A time-space resolution multimodal Raman hyperspectral microscopy imaging system and method

Abstract

The present invention belongs to the fields of optical microscope manufacturing technology and spectral imaging technology, and particularly relates to a time-space resolved multi-modal Raman hyperspectral microscopy imaging system and method. The system comprises a light source module, a coaxial dual-objective microscope module, an SPR reflection imaging module, an image rotation module, an enhanced scattering imaging module and an image processing module. The system can provide SPR images, Rayleigh scattering spectrum images and Raman hyperspectral images of multiple target samples in a field of view in real time, reduce the incident laser power density to avoid sample light damage, reduce the time resolution of collecting Raman hyperspectral images from the second level of the prior art to the millisecond level, and significantly improve the spatial resolution.

Description

Technical Field

[0001] The present invention belongs to the field of optical microscope manufacturing technology and spectral imaging technology, and particularly relates to a time-space resolution multi-modal Raman hyperspectral microscope imaging system and method. Background Art

[0002] Due to the structural and functional heterogeneity of individual nanoparticles or multidimensional nanomaterials at different spatial locations, dynamic studies of the chemical activity of individual nanostructures have attracted increasing interest. Optical microscopy, especially surface plasmon resonance (SPR) microscopy and fluorescence microscopy, has enabled the study of the catalytic chemistry of individual nanomaterials. However, the loss of structural information of the reactant molecules limits the identification of intermediates and products in complex reaction processes (such as metal-catalyzed reactions) using these methods. Raman technology is not interfered by other chemicals and provides a convenient method to capture the fingerprint information of the reactant molecules. In particular, Raman spectral imaging achieves high spatial resolution measurement of the fingerprint information of the local area of ​​the nanostructure. Time-resolved Raman spectroscopy can be used to monitor the dynamic changes of the reactant molecules during the reaction process, attribute the source of the Raman shift peak, and clarify the changes in molecular structure, the formation of intermediates, and the reaction mechanism during the reaction. However, the classic scanning-based Raman imaging mode is time-consuming, which limits the dynamic study of multiple nanostructures at the same time. Secondly, due to the tailing of the irradiated spot, the sample may suffer irreversible damage such as photobleaching or photoreaction before being observed. Wide-field illumination Raman imaging technology provides a potential solution, which mainly realizes hyperspectral image acquisition through wavelength-tunable color filters or wavelength-tunable laser scanning. Therefore, its spectral resolution is lower than that of grating monochromators, and the wavelength scanning method will also lose the information of dynamic changes of the full spectrum. Summary of the invention

[0003] The present invention solves the above-mentioned technical problems existing in the prior art and provides a novel time-space resolution multimodal Raman hyperspectral imaging system, which can provide SPR images, Rayleigh scattering spectrum images and Raman spectrum hyperspectral images of multiple targets in a micro-area in real time.

[0004] The technical solution of the present invention is as follows:

[0005] A time-space resolution multimodal Raman hyperspectral imaging system, comprising a light source module, a coaxial dual-objective microscope module, an SPR reflection imaging module, an image rotation module, an enhanced scattering imaging module and an image processing module;

[0006] The light source module includes an incident laser light source;

[0007] The coaxial dual-objective microscope module and the sample to be tested are arranged on the output light path of the light source module;

[0008] The SPR reflection imaging module is arranged on the output optical path of the reflected light of the sample to be tested, and performs SPR reflection imaging on the reflected light of the sample to be tested;

[0009] The image rotation module and the enhanced scattering imaging module are sequentially arranged on the output optical path of the scattered light of the sample to be tested;

[0010] The image rotation module comprises a rotatable Dove prism; the central axis of the rotatable Dove prism coincides with the central axis of the scattered light output optical path;

[0011] The image processing module extracts the image obtained by the enhanced scattering imaging module, and obtains the Rayleigh scattering spectrum and Raman scattering spectrum of the sample to be tested by performing a deconvolution operation on the scattered light intensity image with spatial position information.

[0012] Preferably, in the light source module, a first convex lens, a polarizing plate, a second convex lens, and a bandpass filter are sequentially arranged on the output light path of the incident laser light source; and the emission light beam angle of the incident laser light source is adjustable.

[0013] Preferably, the coaxial dual-objective lens microscopy module comprises an inverted objective lens, a stage, and a three-dimensionally movable upright objective lens, which are sequentially arranged on the output light path of the incident angle adjustable light source module.

[0014] Preferably, the inverted objective lens is an oil objective lens, and the upright objective lens is a water immersion objective lens.

[0015] Preferably, the SPR reflection imaging module comprises a semi-transparent and semi-reflective mirror, a third convex lens, and a first camera, which are sequentially arranged on the output light path of the reflected light of the sample to be measured.

[0016] Preferably, the enhanced scattering imaging module includes a long-pass filter, a fourth convex lens, a first reflector, a slit, a second reflector, a concave mirror, a reflection grating, a concave mirror, and a second camera, which are sequentially arranged on the output light path of the scattered light of the sample to be measured.

[0017] Preferably, the emission beam of the light source module is a single-frequency laser beam or a broadband beam. More preferably, the wavelength of the single-frequency laser beam is adjustable within 500-700nm; the wavelength of the broadband laser beam is adjustable within a range of 200 to 2000nm.

[0018] Preferably, the substrate carrier of the sample to be tested is any one of a cover glass, glass, and conductive glass with a smooth and uniform surface coated with a metal nanofilm. More preferably, the metal nanofilm is a gold nanofilm or a silver nanofilm.

[0019] Preferably, the field of view and focal length of each objective lens of the coaxial dual-objective lens microscope module can be adjusted independently.

[0020] A time-space resolution multimodal Raman hyperspectral imaging method comprises the following steps:

[0021] The light source module emits an incident light beam, which enters the coaxial dual-objective microscope module and irradiates the sample to be tested at a specific angle greater than 70°, generating total internal reflection, inducing surface plasmon resonance in the metal nanofilm on the substrate carrier of the sample to be tested, and coupling the nanoparticles on the surface to enhance Raman scattering. The scattered light signal is collected by the upright objective lens in the coaxial dual-objective microscope module and enters the enhanced scattering imaging module for scattering imaging and spectral analysis;

[0022] The upright objective lens is adjusted through a three-dimensional translation stage to observe the same field of view as the inverted objective lens;

[0023] The slit of the enhanced scattering imaging module is in a fully open state, the scattered light beam of the sample to be measured is split and expanded in one dimension by the reflection grating, the central wavelength of the grating is adjusted, and the camera obtains the zero-order diffraction image of the sample nanoparticles and the first-order diffraction image that is dispersed and expanded in the horizontal direction respectively;

[0024] The image rotation module rotates and splits the overlapping first-order diffraction spectrum images to obtain a zero-order diffraction image sequence 1 and a first-order diffraction spectrum image sequence 2 of the bright spots of the nanoparticles in the field of view respectively;

[0025] The image processing module captures and cuts the zero-order diffraction image sequence 1 and the first-order diffraction spectrum image sequence 2 obtained by the enhanced scattering imaging module into a zero-order image sequence 3 and a first-order image sequence 4 of the region of interest respectively; for target samples at randomly appearing positions, the image processing module uses an algorithm to capture the horizontal spacing d of the samples in the sample image sequence 3 and the image sequence 4, and calculates the effective dispersion distance through the following relationship:

[0026] d eff =α(x)d,

[0027] Where α(x) is the spectral expansion correction coefficient of the image spacing d between the zero-order and first-order diffraction spectra of the sample nanoparticles, which is linearly related to the horizontal position of the pixel in the image sequence 2;

[0028] The spatial position of each pixel in the image sequence 4 is calculated to generate the corresponding Raman wave number array 5 through the following relationship:

[0029] v=1 / λ ex -1 / (kd eff +λ 0 )

[0030] in

[0031] ν represents the Raman shift value of each pixel in the photo in sequence 3;

[0032] d eff Represents the effective dispersion constant of each pixel in the photo in sequence 3;

[0033] k represents the wavelength resolution of the imaging module, and its value is 0.2071, and its unit is nanometers per pixel;

[0034] λ 0 Indicates setting the grating center position;

[0035] λ ex represents the incident laser wavelength;

[0036] The intensity value of each pixel in the picture sequence 4 is the Raman scattering intensity at the corresponding Raman shift wavenumber, and the Raman scattering spectrum can be obtained by plotting the Raman shift wavenumber and intensity of the pixels in the picture sequence 4 as the horizontal and vertical coordinates.

[0037] Preferably, the image processing module can capture the Raman scattering intensity of multiple sample targets for a specified specific Raman shift wave number, and generate a Raman intensity spatial distribution image at the specified Raman wave number through calculation.

[0038] Preferably, the λ 0 750nm; ex It is 660nm.

[0039] Compared with the prior art, the advantages of the present invention are as follows:

[0040] The spatiotemporal resolution multimodal Raman hyperspectral imaging system of the present invention converts the image directly obtained by the enhanced scattering imaging module in the slit-free state into a Raman hyperspectral data set through an image processing module, and can simultaneously detect the Raman scattering spectra of all target samples in the field of view, and can obtain the Rayleigh scattering spectrum of the target sample by using a broadband light source; it overcomes the lack of time or full-spectrum dynamic change information caused by the traditional Raman microscope point-by-point / line scanning or wavelength-by-wavelength scanning method;

[0041] The present invention simultaneously obtains the SPR reflection image and enhanced scattering image of the sample through a coaxial dual-objective microscope module, uses a laser light source module with an adjustable incident angle to adjust the laser incident angle, irradiates the sample with a wide-field parallel beam, and can excite the surface plasma resonance of the gold nanofilm at a specific angle, generate a great local electromagnetic field enhancement, amplify the Raman scattering signal of the organic molecules on the target sample, effectively reduce the laser power density required for Raman imaging, and avoid damage to the sample caused by high-intensity laser;

[0042] The present invention is a novel multimodal Raman microscopic spectral imaging method, which has significant improvements in function and performance compared with the current wide-field Raman microscopic imaging technology: the spectral resolution of the time-space resolution multimodal Raman hyperspectral imaging system reaches 3 wavenumbers; the time resolution of the spectrum of all particles in the acquisition field is reduced from the seconds level in the prior art to the millisecond level; the spatial resolution is significantly improved to 300nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of modules of the spatiotemporal resolution multimodal Raman hyperspectral microscopic imaging system of the present invention;

[0044] Figure 2 The schematic diagram of the structure of the time-space resolution multi-modal Raman hyperspectral microscopy imaging system of the present invention; 1 is an incident laser light source, 2 is a first convex lens, 3 is a polarizer, 4 is a second convex lens, 5 is a bandpass filter, 6 is a semi-transparent and semi-reflective mirror, 7 is a positive objective lens, 8 is a stage, 9 is a third convex lens, 10 is a first camera, 11 is a three-dimensionally movable inverted objective lens, 12 is a rotatable Dove prism, 13 is a long-pass filter, 14 is a fourth convex lens, 15 is a first reflector, 16 is a slit, 17 is a second reflector, 18 is a concave mirror, 19 is a reflection grating, 20 is a concave mirror, 21 is a second camera, and 22 is an image processing module. The incident angle adjustable light source module is composed of 1 to 5, the coaxial dual objective microscopy module is composed of 7, 8, and 11, the SPR reflection imaging module is composed of 6, 9, and 10, the image rotation module is 12, and the enhanced scattering imaging module is composed of 13 to 21;

[0045] Figure 3 a. Figure 3 b is a dual-field simultaneous imaging diagram of the same sample micro-area in Example 2 of the present invention; and the changes in the Raman scattered light intensity and the SPR reflected light intensity of the same sample micro-area obtained by scanning the laser incident angle. The obtained scattered light intensity and reflected light intensity have opposite trends with the incident angle, and obtain extreme values ​​at the same incident angle;

[0046] Figure 4 a. Figure 4 b are respectively the zero-order and first-order Raman diffraction images of gold nanoparticles at different spatial positions in the field of view of Example 2 in the embodiment of the present invention; Figure 4 c Raman spectrum of nanoparticles obtained by data conversion; Figure 4 d Rayleigh scattering spectrum of nanoparticles obtained by data conversion;

[0047] Figure 5 The present invention is an embodiment of the present invention. In embodiment 3, a 40-degree rotation is applied to avoid the overlap of the primary dispersion images of the gold nanoparticles marked by multiple dotted boxes;

[0048] Figure 6 The spatial distribution of Raman scattering intensity of the target sample at different Raman wave numbers obtained by applying the image processing module in Example 3;

[0049] Figure 7 a is a hyperspectral Raman mapping sequence of a sample micro-area at 0, 60, 560 and 3060 seconds during the reaction of Example 4 in the present invention; Figure 7 b is the scanning electron microscope image of the same area to locate the spatial distribution of nanoparticles; Figure 7 ce are the time-resolved intensity traces of the νNO(4-NBT) and ν3(DMAB) bands of particles 2 (b), 5 (c), and 6 (d) extracted from the real-time acquired hyperspectral spectra; the intensities are normalized to the initial intensity of the νNO(4-NBT) band. DETAILED DESCRIPTION

[0050] Embodiment 1:

[0051] A time-space resolution multimodal Raman hyperspectral imaging system, comprising a light source module, a coaxial dual-objective microscope module, an SPR reflection imaging module, an image rotation module, an enhanced scattering imaging module and an image processing module;

[0052] The light source module comprises an incident laser light source; a first convex lens, a polarizing plate, a second convex lens, and a bandpass filter are sequentially arranged on the output light path of the incident laser light source; the emission beam angle of the incident laser light source is adjustable (e.g., 0 degrees to 80 degrees); the emission beam of the light source module is a single-frequency laser beam or a broadband beam; the wavelength of the single-frequency laser beam is adjustable within 500-700nm; the wavelength of the broadband laser beam is adjustable in the range of 200 to 2000nm

[0053] The coaxial dual-objective microscope module and the sample to be tested are arranged on the output light path of the light source module;

[0054] The coaxial dual-objective microscope module comprises an inverted objective lens, a stage, and a three-dimensionally movable upright objective lens, which are sequentially arranged on the output light path of the incident angle adjustable light source module; the inverted objective lens is an oil objective lens, and the upright objective lens is a water immersion objective lens; the field of view and focal length of each objective lens of the coaxial dual-objective microscope module can be adjusted independently;

[0055] The SPR reflection imaging module is arranged on the output optical path of the reflected light of the sample to be tested, and performs SPR reflection imaging on the reflected light of the sample to be tested; the SPR reflection imaging module comprises a semi-transparent and semi-reflective mirror, a third convex lens, and a first camera, which are arranged in sequence on the output optical path of the reflected light of the sample to be tested;

[0056] The image rotation module and the enhanced scattering imaging module are sequentially arranged on the output optical path of the scattered light of the sample to be tested;

[0057] The image rotation module comprises a rotatable Dove prism; the central axis of the rotatable Dove prism coincides with the central axis of the scattered light output optical path;

[0058] The enhanced scattering imaging module includes a long-pass color filter, a fourth convex lens, a first reflector, a slit, a second reflector, a concave mirror, a reflection grating, a concave mirror, and a second camera, which are sequentially arranged on the output light path of the scattered light of the sample to be measured;

[0059] The image processing module extracts the image obtained by the enhanced scattering imaging module, and obtains the Rayleigh scattering spectrum and Raman scattering spectrum of the sample to be tested by performing a deconvolution operation on the scattered light intensity image with spatial position information.

[0060] The substrate carrier of the sample to be tested is any one of a cover glass, glass, and conductive glass with a smooth and uniform surface coated with a metal nanofilm. The metal nanofilm is a gold nanofilm or a silver nanofilm.

[0061] Embodiment 2:

[0062] like Figure 2 The incident angle adjustable light source module emits a single-frequency 660nm laser beam or a broadband beam in the range of 200 to 2000nm; the incident light beam passes through a high numerical aperture oil objective and irradiates a sample (such as "glass-gold nanofilm-sample") at a specific angle greater than 70°, generating total internal reflection, inducing surface plasmon resonance of the gold nanofilm, and coupling the nanoparticles on the surface to enhance Raman scattering. The scattered light signal is collected by the water immersion objective and enters the scattering imaging module for scattering imaging and spectral analysis.

[0063] The incident angle adjustable light source module is provided with a polarizing plate and a bandpass filter.

[0064] The sample substrate carrier is any one of a cover glass (gold nanofilm, silver nanofilm) coated with a metal nanofilm with a smooth and uniform surface, glass, and conductive glass ITO.

[0065] The coaxial dual objective microscope module uses an inverted oil objective lens with a numerical aperture of 1.51 and an upright water immersion objective lens with a numerical aperture of 1.0 of the same magnification. The upright objective lens is adjusted through a three-dimensional translation stage to observe the same field of view as the inverted objective lens. The observation results are as follows: Figure 3 .

[0066] The field of view and focal length of each objective lens of the coaxial dual-objective lens microscopy module can be adjusted independently.

[0067] The slit of the enhanced scattering imaging module is in a fully open state, the sample scattered light beam is split and expanded in one dimension by the reflection grating, the central wavelength of the grating is adjusted, and the camera obtains the zero-order diffraction image of the sample nanoparticles and the first-order diffraction image that is dispersed and expanded in the horizontal direction.

[0068] The image rotation module is composed of a dove prism and a rotating stage, which is installed above the upright objective lens, and its central axis coincides with the central axis of the scattered light output optical path. The image rotation module rotates and splits the overlapping first-order diffraction spectrum images to obtain a zero-order diffraction image sequence 1 and a first-order diffraction spectrum image sequence 2 of the bright spots of the nanoparticles in the field of view. The results are as follows: Figure 4 ab.

[0069] The image processing module captures and cuts the zero-order diffraction image sequence 1 and the first-order diffraction spectrum image sequence 2 obtained by the enhanced scattering imaging module into a zero-order image sequence 3 and a first-order image sequence 4 of the region of interest respectively; for target samples at randomly appearing positions, the image processing module uses an algorithm to capture the horizontal spacing d of the samples in the sample image sequence 3 and the image sequence 4, and calculates the effective dispersion distance through the following relationship:

[0070] d eff =α(x)d,

[0071] Where α(x) is the spectral expansion correction coefficient of the image spacing d between the zero-order and first-order diffraction spectra of the sample nanoparticles, which is linearly related to the horizontal position of the pixel in the image sequence 2;

[0072] The spatial position of each pixel in the image sequence 4 is calculated to generate the corresponding Raman wave number array 5 through the following relationship:

[0073] v=1 / λ ex -1 / (kd eff +λ 0 )

[0074] in

[0075] v represents the Raman shift value of each pixel in the photo of sequence 3;

[0076] d eff Represents the effective dispersion constant of each pixel in the photo in sequence 3;

[0077] k represents the wavelength resolution of the imaging module, and its value is 0.2071, and its unit is nanometers per pixel;

[0078] λ 0 Indicates setting the grating center position, preferably 750nm;

[0079] λ ex represents the incident laser wavelength, preferably 660nm;

[0080] The intensity value of each pixel in the image sequence 4 is the Raman scattering intensity at the corresponding Raman shift wavenumber. The Raman shift wavenumber and intensity of the pixels in the image sequence 4 are used as the horizontal and vertical coordinates to plot the Raman scattering spectrum. The result is as follows: Figure 4 c;

[0081] The image processing module can capture the Raman scattering intensity of multiple sample targets for a specified specific Raman shift wave number, and generate a Raman intensity spatial distribution image at the specified Raman wave number by calculation;

[0082] When the light source module uses a broadband light source in the range of 200 to 2000 nm, the Rayleigh scattering spectrum data of all target samples in the field of view can be obtained through the image processing module. The results are as follows: Figure 4 d.

[0083] Embodiment 3:

[0084] Same as Example 2, except that the image rotation module uses the Dove prism to rotate at different angles to obtain zero-order and first-order diffraction images of the target sample at different angles;

[0085] like Figure 5 In order to avoid the overlap of the horizontal dispersion spectrum images in the first-order diffraction image, which affects the signal collection flux, the Dove prism is rotated at a certain angle to obtain a first-order diffraction spectrum image that does not overlap in the horizontal direction. For the specified Raman shift wave number, the Raman scattering intensity of multiple sample targets in the field of view can be captured, and the Raman intensity spatial distribution image at the specified Raman wave number is generated through the deconvolution operation of the data processing module. The result is as follows: Figure 6 .

[0086] Embodiment 4:

[0087] A spatiotemporally resolved multimodal Raman hyperspectral microscopy system was used to study the dynamics of the gold nanoparticle-catalyzed reduction of p-nitrothiophenol molecules (4-NBT) to form p-aminothiophenol (4-ABT) and the generation of the reaction intermediate p-dimercaptoazobenzene (DMAB).

[0088] The enhanced Raman scattering spectra of organic molecules on the surface of gold nanoparticles during the reaction were dynamically detected using the spatiotemporal resolution multimodal Raman hyperspectral microscopy imaging system described in Example 3. The image processing module was used to output specific wavenumbers of 1344 cm-1, which were respectively assigned to the characteristic vibration of 4-NBT molecules. -1 and 1445cm, which is the characteristic vibration of DMAB molecule -1 The spatial distribution image of the peak intensity, the two sets of images change dynamically with the reaction time, the results Figure 7 As the reaction proceeds, the 1344cm -1The peak intensity gradually weakened, indicating that the peak at 1445 cm -1 The peak intensity fluctuates and varies between different particles, revealing the heterogeneity of the catalytic performance of the sample nanoparticles.

[0089] Based on the above experiments, the spatiotemporal resolution multimodal Raman hyperspectral microscopy imaging system of the present invention can be used to simultaneously and dynamically detect the SPR reflection images, Rayleigh scattering spectrum signals and Raman scattering spectrum signals of a large number of target nanoparticles in the microscopic field of view with high throughput.

[0090] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Equivalent replacements or substitutions made on the basis of the above embodiments all fall within the protection scope of the present invention.

Claims

1. A temporally and spatially resolved multimodal Raman hyperspectral imaging method, It is characterized in that Using a spatiotemporally resolved multimodal Raman hyperspectral imaging system; The time-space resolution multimodal Raman hyperspectral imaging system comprises a light source module, a coaxial dual-objective microscope module, an SPR reflection imaging module, an image rotation module, an enhanced scattering imaging module and an image processing module; The light source module includes an incident laser light source; The coaxial dual-objective microscope module and the sample to be tested are arranged on the output light path of the light source module; The SPR reflection imaging module is arranged on the output optical path of the reflected light of the sample to be tested, and performs SPR reflection imaging on the reflected light of the sample to be tested; The image rotation module and the enhanced scattering imaging module are sequentially arranged on the output optical path of the scattered light of the sample to be tested; The image rotation module comprises a rotatable Dove prism; the central axis of the rotatable Dove prism coincides with the central axis of the scattered light output optical path; The image processing module extracts the image obtained by the enhanced scattering imaging module, and obtains the Rayleigh scattering spectrum and Raman scattering spectrum of the sample to be tested by performing a deconvolution operation on the scattered light intensity image with spatial position information; The light source module emits an incident light beam, which enters the coaxial dual-objective microscope module and irradiates the sample to be tested at a specific angle greater than 70°, generating total internal reflection, inducing surface plasmon resonance in the metal nanofilm on the substrate carrier of the sample to be tested, and coupling the nanoparticles on the surface to enhance Raman scattering. The scattered light signal is collected by the upright objective lens in the coaxial dual-objective microscope module and enters the enhanced scattering imaging module for scattering imaging and spectral analysis; The upright objective lens is adjusted through a three-dimensional translation stage to observe the same field of view as the inverted objective lens; The slit of the enhanced scattering imaging module is in a fully open state, the scattered light beam of the sample to be measured is split and expanded in one dimension by the reflection grating, the central wavelength of the grating is adjusted, and the camera obtains the zero-order diffraction image of the sample nanoparticles and the first-order diffraction image that is dispersed and expanded in the horizontal direction respectively; The image rotation module rotates and splits the overlapping first-order diffraction spectrum images to obtain a zero-order diffraction image sequence 1 and a first-order diffraction spectrum image sequence 2 of the bright spots of the nanoparticles in the field of view respectively; The image processing module grabs and cuts the zero-order diffraction image sequence 1 and the first-order diffraction spectrum image sequence 2 obtained by the enhanced scattering imaging module into a zero-order image sequence 3 and a first-order image sequence 4 of the region of interest respectively; for target samples at random positions, the image processing module uses an algorithm to grab the horizontal spacing d of the samples in the zero-order image sequence 3 and the first-order image sequence 4 of the region of interest of the sample, and calculates the effective dispersion distance through the following relationship: d eff =α(x)d, Where α(x) is the spectral expansion correction coefficient of the spacing d between the zero-order and first-order diffraction spectrum images of the sample nanoparticles, which is linearly related to the horizontal position of the pixel in the first-order diffraction spectrum image sequence 2; The spatial position of each pixel in the primary image sequence 4 is calculated to generate the corresponding Raman wave number array 5 through the following relationship: n=1 / λ ex -1 / (kd eff +λ 0 ) in v represents the Raman shift value of each pixel in the zero-level image sequence 3 of the region of interest; d eff represents the effective dispersion constant of each pixel in the zero-level image sequence 3 of the region of interest; k represents the wavelength resolution of the imaging module, and its value is 0.2071, and its unit is nanometers per pixel; λ 0 Indicates setting the grating center position; λ ex represents the incident laser wavelength; The intensity value of each pixel in the primary image sequence 4 is the Raman scattering intensity at the corresponding Raman shift wavenumber, and the Raman scattering spectrum can be obtained by plotting the Raman shift wavenumber and intensity of the pixels in the primary image sequence 4 as the horizontal and vertical coordinates.

2. The method according to claim 1, It is characterized in that In the light source module, a first convex lens, a polarizing plate, a second convex lens, and a bandpass filter are sequentially arranged on the output light path of the incident laser light source; and the emission light beam angle of the incident laser light source is adjustable.

3. The method according to claim 1, It is characterized in that The coaxial dual-objective lens microscopy module comprises an inverted objective lens, an objective stage, and an upright objective lens which can move in three dimensions and which are sequentially arranged on the output light path of a light source module with adjustable incident angle.

4. The method according to claim 1, It is characterized in that The SPR reflection imaging module comprises a semi-transparent and semi-reflective mirror, a third convex lens, and a first camera which are sequentially arranged on the output light path of the reflected light of the sample to be tested.

5. The method according to claim 1, It is characterized in that The enhanced scattering imaging module includes a long-pass color filter, a fourth convex lens, a first reflector, a slit, a second reflector, a concave mirror, a reflection grating, a concave mirror, and a second camera, which are sequentially arranged on the output light path of the scattered light of the sample to be tested.

6. The method according to claim 1, It is characterized in that The emission light beam of the light source module is a single-frequency laser beam or a broadband laser beam; the wavelength of the single-frequency laser beam is adjustable within 500-700nm; the wavelength of the broadband laser beam is adjustable in the range of 200 to 2000nm.

7. The method according to claim 1, It is characterized in that The substrate carrier of the sample to be tested is any one of a cover glass, glass, and conductive glass with a smooth and uniform surface coated with a metal nanofilm.

8. The method according to claim 1, It is characterized in that The field of view and focal length of each objective lens of the coaxial dual-objective lens microscopy module can be adjusted independently.

9. The method according to claim 1, It is characterized in that The lambda 0 750nm; ex It is 660nm.

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