can be generated and the purpose that can reach, it should all still fall in disclosed skill
Art content obtains in the range of capable of covering.Meanwhile in this specification it is cited as "upper", "lower", "left", "right", " centre " and
The term of " one " etc. is merely convenient to being illustrated for narration, rather than to limit the scope of the invention, relativeness
It is altered or modified, under the content of no substantial changes in technology, when being also considered as the enforceable scope of the present invention.
For unimolecule location technology for the scope of application to be small, imaging time is long in Raman super-resolution imaging, SERS flashing
The problems such as behavior is uncontrollable is used the purpose of the present embodiment is that providing a kind of super-resolution Raman spectrum imaging system 100 and method
In the super-resolution imaging of the unmarked super-resolution imaging and Raman spectrum of biological sample and chemical sample, effectively the solution prior art
The problems such as scope of application is small in middle Raman super-resolution imaging, imaging time is long, SERS flicker behavior is uncontrollable.
The present embodiment is a kind of surface plasmons Raman enhancing super-resolution Raman spectrum imaging based on Polarization Modulation
System 100 and method, be using surface plasmons Raman enhancement effect a kind of unmarked super-resolution imaging system and
Raman spectrum imaging method based on the system.The super-resolution Raman spectrum imaging system 100 of the present embodiment includes excitation light source
Module 110, Polarization Modulation module 120, vibration mirror scanning module 130, microscopic system module 140, super-resolution imaging module 150 and drawing
Graceful spectral analysis module 160.The imaging method of super-resolution Raman spectrum imaging system 100 based on the present embodiment is to pass through tune
The polarization of exciting light processed makes the surface Raman enhancement hot spot with random excitation polarization independent, generates sample Raman signal inclined
The scintillation effect that vibration relies on carries out random optical reconstruct super-resolution imaging using the flashing Raman signal of acquisition and Raman spectrum is super
Resolution imaging.
The super-resolution Raman spectrum imaging system 100 of the present embodiment described in detail below and principle and the embodiment party of method
Formula makes those skilled in the art not need the super-resolution Raman spectrum imaging system that creative work is appreciated that the present embodiment
100 and method.
The embodiment provides a kind of super-resolution Raman spectrum imaging system 100, the super-resolution Raman spectrums
Imaging system 100 includes: excitation light source module 110, Polarization Modulation module 120, vibration mirror scanning module 130, microscopic system module
140, excite optical coupler module 170, super-resolution imaging module 150 and Raman spectrum analysis module 160.
The super-resolution Raman spectrum imaging system 100 of the present embodiment is specifically described in detail below.
In this present embodiment, the excitation light source module 110 is for generating excitation light source.
Specifically, in this present embodiment, as shown in Fig. 2, the excitation light source module 110 includes: Raman excitation laser
111, generate the excitation light source；Optical fiber collimator 112 is connected, to described with the Raman excitation laser 111 by optical fiber
The excitation light source that Raman excitation laser 111 exports is output to the Polarization Modulation module 120 after being collimated.
The optical maser wavelength that Raman excitation laser 111 exports includes but is not limited to common Raman excitation wavelength:
405nm, 488nm, 532nm, 632nm, 785nm etc., optical fiber output bore meet cofocus scanning micro-imaging requirement, make system
With confocal microscopic imaging ability, Raman excitation laser 111 is collimated by optical fiber output laser by optical fiber collimator 112
Enter Polarization Modulation module 120 afterwards.
In this present embodiment, the Polarization Modulation module 120 is polarizer, for modulating the polarization side of the excitation light source
To.
Specifically, the polarization that the Polarization Modulation module 120 makes incident linearly polarized laser generate zero to 2 π rotates.
Specifically, in this present embodiment, it as shown in Fig. 2, the Polarization Modulation module 120 includes: polarizing film 121, receives
The excitation light source that the optical fiber collimator 112 exports；Polarization Controller 122 controls the rotation of laser polarization in the polarizing film 121
Gyration and rotation speed.
The rotation angle and rotation speed of laser polarization by it is specific in Polarization Controller 122 and Polarization Controller 122 when
The control of sequence control program.Wherein, the device for providing polarization is polarized including but not limited to liquid crystal polarization rotator and 1/2 wave plate
Rotator.
In this present embodiment, the vibration mirror scanning module 130 completes the excitation light source in sample imaging area for scanning
Different location focus.
Specifically, in this present embodiment, it as shown in Fig. 2, the vibration mirror scanning module 130 includes: laser galvanometer 131, connects
The excitation light source after the polarizing film 121 carries out Polarization Modulation is received, scanning completes the excitation light source in sample imaging area
Different location focuses；Galvanometer controller 132 controls the scanning angle range and angulation change rate of the laser galvanometer 131.
It is realized by laser galvanometer 131 and the fast two-dimensional of sample imaging area in confocal microscope system module 140 is scanned, swashed
The scanning angle range and angulation change rate of light galvanometer 131 are simultaneously by galvanometer controller 132 and galvanometer controller 132
The control of timing control program.
Specifically, in this present embodiment, to a kind of mode of the control of the polarizing film 121 and the laser galvanometer 131
Are as follows: it keeps 131 angle of laser galvanometer constant, constantly changes the polarization angle of the polarizing film 121, when the polarizing film
121 vibrations change 131 angle of laser galvanometer after rotating a circle, and rotate the polarizing film again 121 1 weeks, repeat above-mentioned mistake
Journey is until completing the scanning to the sample imaging region.
To the another way of the control of the polarizing film 121 and the laser galvanometer 131 are as follows: keep the polarizing film
121 angles are constant, control the scanning of the complete paired samples imaging region of the laser galvanometer 131, and scanning completes back spin and turns described inclined
Shake piece 121, then scanning of the laser galvanometer 131 to sample imaging region described in secondary control, repeats the above process until the polarization
Piece 121 terminates after rotating a circle.
In this present embodiment, the excitation optical coupler module 170 is installed in the laser galvanometer 131 and the microscopic system
Between module 140, the excitation light source that the laser galvanometer 131 exports is exported to the microscopic system module 140.
Specifically, in this present embodiment, as shown in Fig. 2, the excitation optical coupler module 170 include reflecting mirror 171, two to
One or more combinations in Look mirror 172, beam splitter 173 or tilting mirror.
In this present embodiment, the microscopic system module 140 is used to focus the excitation light source simultaneously by sample imaging area
Excitation generates Raman signal, wherein as shown in Fig. 2, the sample imaging area includes Surface enhanced Raman spectroscopy substrate 141 and position
In the test sample 142 on Surface enhanced Raman spectroscopy substrate 141.
Specifically, in this present embodiment, the Surface enhanced Raman spectroscopy substrate 141 is by receiving with polarization independent characteristic
Rice grain dimer, nano wire and nanoparticle system, nano-grain array-nano wire system, nanocube have
One or more mixed systems of nanocube core-shell structure system.
Enter object lens after laser galvanometer 131, plane mirror and dichroscope 172 by the excitation light source of Polarization Modulation
143, it focuses in the test sample 142 on SERS substrate.Wherein, test sample 142 can be by the shapes such as suspending, adsorbing
Formula is fixed on Surface enhanced Raman spectroscopy substrate 141 (SERS substrate), between the excitation light source after focusing and test sample 142
Inelastic Raman scattering occurs, generates Raman signal.
Polarization side of the hot spot only with excitation light source since excitation light source has the polarization of a direction, in SERS substrate
It can be only achieved maximum Raman reinforcing effect to consistent polarization polarization plasmon modes, enable the Raman signal on SERS substrate
The regulation of the polarizability of hard to bear exciting light.The Raman signal that sample molecule near the hot spot being excited generates is strengthened, and
Raman signal near unawakened hot spot is not reinforced.Fig. 3 is illustrated by taking silver nano-grain dimer as an example: dimer
Polarization direction it is related with the long axis direction of dimer, a kind of form of random arrangement is presented on substrate.As exciting light such as Fig. 3
Middle long arrow form polarization.It can be seen that the consistent hot spot in the polarization direction of only long axis direction and exciting light is excited.In turn
Surface-enhanced raman scattering enhancement effect is generated to reinforce the Raman signal in sample signal.And unawakened dimer can not be to sample
Raman signal in product is reinforced.
Specifically, as shown in Fig. 2, the microscopic system module 140 includes: object lens 143, described in the excitation light source warp of input
Object lens 143 focus on the sample imaging area, to excite generation Raman signal；Spectral module 144 is used for the Raman signal
It is divided into two-way, enters the super-resolution imaging module 150 all the way, another way enters the Raman spectrum analysis module 160., its
In, the spectral module 144 is electronic tilting mirror or beam splitter.
In this present embodiment, the super-resolution imaging module 150 is used to generate test sample 142 according to the Raman signal
Super resolution image.
Specifically, in this present embodiment, as shown in Fig. 2, the super-resolution imaging module 150 includes: the drawing set gradually
Graceful signal filter plate 151, band pass filter 152, imaging len 153 and detector array 154；Wherein, the detector array
154 record the luminous state of the test sample 142 in real time, carry out multiple frame acquisitions simultaneously to 142 same position of test sample
The flicker effect generated using Raman signal carries out the positioning reconstruct of Raman hot spot to image, generates super resolution image.
Specifically, in this present embodiment, the detector array 154 is ccd array detector, EMCCD detector array
Or CMOS array detector.
Raman signal after being reinforced enters CCD/EMCCD/CMOS detector array 154 all the way and is imaged.Due to light
It is that a kind of electromagnetic wave has the characteristics that diffraction, the Raman letter that we are collected into CCD/EMCCD/CMOS detector array 154
It number is the diffraction pattern after the point spread function that is determined by imaging system is modulated.Diffraction pattern light intensity substantially has Gaussian Profile
The characteristics of, the nano-precision hotspot location information of Raman hot spot can be found by the method for fitting positioning.In this system
The localization method of use includes but is not limited to Gauss Distribution Fitting reconstructing method, more hot spot super-Gaussian fitting reconfiguration methods and pressure
Contracting perception data reconstructing method.
It keeps galvanometer constant, changes light polarization modulator in real time, the continuous polarization direction for changing exciting light makes polarization side
It can be excited to the hot spot of random distribution as the change of the polarization direction of laser is random.Change the polarization of exciting light every time
Direction, CCD/EMCCD/CMOS detector array 154 carries out every image frame grabber, positioning obtains the image of hotspot location information.
All image superpositions that this process is obtained realize super-resolution imaging.Fig. 3 is right by taking the substrate of nano particle dimer as an example
The process is described in detail: the polarization direction angle of exciting light is 0 ° in Fig. 3 A, some nano particle shown in Fig. 3 A
Dimer (black dimer particle in Fig. 3 A) Raman enhancement effect reaches most strong, and the Raman signal of enhancing is by detector array
After 154 acquisitions, diffraction pattern image is obtained.By above-mentioned localization method, and then obtain the location information of Raman signal.Continuously
Change the polarization direction angle of exciting light, (Fig. 3 B=30 °, Fig. 3 C=45 °, Fig. 3 D=90 °) obtains the Raman signal of different location
The super resolution image (Fig. 3 E) that sample can be obtained is reconstructed in all images of acquisition by information.Constantly regulate laser galvanometer
131 change exciting lights focus on the position on sample, obtain the super resolution image of the detection sample of a number of other positions.
In this present embodiment, the Raman spectrum analysis module 160 is used to generate Raman spectrum according to the Raman signal
And analysis detection sample.
Specifically, in this present embodiment, as shown in Fig. 2, the Raman spectrum analysis module 160 includes: Raman signal filter
Wave plate 162, fiber coupler 163 and the spectrometer 161 for generating the Raman spectrum super resolution image.Optical fiber bore is coupled to meet
Requirement of the cofocus scanning micro-imaging to optical signal is collected, cooperates with output optical fibre bore in excitation light source module 110, makes system
With cofocus scanning micro-imaging ability.
Raman signal enters spectrometer 161 by fiber coupling, obtain Raman spectral information under a certain angle of polarization and by
The location information of the hot spot of excitation.Raman spectrogram is in different wave length (λ1, λ2, λ3, λ4...) on can show Raman peaks.It is logical
Cross the Same Wavelength (λ for extracting different loci1) Raman peak intensity is reconstructed with Raman signal location information, it obtains at this
(λ under wavelength1) site that is excited Raman spectrum imaging figure.Similarly obtain other wavelength (λ2, λ3, λ4...) under Raman light
Compose image.
The Raman spectrum imaging figure acquisition process is described in detail by taking Fig. 4 as an example.
When the angle of polarization of exciting light is 0 ° (the black double arrows direction of Fig. 3 A), swashed at this time by the acquisition of spectrometer 161
Shine the location information (Fig. 4 A) and Raman spectrum (Fig. 4 A focused5).Raman spectrum on these positions is respectively in λ1, λ2, λ3, λ4
The upper Raman peaks with varying strength, by λ1The light source position of Raman peak intensity and Raman signal under wavelength is reconstructed such as Fig. 4 A1
Raman spectrum imaging figure.Fig. 4 A1The location information of Raman signal, the gray value of image represent in the dimension of the direction x, y
The angle of polarization is 0 °, λ1Wavelength condition under Raman peaks strength information (Fig. 4 A1In black point indicate this position have draw
Man Feng).It similarly can get λ2, λ3, λ4Raman spectrum imaging figure (Fig. 4 A under wavelength2, Fig. 4 A3, Fig. 4 A4).Rotatory polarization piece 121,
The polarization direction for constantly changing exciting light, repeats the above process, and obtains the Raman spectrum imaging figure under different polarization angle.Such as Fig. 4 B
Shown, when direction of polarized light changes θ, Raman spectrum changes.The Raman spectrum imaging figure of reconstruct such as Fig. 4 B1, Fig. 4 B2, figure
4B3, Fig. 4 B4It is shown.
The Raman spectrum imaging figure that will acquire, which is reconstructed, obtains Raman spectrum super resolution image.Reconstructing method: will be same
The Raman spectrum imaging figure superposition of wavelength, different polarization angle.Fig. 4 C is λ1The Raman spectrum super resolution image of condition, the figure be by
Different polarization angle (θ1, θ2, θ3...), Same Wavelength (λ1) Raman spectrum distribution map (Fig. 4 A1, Fig. 4 B1...) reconstruct acquisition
's.The Fig. 4 is the location information of Raman signal in the dimension of the direction x, y, and the gray scale of image represents different polarization, λ1Wavelength item
The intensity (indicating different Raman peak intensities with different filling patterns in Fig. 4 C, Fig. 4 D) of Raman peaks under part.
The course of work of super-resolution Raman spectrum imaging system 100 in the present embodiment is as follows:
111 optical fiber output laser of Raman excitation laser forms directional light by the collimation of optical fiber collimator 112 and enters by soft
The polarizing film 121 of the continuous rotation of part control generates the polarised light that polarization direction continuously changes.Subsequent polarised light shakes by laser
Enter object lens 143 after mirror 131 and reflecting mirror 171 to focus on SERS substrate, excitation-detection sample emission Raman signal.Raman
Signal is divided after the collection of object lens 143 by electronic tilting mirror or beam splitter 173 for two-way: the first via enters CCD/EMCCD/CMOS times
Row detector 154 is imaged, and the super-resolution imaging method of use is Raman hot spot positioning reconstruct super-resolution imaging method.It is another
Road enters spectrometer 161 and carries out Raman spectrum super-resolution imaging.
As shown in figure 5, the present embodiment also provides a kind of super-resolution Raman spectrum imaging method, the super-resolution Raman spectrum
Imaging method includes:
Step S110 generates excitation light source and modulates the polarization direction of the excitation light source；
The excitation light source is focused on sample imaging area by laser galvanometer 131, and is imaged by sample by step S120
Area's excitation generates Raman signal；Wherein, the sample imaging area includes Surface enhanced Raman spectroscopy substrate 141 and increases positioned at surface
Test sample 142 on strong Raman spectrum substrate 141；
Step S130 constantly adjusts the polarization direction of the laser excitation light source or adjusts sweeping for the laser galvanometer 131
Direction is retouched, the different excitation positions that the sample imaging area is completed in control generate Raman signal；
Step S140 generates the super resolution image of test sample 142 according to the Raman signal respectively and Raman spectrum surpasses
Resolution image.
In this present embodiment, a kind of process for obtaining the super resolution image includes: to keep the laser galvanometer 131
Angle is constant, constantly adjusts the polarization direction of the laser excitation light source, obtains sample imaging area under each polarization direction respectively
The hotspot location information image of the Raman signal of generation, and the hotspot location information image obtained under each polarization direction is carried out
Superposition forms the super resolution image of test sample 142；131 angle of laser galvanometer is adjusted to change the excitation light source and swash
Position of the Raman signal in test sample 142 is sent out, and is repeated the above process, different location on the sample imaging area is obtained
The super resolution image of multiple test samples 142.
Change the polarization characteristic of excitation light source by adjusting polarization rotator.Excite the enhancing of SERS substrate plasma excimer
With the excitation consistent hot spot of light polarization direction in mode, maximum Raman enhancement effect is generated.Raman signal is by detector array 154
Acquisition, obtains random hotspot location information.Constantly change the polarization direction of exciting light, control CCD/EMCCD/CMOS array is visited
The acquisition rate of device 154 is surveyed, a series of random hotspot location informations are obtained.To it is collected as carry out the positioning of Raman hot spot,
Reconstruct obtains the super resolution image of test sample 142.
Specifically, in this present embodiment, the complete procedure for generating the super resolution image of test sample 142 is as follows:
1) imageable target is transferred on the Surface enhanced Raman spectroscopy with strong polarization dependence (SERS) substrate.Partially
Vibration relies on SERS substrate can be by having metal dimer, polymer, cube, the nucleocapsid knot of polarization polarized plasma mode
One or more of structure, nano particle-nanowire structure mix random distribution composition.
2) after the exciting light generated by excitation light source module 110 generates directional light after optical fiber and optical fiber collimator 112
Into Polarization Modulation module 120.In Polarization Modulation module 120,121 direction of rotation of polarizing film and rotation speed can be controlled
Device is adjusted.Rotation mode is polarized including but not limited to liquid crystal polarization rotator and 1/2 wave plate polarization rotator.Keep laser vibration
Mirror 131 is motionless, rotatable polarizer, modulates the polarization direction of excitation light source, controls the polarization angle theta of exciting light, to excite and swash
Polarized plasmon modes is polarized in the consistent SERS substrate in light emitting source polarization direction (definition is as shown in Figure 3).
3) exciting light after Polarization Modulation enters object lens by laser galvanometer 131, reflecting mirror 171 and dichroscope 172
143, focus on the sample on SERS substrate.Test sample 142 can be fixed on SERS substrate by the forms such as suspending, adsorbing
On.Inelastic Raman scattering occurs between exciting light and sample analytes after focusing, generates Raman signal.Due to SERS substrate
With very strong polarization dependence, only can be only achieved with the consistent polarization polarization plasmon modes in the polarization direction of excitation light source
Maximum Raman reinforcing effect.The detection target in these sites issues detectable Raman signal, and remaining polarization is polarized etc.
Ion mode site does not have reinforcing effect.Realize SERS hot spot light and shade flicker effect in different loci.
Since exciting light has the polarization of a direction, the hot spot in SERS substrate only with the polarization direction of excitation light source
Consistent polarization polarization plasmon modes can be only achieved maximum Raman reinforcing effect, enable the Raman signal on SERS substrate
The regulation of the polarizability of stimulated luminescence.The Raman signal that sample molecule near the hot spot being excited generates is strengthened, and not
The Raman signal near hot spot being excited is not reinforced.Fig. 3 is illustrated by taking silver nano-grain dimer as an example: dimer
Polarization direction is related with the long axis direction of dimer, and a kind of form of random arrangement is presented on substrate.When in exciting light such as Fig. 3
Long arrow form polarization.It can be seen that the consistent hot spot in the polarization direction of only long axis direction and exciting light is excited.And then it produces
Surface-enhanced raman scattering enhancement effect is given birth to reinforce the Raman signal in sample signal.And unawakened dimer can not be to sample
In Raman signal reinforced.
4) polarization direction that rotatable polarizer continuously changes laser is adjusted, thus Stochastic Modulation different location not same polarization side
It shines to the Raman of hot spot, generates the flicker effect of SERS detection target.
In this present embodiment, the excitation light source is modulated by liquid crystal polarization rotator or 1/2 wave plate polarization rotator
Polarization direction.
5) SERS signal of imageable target enters CCD/EMCCD/CMOS detector array 154 after entering object lens 143,
The state that record sample shines in real time of CCD/EMCCD/CMOS detector array 154, carries out multiple frame acquisitions to sample same position.
The positioning reconstruct of Raman hot spot is carried out to image using the flicker effect of generation, generates super resolution image.
Specifically, the Raman signal after being reinforced enters CCD/EMCCD/CMOS detector array 154 all the way and is imaged.
Since light is that a kind of electromagnetic wave has the characteristics that diffraction, what we were collected into CCD/EMCCD/CMOS detector array 154
Raman signal is the diffraction pattern after the point spread function that is determined by imaging system is modulated.Diffraction pattern light intensity substantially has height
The characteristics of this distribution, can find the nano-precision location information of Raman hot spot by the method for fitting positioning.It is at this
System use localization method include but is not limited to Gauss Distribution Fitting reconstructing method, more hot spot super-Gaussian fitting reconfiguration methods and
Compressed sensing data reconstruction method.
It keeps galvanometer constant, changes light polarization modulator in real time, the continuous polarization direction for changing exciting light makes polarization side
It can be excited to the hot spot of random distribution as the change of the polarization direction of laser is random.Change the polarization of exciting light every time
Direction, CCD/EMCCD/CMOS detector array 154 carries out every image frame grabber, positioning obtains the image of hotspot location information.
All image superpositions that this process is obtained realize super-resolution imaging.Fig. 3 is right by taking the substrate of nano particle dimer as an example
The process is described in detail: the polarization direction angle θ of exciting light is 0 ° in Fig. 3 A, some nano particle shown in Fig. 3
Dimer (black dimer particle in Fig. 3 A) Raman enhancement effect reaches most strong, and the Raman signal of enhancing is by CCD/EMCCD/
After CMOS array detector 154 acquires, diffraction pattern image is obtained.By the localization method described in step 5), and then drawn
The location information of graceful signal.The continuous polarization direction angle θ, (Fig. 3 B θ for changing exciting light1=30 °, Fig. 3 C θ2=45 °, Fig. 3 D θ3
=90 °) obtain different location Raman signal information, all images of acquisition are reconstructed to the super-resolution that sample can be obtained
Image (Fig. 3 E).
6) laser galvanometer 131 is adjusted, i.e. adjusting laser galvanometer 131 change exciting light focuses on the position on sample, changes
The focal position of exciting light repeats step 2) to the super resolution image for 5) obtaining another position.
In this present embodiment, the hotspot location information in hotspot location information image is obtained by fitting localization method；Its
In, the localization method is Gauss Distribution Fitting reconstructing method, more hot spot super-Gaussian fitting reconfiguration methods or compressed sensing data
Reconstructing method.
In this present embodiment, a kind of process for obtaining the Raman spectrum super resolution image includes: to keep the exciting light
The polarization direction in source is constant, constantly adjusts the focal position of the laser galvanometer 131, and adjusting the laser galvanometer every time
The hotspot location information and Raman spectrum corresponding with hotspot location information letter of the position that is excited are acquired when 131 focal position
Breath；Raman spectrum is constructed by the hotspot location information of acquisition and Raman spectral information corresponding with the hotspot location information
Image；The polarization direction for changing the excitation light source, repeating the above process terminates after laser polarization rotates a circle, and obtains
Multiple Raman spectrum imaging figures under different polarization direction；The Raman spectrum imaging figure reconstruct that will acquire obtains the Raman light
Compose super resolution image.
Change the polarization characteristic of excitation light source by adjusting polarization rotator.Polarised light excites SERS substrate plasma to swash
With the excitation consistent hot spot of light polarization direction in first enhancement mode, maximum Raman enhancement effect is generated.Raman signal passes through optical fiber
It is coupled to spectrometer 161, Raman signal is acquired by spectrometer 161, obtains Confocal Raman microspectroscopy.It is completed by galvanometer to sample
The confocal quick scanning of two dimension of product.During the scanning process, galvanometer changes scan position each time, and spectrometer 161 just acquires primary
Raman spectrum.Raman spectrum imaging figure is constructed by the location information of scanning and the Raman spectrum of the position.Change exciting light
Polarization direction, carry out obtaining Raman image figure with region cofocus scanning Raman spectrum imaging.The process is repeated until laser is inclined
Vibration terminates after rotating a circle.Same Wavelength Raman spectrum distribution map is reconstructed and obtains Raman spectrum super-resolution figure.
In this present embodiment, a kind of mode of the construction Raman spectrum imaging figure are as follows: extract Same Wavelength Raman peak intensity
Degree is overlapped with hotspot location information of the excitation light source Raman emission in test sample 142, obtains the wavelength
Under be excited the Raman spectrum imaging figure of position；Wherein, the Raman spectrum imaging figure is excitation light source in the dimension of the direction x, y
Location information of the Raman emission in test sample 142, the gray value of image are the strength informations of wavelength Raman peaks.
Since Raman spectrogram is in different wave length (λ1, λ2, λ3, λ4...) on can show Raman peaks.It can extract same
The location information that one wavelength period Raman peak intensity focuses on sample with laser is reconstructed, i.e., (the λ under this wavelength period1) quilt
Excite the Raman spectrum imaging figure in site.Raman spectrum imaging figure is that laser focuses on the position on sample in the dimension of the direction x, y
Confidence breath, the gray scale of image is the strength information of wavelength period Raman peaks.
In this present embodiment, obtain a kind of mode of the Raman spectrum super resolution image are as follows: by Same Wavelength, it is all partially
The Raman spectrum imaging figure reconstruct shaken under direction obtains the Raman spectrum super resolution image；It is super to obtain the Raman spectrum
Resolution image is the location information of Raman signal in the dimension of the direction x, y, and the gray scale of image is different polarization, a certain wavelength condition
The intensity of lower Raman peaks integrates.
Specifically, the process for obtaining the Raman spectrum super resolution image is as follows:
1) imageable target is transferred to the SERS substrate with polarization dependence, the SERS substrate knot with polarization dependence
Structure includes but is not limited to: nano particle dimer, nano wire and nanoparticle system, are received nano-grain array-nano wire system
Rice cube has nanocube core-shell structure system.
2) exciting light enters Polarization Modulation module 120 and generates polarization by optical fiber and the generation directional light of optical fiber collimator 112
Light.
3) polarised light focuses on SERS substrate after laser galvanometer 131, plane mirror, dichroscope 172 and object lens 143
On, controller controls 131 two-dimensional scanning of laser galvanometer, quickly scans sample confocal microscopic imaging region.
4) polarization side of the hot spot only with excitation light source since exciting light has the polarization of a direction, in SERS substrate
It can be only achieved maximum Raman reinforcing effect to the related plasmon modes of consistent polarization, the sample near the hot spot being excited produces
Raw Raman signal is strengthened, and the Raman signal near unawakened hot spot is not reinforced.
5) Raman signal enters spectrometer 161 by fiber coupling, obtain Raman spectral information under a certain angle of polarization and
The location information for the hot spot being excited.Raman spectrogram is in different wave length (λ1, λ2, λ3, λ4...) on can show Raman peaks.
By the Same Wavelength (λ for extracting different loci1) Raman peak intensity is reconstructed with Raman signal location information, it obtains at this
(λ under one wavelength1) site that is excited Raman spectrum imaging figure.Similarly obtain other wavelength (λ2, λ3, λ4...) under Raman
Light spectrum image-forming figure.The Raman spectrum imaging picture capturing method is described in detail by taking Fig. 4 as an example.When the angle of polarization of exciting light is 0 °
(the black double arrows direction of Fig. 3 A) obtains the location information (Fig. 4 A) and Raman that exciting light focuses at this time by spectrometer 161
Spectrum (Fig. 4 A5).Raman spectrum on these positions is respectively in λ1, λ2, λ3, λ4The upper Raman peaks with varying strength, by λ1Wave
The light source position of Raman peak intensity and Raman signal under long is reconstructed such as Fig. 4 A1Raman spectrum imaging figure.The figure is in x, y
The location information of Raman signal in the dimension of direction, the gray scale of image are that the angle of polarization is 0 °, λ1Wavelength condition under Raman peaks it is strong
Spend information (Fig. 4 A1In black point indicate this position have Raman peaks).It similarly can get λ2, λ3, λ4Raman light under wavelength
Compose image (Fig. 4 A2, Fig. 4 A3, Fig. 4 A4)。
6) rotatory polarization piece 121 constantly changes the polarization direction of exciting light, repeats step 5) and obtains under different polarization angle
Raman spectrum imaging figure.As shown in Figure 4 B, when direction of polarized light changes θ, Raman spectrum changes.The Raman spectrum of reconstruct at
As figure such as Fig. 4 B1, Fig. 4 B2, Fig. 4 B3, Fig. 4 B4It is shown.
7) the Raman spectrum imaging figure that will acquire, which is reconstructed, obtains Raman spectrum super resolution image.Reconstructing method: will be same
The Raman spectrum imaging figure superposition of one wavelength, different polarization angle.Fig. 4 C is λ1The Raman spectrum super resolution image of condition, the figure are
By different polarization angle (θ1, θ2, θ3...), Same Wavelength (λ1) Raman spectrum distribution map (Fig. 4 A1, Fig. 4 B1...) reconstruct acquisition
's.The Fig. 4 is the location information of Raman signal in the dimension of the direction x, y, and the gray scale expression of image is different polarization, λ1Wavelength
Under the conditions of Raman peaks intensity integrate (indicating different Raman peak intensities with different filling patterns in Fig. 4 C, Fig. 4 D).
In conclusion the present invention is based on Polarization Modulations and Raman hot spot positioning super resolution technology by actively modulating exciting light
It polarizes to adjust the blinking characteristic of SERS, control SERS flashing rate simultaneously combines with efficient quickly reconfiguration technique, not only exists
The use scope of super-resolution imaging sample can be extended, additionally it is possible to which effective acquisition is unmarked for biological sample and chemical sample
The super-resolution imaging of super-resolution imaging and Raman spectrum largely increases the information content of sample Raman spectrum, effectively solves
The problems such as scope of application is small in Raman super-resolution imaging in the prior art, imaging time is long, SERS flicker behavior is uncontrollable, effectively
The analytic ability for sample composition is improved, the influence for research polarization for sample Raman spectrum plays an important role.So this
Invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, includes that institute is complete without departing from the spirit and technical ideas disclosed in the present invention for usual skill in technical field such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (18)
1. a kind of super-resolution Raman spectrum imaging system, which is characterized in that the super-resolution Raman spectrum imaging system includes:
Excitation light source module, for generating excitation light source；
Polarization Modulation module, for modulating the polarization direction of the excitation light source；
Vibration mirror scanning module completes the excitation light source in the different location focusing of sample imaging area for scanning；
Microscopic system module, for focusing the excitation light source by sample imaging area and exciting generation Raman signal, wherein institute
Stating sample imaging area includes Surface enhanced Raman spectroscopy substrate and the test sample on Surface enhanced Raman spectroscopy substrate；
Super-resolution imaging module, for generating the super resolution image of test sample according to the Raman signal；
Raman spectrum analysis module, for generating Raman spectrum and analysis detection sample according to the Raman signal.
2. super-resolution Raman spectrum imaging system according to claim 1, which is characterized in that the excitation light source module packet
It includes:
Laser generates the excitation light source；
Optical fiber collimator is connected by optical fiber with the laser, after collimating to the excitation light source of laser output
It is output to the Polarization Modulation module.
3. super-resolution Raman spectrum imaging system according to claim 2, which is characterized in that the Polarization Modulation module packet
It includes:
Polarizing film receives the excitation light source of the optical fiber collimator output；
Polarization Controller controls the rotation angle and rotation speed of laser polarization in the polarizing film.
4. super-resolution Raman spectrum imaging system according to claim 3, which is characterized in that the vibration mirror scanning module packet
It includes:
Laser galvanometer, receives the excitation light source after the polarizing film carries out Polarization Modulation, scanning complete the excitation light source in
The different location of sample imaging area focuses；
Galvanometer controller controls the scanning angle range and angulation change rate of the laser galvanometer.
5. super-resolution Raman spectrum imaging system according to claim 4, it is characterised in that:
To a kind of mode of the control of the polarizing film and the laser galvanometer are as follows: keep the laser galvanometer angle constant, no
The disconnected polarization angle for changing the polarizing film changes the laser galvanometer angle after polarizing film vibration rotates a circle, again
It rotates the polarizing film one week, repeats the above process until completing the scanning to the sample imaging region；
To the another way of the control of the polarizing film and the laser galvanometer are as follows: keep the polarizing film angle constant, control
The scanning of the complete paired samples imaging region of the laser galvanometer is made, scanning completes back spin and turns the polarizing film, then described in secondary control
Scanning of the laser galvanometer to sample imaging region, repeating the above process terminates after the polarizing film rotates a circle.
6. super-resolution Raman spectrum imaging system according to claim 4, which is characterized in that the super-resolution Raman spectrum
Imaging system further include: excitation optical coupler module swashs between the laser galvanometer and the microscopic system module by described
The excitation of light galvanometer output is optically coupled to the microscopic system module；
The microscopic system module includes:
The excitation light source of object lens, input focuses on the sample imaging area through the object lens, to excite generation Raman signal；
Spectral module enters the super-resolution imaging module for the Raman signal to be divided into two-way all the way, and another way enters
The Raman spectrum analysis module.
7. super-resolution Raman spectrum imaging system according to claim 6, which is characterized in that the excitation optical coupler module
Including the one or more combination in reflecting mirror, dichroscope, beam splitter or tilting mirror.
8. super-resolution Raman spectrum imaging system according to claim 1, which is characterized in that the super-resolution imaging module
It include: Raman signal filter plate, band pass filter, imaging len and the detector array set gradually；Wherein, the array is visited
It surveys device and records the luminous state of the test sample in real time, multiple frame acquisitions are carried out to the test sample same position and utilize drawing
The flicker effect that graceful signal generates carries out the positioning reconstruct of Raman hot spot to image, generates super resolution image.
9. super-resolution Raman spectrum imaging system according to claim 8, which is characterized in that the detector array is
Ccd array detector, EMCCD detector array or CMOS array detector.
10. super-resolution Raman spectrum imaging system according to claim 1, which is characterized in that the Raman spectrum analysis
Module includes: Raman signal filter plate, fiber coupler and the spectrometer for generating the Raman spectrum super resolution image.
11. super-resolution Raman spectrum imaging system according to claim 1, which is characterized in that the surface-enhanced Raman
Spectrum substrate is by with nano particle dimer, nano wire and the nanoparticle system of polarization independent characteristic, nano-grain array-
Nano wire system, nanocube or one or more mixed systems with nanocube core-shell structure system.
12. a kind of super-resolution Raman spectrum imaging method, which is characterized in that the super-resolution Raman spectrum imaging method includes:
It generates excitation light source and modulates the polarization direction of the excitation light source；
The excitation light source is focused on into sample imaging area by laser galvanometer, and Raman letter is generated by the excitation of sample imaging area
Number；Wherein, the sample imaging area includes Surface enhanced Raman spectroscopy substrate and on Surface enhanced Raman spectroscopy substrate
Test sample；
It constantly adjusts the polarization direction of the laser excitation light source or institute is completed in the scanning direction of the adjustment laser galvanometer, control
The different excitation positions for stating sample imaging area generate Raman signal；
The super resolution image and Raman spectrum super resolution image of test sample are generated according to the Raman signal respectively.
13. super-resolution Raman spectrum imaging method according to claim 12, which is characterized in that obtain the super-resolution figure
A kind of process of picture includes:
It keeps the laser galvanometer angle constant, constantly adjusts the polarization direction of the laser excitation light source, obtain respectively
The hotspot location information image for the Raman signal that sample imaging area generates under each polarization direction, and will be obtained under each polarization direction
The hotspot location information image taken is overlapped, and forms the super resolution image of test sample；
The laser galvanometer angle is adjusted to change position of the excitation light source Raman emission in test sample, is laid equal stress on
The multiple above process, obtains the super resolution image of multiple test samples of different location on the sample imaging area.
14. super-resolution Raman spectrum imaging method according to claim 13, which is characterized in that by being fitted localization method
Obtain the hotspot location information in hotspot location information image；Wherein, the localization method be Gauss Distribution Fitting reconstructing method,
More hot spot super-Gaussian fitting reconfiguration methods or compressed sensing data reconstruction method.
15. super-resolution Raman spectrum imaging method according to claim 12, which is characterized in that obtain the Raman spectrum
A kind of process of super resolution image includes:
It keeps the polarization direction of the excitation light source constant, constantly adjusts the focal position of the laser galvanometer, and adjusting every time
Acquisition is excited the hotspot location information of position and corresponding with the hotspot location information when focal position of the whole laser galvanometer
Raman spectral information；
Raman light is constructed by the hotspot location information of acquisition and Raman spectral information corresponding with the hotspot location information
Compose image；
The polarization direction for changing the excitation light source, repeating the above process terminates after laser polarization rotates a circle, and obtains not
With the Raman spectrum imaging figures multiple under polarization direction；
The Raman spectrum imaging figure reconstruct that will acquire obtains the Raman spectrum super resolution image.
16. super-resolution Raman spectrum imaging method according to claim 15, which is characterized in that the Raman spectrum imaging
A kind of mode of figure are as follows: extract Same Wavelength Raman peak intensity and the excitation light source Raman emission in test sample
Hotspot location information is overlapped, and obtains the Raman spectrum imaging figure for the position that is excited under the wavelength；Wherein, the Raman spectrum
Image is location information of the excitation light source Raman emission in test sample, the gray scale of image in X, Y-direction dimension
Value is the strength information of the Raman peaks under the wavelength.
17. super-resolution Raman spectrum imaging method according to claim 15, which is characterized in that obtain the Raman spectrum
A kind of mode of super resolution image are as follows: the Raman spectrum reconstruct under Same Wavelength, all polarization directions is obtained into the drawing
Graceful spectrum super resolution image；Obtain the position letter that the Raman spectrum super resolution image is Raman signal in the dimension of the direction x, y
Breath, the gray scale of image indicate the intensity integral of Raman peaks under different polarization, a certain wavelength condition.
18. super-resolution Raman spectrum imaging method according to claim 12, which is characterized in that pass through liquid crystal polarized rotation
Device or 1/2 wave plate polarization rotator modulate the polarization direction of the excitation light source.
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