Linear scanning microscopic Raman spectrum imaging method and system, electronic equipment and storage medium

Abstract

The invention provides a linear scanning microscopic Raman spectrum imaging method and system, electronic equipment and a storage medium. The method comprises the following steps: obtaining Raman spectrums of the same sample in different directions; spectral feature maps are extracted from the Raman spectrums in different directions respectively; processing the spectral feature map to obtain reinforced features corresponding to different directions so as to make up that the spectral acquisition resolution in the line length direction is lower than the spectral acquisition resolution in the line width direction; and fusing the enhanced features to obtain a fused spectral feature map for qualitative or quantitative analysis. According to the invention, the uniformity of linear exciting light is improved by using a micro-cylindrical lens array or a DOE element, and an omnidirectional high-resolution spectral image in a sample plane is obtained by combining orthogonal double-frame sampling with an atlas fusion algorithm. The device is simple in structure and low in cost, linear exciting light energy spatial distribution uniformity and in-plane omnidirectional horizontal spatial resolution are improved, and a powerful tool is provided for rapid research of cell space metabolome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of line-scan microscopic Raman spectrum imaging, in particular to a line-scan microscopic Raman spectrum imaging method, system, electronic device and storage medium. BACKGROUND

[0002] The existing line-scan Raman system uses a cylindrical lens to shape a laser beam with a wavelength of 532 nm into a linear beam, transmits the excitation light beam to the sample surface through a lens group and an objective lens, and the objective lens also functions to collect Raman scattered light. A high numerical aperture objective lens is provided in the process of cell research. A low reflection long-pass filter with a small incident angle is used to separate the excitation light path and the Raman light path. The Raman signal is dispersed into a one-dimensional spectrum by a grating, and is collected by a high quantum efficiency refrigerated CCD. The number of spatial sampling points is determined by the line length and the number of pixels. A high-speed galvanometer is placed on the Fourier plane of the excitation light modulation light path, and the high-speed scanning of the galvanometer can realize the high-speed acquisition of two-dimensional hyperspectral data, thereby improving the Raman data throughput per unit time. The structure is shown in Figure 1

[0003] However, when this technology is applied to spatial group metabolic analysis of complex biological samples such as tumor tissues, there are technical problems such as insufficient uniformity, limited and inconsistent spatial resolution in the line length direction and the line width direction, trade-off between imaging speed and resolution, and poor applicability to large-area samples. These problems limit the wide application of this technology under the requirements of high precision and high speed imaging, and it is urgent to upgrade the technology to improve its performance in the rapid Raman acquisition and analysis of biological large samples. SUMMARY

[0004] In order to achieve the above-mentioned purposes and other advantages of the present application, the first object of the present application is to provide a line-scan microscopic Raman spectrum imaging method, comprising the following steps:

[0005] Rotating the sample to realize the acquisition of linear Raman signals in different directions of the sample, and obtaining Raman spectra in different directions of the same sample;

[0006] Extracting spectral feature maps from Raman spectra in different directions, respectively;

[0007] Processing the spectral feature maps to obtain enhanced features corresponding to different directions;

[0008] Fusing the enhanced features to obtain a fused spectral feature map for qualitative or quantitative analysis.

[0009] Further, before the step of rotating the sample to realize the acquisition of linear Raman signals in different directions of the sample, the method further comprises:

[0010] ​A wide-field image of the sample is taken to identify the region of interest, and then the direction of the linear laser linewidth at this point is determined as the X direction.

[0011] Furthermore, the step of rotating the sample to acquire linear Raman signals in different directions includes:

[0012] In the X direction, the linear Raman signal is scanned and acquired sequentially using the highest spatial resolution scale as the single step size.

[0013] The sample is rotated 90° to determine the direction of the linear laser linewidth as the Y direction. Using the highest spatial resolution scale as the single-step size, the linear Raman signals are scanned and acquired sequentially to obtain the Raman spectra of the same sample in two orthogonal directions. X ∈R B×C×L and Z Y ∈R B×C×L Where B represents the movement dimension in the X or Y direction, C represents the number of linear scans, and L represents the spectral wavelength.

[0014] Furthermore, the step of extracting spectral feature maps from Raman spectra from different directions includes:

[0015] The X-direction spectral feature map Z was extracted using a feature extractor. X ∈R B / 2×C×L and Y-direction spectral characteristic map Z Y ∈R B / 2×C×L .

[0016] Furthermore, the step of processing the spectral feature map to obtain the enhancement features corresponding to different directions includes:

[0017] By processing the spectral feature maps using a cross-spectral relationship model, enhanced feature maps with spectral information in different directions are obtained.

[0018] Furthermore, the step of fusing the enhanced features to obtain a fused spectral feature map includes:

[0019] via concat(Z) X Z Y )∈R B / 2×C×L The fused spectral feature maps in the X and Y directions are obtained.

[0020] Furthermore, the step of processing the spectral feature map using a cross-spectral relationship model includes:

[0021] X-direction spectral feature map Z X and Y-direction spectral characteristic map Z Y As input to the cross-graph relation model;

[0022] X-direction spectral feature map Z X Z with Y-direction spectral characteristics Y Transformed into R B / 2×N The matrix; where N = C × L represents the number of spectral points in any displacement dimension;

[0023] By analyzing the X-direction spectral feature map Z after deformation X Z with Y-direction spectral characteristics Y Perform matrix multiplication between them to obtain the cross-graph relation measurement matrix A∈R R×R The feature representation of each position in A is as follows:

[0024]

[0025] Where i and j represent the deformed feature map Z X and Z Y Feature location index, and Representing feature maps Z respectively X and Z Y The eigenvalues ​​at positions i and j;

[0026] The cross-graph relation measurement matrix A is normalized by applying a softmax layer to ensure that the value at each position in the graph relation measurement matrix is ​​between 0 and 1. The specific operation is as follows:

[0027]

[0028] In the spectral relation measurement matrix A and the transformed feature matrix Z X Matrix multiplication is performed between the transposes of the original feature map to enhance attention to the original feature map, transforming the result into R. B / 2×C×L ;

[0029] The deformation result is multiplied by the scaling parameter λ, and the original feature map is summed element-wise to obtain the final enhanced feature map. Its definition is as follows:

[0030]

[0031] A second objective of this invention is to provide a line-scanning micro-Raman spectroscopy imaging system, which implements the above-mentioned method, comprising a laser source, a laser shaping module, an excitation light modulation module, an objective lens, a turntable, a Raman signal collection module, and a controller; wherein,

[0032] The turntable is used to place and rotate the sample;

[0033] The laser source is used to output excitation light to irradiate the sample;

[0034] The laser shaping module is used to perform beam splitting and high uniformity processing on the excitation light;

[0035] The excitation light modulation module is used to guide the processed linear excitation beam into the pupil of the objective lens, and then focus it onto the sample through the objective lens to excite Raman light.

[0036] The Raman signal collection module is used to collect linear Raman signals before and after sample rotation in order to obtain Raman spectra of the same sample in different directions.

[0037] The controller is used to acquire Raman spectra of the same sample in different directions, and to process the Raman spectra to obtain a fused spectral feature map for qualitative or quantitative analysis.

[0038] Furthermore, the laser shaping module includes a micropillar lens array or DOE element, as well as a beam expander and a beam splitter cube; the laser beam emitted by the laser source is expanded in diameter by the beam expander, then split by the beam splitter cube, and then subjected to high uniformity processing by the micropillar lens array or the DOE element.

[0039] Furthermore, the excitation light modulation module includes a Fourier lens, a first plane mirror, a notch filter, a second plane mirror, a first lens, a first beam splitter, a second beam splitter, and a third plane mirror arranged in sequence. The light beam processed by the micropillar lens array or the DOE element is parallelized by the Fourier lens. The parallel light is reflected by the first plane mirror to the notch filter, and then reflected by the notch filter in sequence through the second plane mirror, the first lens, the first beam splitter, the second beam splitter, and the third plane mirror to be focused on the back focal plane of the objective lens.

[0040] Furthermore, it also includes a bright-field imaging module, which is used to perform wide-field imaging of the sample to identify the region of interest.

[0041] Furthermore, the bright-field imaging module includes a white light camera and a white light source. The white light camera is used to image the beam split by the first beam splitter, and the white light source is used to illuminate the sample with white light through the second beam splitter.

[0042] Furthermore, the Raman signal collection module includes a fourth plane mirror, a second lens, a third lens, a slit, and a spectrometer arranged sequentially. The excited Raman light passes sequentially through the objective lens, the third plane mirror, the second beam splitter, the first beam splitter, the first lens, the second plane mirror, the notch filter, the fourth plane mirror, the second lens, the third lens, and the slit before entering the spectrometer.

[0043] A third object of the present invention is to provide an electronic device comprising: a memory having program code stored thereon; and a processor connected to the memory, wherein the above-described method is implemented when the program code is executed by the processor.

[0044] A fourth objective of this invention is to provide a computer-readable storage medium having program instructions stored thereon, which, when executed by a processor, implement the method described above.

[0045] Compared with the prior art, the beneficial effects of the present invention are:

[0046] This invention provides a linear scanning micro Raman spectroscopy imaging method, system, electronic device, and storage medium. It utilizes a micropillar lens array or DOE element to improve the uniformity of linear excitation light and obtains a high-resolution spectral image of the sample plane in all directions through orthogonal dual-frame sampling combined with a spectrum fusion algorithm.

[0047] This invention features a simple structure, low cost, and improves the uniformity of spatial distribution of linear excitation light energy and the horizontal spatial resolution in all directions within a plane. Compared with point scanning Raman spectroscopy, it greatly increases the scanning throughput per unit time, and compared with line scanning Raman, it greatly improves the spectral acquisition resolution along the line length direction. This enhances the speed and quality of high-throughput line scanning Raman spectroscopy imaging, providing a powerful tool for rapid research on cellular spatial metabolomics.

[0048] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0049] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0050] Figure 1 A schematic diagram of an existing line scan Raman system;

[0051] Figure 2 Schematic diagram of the line-scanning micro Raman spectroscopy imaging system of Example 1 Figure 1 ;

[0052] Figure 3 Schematic diagram of the line-scanning micro Raman spectroscopy imaging system of Example 1 Figure 2 ;

[0053] Figure 4Line scan micro Raman spectra in two orthogonal directions;

[0054] Figure 5 This is a schematic diagram of the cross-graph relational grid structure;

[0055] Figure 6 Here is a flowchart of the line-scan micro Raman spectroscopy imaging method in Example 2;

[0056] Figure 7 This is a flowchart of the linear Raman signal acquisition process for the sample in different directions in Example 2;

[0057] Figure 8 This is a schematic diagram of a cross-graph relational model;

[0058] Figure 9 This is a flowchart of Example 2 showing the processing of spectral feature maps using a cross-spectral relationship model;

[0059] Figure 10 This is a schematic diagram of the electronic device in Example 3;

[0060] Figure 11 This is a schematic diagram of the storage medium in Example 4.

[0061] In the diagram: 1. Laser source; 2. Beam expander; 3. Beam splitter cube; 4. Microlens array; 5. Fourier lens; 6. First plane mirror; 7. Second plane mirror; 8. First lens; 9. First beam splitter; 10. Second beam splitter; 11. White light camera; 12. White light source; 13. Third plane mirror; 14. Objective lens; 15. Turntable; 16. Notch filter; 17. Fourth plane mirror; 18. Second lens; 19. Third lens; 20. Slit; 21. Spectrometer. Detailed Implementation

[0062] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0063] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0064] The drawing numbers in this application are only used to distinguish the steps in the scheme and are not used to limit the execution order of the steps. The specific execution order is as described in the specification.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0066] Example 1

[0067] A line-scan micro Raman spectroscopy imaging system, such as Figure 2 , Figure 3 As shown, it includes a laser source 1, a laser shaping module, an excitation light modulation module, an objective lens 14, a turntable 15, a Raman signal collection module, and a controller; among which,

[0068] The turntable is used to place and rotate the sample;

[0069] The laser source is used to output excitation light to irradiate the sample; for example, the laser source can be a laser.

[0070] The laser shaping module is used to perform beam splitting and high uniformity processing on the excitation light;

[0071] The excitation light modulation module is used to guide the processed linear excitation beam into the pupil of the objective lens, and then focus it onto the sample through the objective lens to excite Raman light.

[0072] The Raman signal collection module is used to collect linear Raman signals before and after sample rotation in order to obtain Raman spectra of the same sample in different directions.

[0073] The controller is used to acquire Raman spectra of the same sample in different directions, and to process the Raman spectra to obtain a fused spectral feature map for qualitative or quantitative analysis.

[0074] In some embodiments, the laser shaping module includes a micropillar lens array 4 or a DOE element, a beam expander 2, and a beam splitter 3; the laser beam emitted by the laser source is expanded in diameter by the beam expander, then split by the beam splitter, and then subjected to high uniformity processing by the micropillar lens array or the DOE element.

[0075] In some embodiments, the excitation light modulation module includes a Fourier lens 5, a first plane mirror 6, a notch filter 16, a second plane mirror 7, a first lens 8, a first beam splitter 9, a second beam splitter 10, and a third plane mirror 13 arranged sequentially. The light beam processed by the micropillar lens array or the DOE element is parallelized by the Fourier lens. The parallel light is reflected by the first plane mirror to the notch filter, and then reflected by the notch filter sequentially through the second plane mirror, the first lens, the first beam splitter, the second beam splitter, and the third plane mirror to be focused onto the back focal plane of the objective lens.

[0076] In some embodiments, a bright-field imaging module is further included, which is used to perform wide-field imaging of the sample to clearly define the region of interest. Specifically, the bright-field imaging module includes a white light camera 11 and a white light source 12. The white light camera is used to image the beam split by the first beam splitter, and the white light source is used to illuminate the sample with white light through the second beam splitter.

[0077] In some embodiments, the Raman signal collection module includes a fourth plane mirror 17, a second lens 18, a third lens 19, a slit 20, and a spectrometer 21 arranged sequentially. The excited Raman light passes sequentially through the objective lens, the third plane mirror, the second beam splitter, the first beam splitter, the first lens, the second plane mirror, the notch filter, the fourth plane mirror, the second lens, the third lens, and the slit before entering the spectrometer.

[0078] The above-described line-scan micro Raman spectroscopy imaging system will be illustrated using two perspectives: top view and side view. The side view perspective is as follows: Figure 2 As shown, the beam emitted by the ultra-narrow linewidth 532nm laser is expanded in diameter by beam expander 2, then subjected to high uniformity processing by micropillar lens array 4, and parallelized by Fourier lens 5. After reflection by a short-wavelength reflector, long-wavelength pass, and high cutoff depth notch filter 16, it is focused by first lens 8 onto the back focal plane of objective lens 14. Finally, from a side view, the laser shape on the sample surface appears as a line, exciting Raman light which is then imaged at the slit 20 position by objective lens 14, first lens 8, notch filter 16, and a series of focusing lenses. Due to conjugate relationships, the line shape on the sample surface corresponds to the line shape at the slit 20 position with a certain magnification ratio, and enters Raman spectrometer 21 for Raman signal dispersion and collection.

[0079] A bird's-eye view, such as Figure 3As shown, the beam emitted by the ultra-narrow linewidth 532nm laser is expanded in diameter by beam expander 2, and then split into two beams by beam splitter cube 3. After passing through micropillar lens array 4 (which does not modulate the light), the beam is focused on the focal plane of Fourier lens 5. Parallel light is then formed by the tube lens and enters the pupil of objective lens 14, where it is focused onto the sample surface. Finally, from a top-down perspective, the laser shape on the sample surface appears as a point, which excites Raman light. This light is then imaged at the slit 20 position by objective lens 14, first lens 8, notch filter 16, and a series of focusing lenses. Due to the conjugate relationship, the point shape on the sample surface corresponds to the point shape at the slit 20 position with a certain magnification ratio, and enters Raman spectrometer 21 for Raman signal dispersion and collection.

[0080] On a constructed line-scanning micro Raman spectroscopy imaging system, the sample is placed and fixed on a high-precision turntable. First, a wide-field image is taken of the sample to identify the region of interest. Then, the direction of the linear laser linewidth is determined as the X-axis, and the highest spatial resolution scale is used as the single-step size. Linear Raman signals are acquired sequentially by scanning. The sample is then rotated 90° using the high-precision turntable. The direction of the linear laser linewidth changes from the original direction (where the linewidth was initially along the linear laser linewidth) to the new direction (where the linewidth is now along the linear laser linewidth). Again, the highest spatial resolution scale is used as the single-step size, and linear Raman signals are acquired sequentially by scanning. This allows the acquisition of Raman spectra of the same sample in two orthogonal directions, such as... Figure 4 As shown.

[0081] Two spectra of the same sample in different directions possess complementary features, connecting relevant regions across the spectrum. These can be propagated to supplement the representation and obtain more complete characteristic information of the sample. To fully utilize the spectral information in different directions of line-scan micro Raman spectroscopy, this invention proposes a cross-spectral relationship network, such as... Figure 5 As shown. Based on cross-spectral relationships, more relevant features can be extracted, resolving some error analyses. Simultaneously, this relationship helps the network learn more consistent feature representations across the entire dataset, facilitating quantitative and qualitative spectral analysis. Specific line-scan micro Raman spectroscopy imaging methods can be found in the corresponding descriptions in the following method embodiments, and will not be repeated here.

[0082] The linear scanning micro Raman spectroscopy imaging system provided by this invention can achieve a spatial resolution of ≤320nm*500nm, a linear Raman spectrum distribution uniformity of better than 90%, a single acquisition of up to 200 spectra, and a Raman spectrum detection speed (Si) of 2400 spectra / minute.

[0083] This embodiment provides a line-scan micro Raman spectroscopy imaging system. It utilizes a micropillar lens array or DOE element to improve the uniformity of the linear excitation light. Through orthogonal dual-frame sampling combined with a spectral fusion algorithm, it obtains a high-resolution spectral image of the sample plane in all directions. This embodiment features a simple structure, low cost, and improves the spatial uniformity of the linear excitation light energy distribution and the horizontal spatial resolution in all directions within the plane. Compared to point-scan Raman spectroscopy, it significantly increases the scanning throughput per unit time, and compared to line-scan Raman, it significantly improves the spectral acquisition resolution along the line length. This enhances the speed and quality of high-throughput line-scan Raman spectroscopy imaging, providing a powerful tool for rapid research in cellular spatial metabolomics.

[0084] Example 2

[0085] Example 1 provides a line-scanning micro Raman spectroscopy imaging system and a corresponding line-scanning micro Raman spectroscopy imaging method. For a detailed description of the system, please refer to the corresponding description in the above system examples, which will not be repeated here.

[0086] A line-scan micro Raman spectroscopy imaging method, such as Figure 6 As shown, it includes the following steps:

[0087] S1. The sample is rotated to acquire linear Raman signals in different directions, thus obtaining Raman spectra of the same sample in different directions, such as... Figure 4 As shown; the acquisition process of linear Raman signals in different directions of the sample can be referred to the corresponding description in the above system embodiment, and will not be repeated here.

[0088] In some embodiments, the method further includes, before rotating the sample to acquire linear Raman signals in different directions:

[0089] On the line-scanning micro Raman spectroscopy imaging system built in Example 1, the sample is placed on a high-precision turntable and fixed. First, a wide-field image is taken of the sample to identify its region of interest. Then, the direction of the linear laser linewidth at this time is determined to be the X direction.

[0090] In some embodiments, such as Figure 7 As shown, the step of rotating the sample to acquire linear Raman signals in different directions includes:

[0091] S11. In the X direction, using the highest spatial resolution scale as the single step size, scan and acquire linear Raman signals sequentially.

[0092] S12. The sample is rotated 90° using a high-precision turntable. Before rotation, the direction along the linear laser line length becomes the direction of the new linear laser linewidth after 90° rotation. The direction of the linear laser linewidth at this point is determined as the Y direction. Using the highest spatial resolution scale as the single-step size, the linear Raman signals are scanned and acquired sequentially to obtain the Raman spectra Z of the same sample in two orthogonal directions. X ∈R B×C×L and Z Y ∈R B×C×L Where B represents the movement dimension in the X or Y direction, C represents the number of linear scans, and L represents the spectral wavelength.

[0093] Two spectra of the same sample in different directions possess complementary features, connecting relevant regions across the spectrum. These can be propagated to supplement the representation and obtain more complete characteristic information of the sample. To fully utilize the spectral information in different directions of line-scan micro Raman spectroscopy, this invention proposes a cross-spectral relationship network, such as... Figure 5 As shown, based on cross-spectral relationships, more relevant features can be extracted, resolving some errors in analysis. Simultaneously, these relationships help the network learn more consistent feature representations across the entire dataset, facilitating quantitative and qualitative analysis of the spectrum.

[0094] S2. Extract spectral feature maps from Raman spectra from different directions;

[0095] In some embodiments, the step of extracting spectral feature maps from Raman spectra from different directions includes:

[0096] The X-direction spectral feature map Z was extracted using a feature extractor. X ∈R B / 2×C×L and Y-direction spectral characteristic map Z Y ∈R B / 2×C×L .

[0097] S3. Process the spectral feature map to obtain the enhancement features corresponding to different directions, so as to compensate for the lower spectral acquisition resolution in the line length direction than in the line width direction.

[0098] In some embodiments, the step of processing the spectral feature map to obtain enhancement features corresponding to different directions includes:

[0099] By processing the spectral feature maps using a cross-spectral relationship model, enhanced feature maps with spectral information in different directions are obtained.

[0100] Specifically, such as Figure 8 , Figure 9 As shown, the step of processing the spectral feature map using a cross-spectral relationship model includes:

[0101] S31, The X-direction spectral feature map Z X ∈R B / 2×C×L and Y-direction spectral characteristic map Z Y ∈R B / 2×C×L As input to the cross-graph relation model;

[0102] S32, The X-direction spectral feature map Z X Z with Y-direction spectral characteristics Y Transformed into R B / 2×N The matrix; where N = C × L represents the number of spectral points in any displacement dimension;

[0103] S33, By analyzing the X-direction spectral characteristic map Z after deformation X Z with Y-direction spectral characteristics Y Perform matrix multiplication between them to obtain the cross-graph relation measurement matrix A∈R R×R The feature representation of each position in A is as follows:

[0104]

[0105] Where i and j represent the deformed feature map Z X and Z Y Feature location index, and Representing feature maps Z respectively X and Z Y The eigenvalues ​​at positions i and j; a i,j The influence of the feature map generated by the online scan Raman spectrum in the Y direction at position j on the feature map generated by the online scan Raman spectrum in the X direction at position i is measured, which is a similarity measure of different positions in the two feature maps.

[0106] S34. Next, the cross-spectral relationship measurement matrix A is normalized by applying a softmax layer to ensure that the value at each position in the spectral relationship measurement matrix is ​​between 0 and 1. The specific operation is as follows:

[0107]

[0108] Excitation at any position in the feature map generated by calculating the online scan Raman spectrum in the Y direction Excitation at any position of the feature map generated by the X-axis online scan Raman spectrum The more similar the feature representations of two positions are, the stronger their correlation, thus obtaining the correlation between the maps. This allows the specific class feature maps generated by the classification network to focus on a more complete target map region and suppress the generation of noise.

[0109] S35. Then, in the spectral relation measurement matrix A and the deformed feature matrix Z...X Matrix multiplication is performed between the transposes of the original feature map to enhance attention to the original feature map, transforming the result into R. B / 2×C×L ;

[0110] S36. Finally, multiply the deformation result by the scaling parameter λ, and perform element-wise summation on the original feature map to obtain the final enhanced feature map. Its definition is as follows:

[0111]

[0112] In this case, λ is set to 0 in the initial stage and gradually learns to be assigned more weights during the training stage. The value at each position is a weighted sum of all positions in the cross-spectral relation measurement matrix generated by the cross-spectral relation model and the features generated from the original spectrum. Therefore, the cross-spectral relation model can capture long-range contextual dependencies between spectra generated on different axes.

[0113] S4. The enhanced features are fused to obtain a fused spectral feature map for qualitative or quantitative analysis.

[0114] In some embodiments, the step of fusing the enhanced features to obtain a fused spectral feature map includes:

[0115] via concat(Z) X Z Y )∈R B / 2×C×L The fused spectral feature maps in the X and Y directions are obtained, and qualitative or quantitative analysis is performed.

[0116] This embodiment provides a linear scanning micro-Raman spectroscopy imaging method. It utilizes a micropillar lens array or DOE element to improve the uniformity of the linear excitation light, and obtains a high-resolution spectral image of the sample plane in all directions through orthogonal dual-frame sampling combined with a spectral fusion algorithm. This embodiment features a simple structure, low cost, and improves the spatial uniformity of the linear excitation light energy distribution and the horizontal spatial resolution in all directions within the plane. Compared to point scanning Raman spectroscopy, it significantly increases the scanning throughput per unit time, and compared to linear scanning Raman, it significantly improves the spectral acquisition resolution along the line length direction. This enhances the speed and quality of high-throughput linear scanning Raman spectroscopy imaging, providing a powerful tool for rapid research on cellular spatial metabolomics.

[0117] Example 3

[0118] An electronic device, such as Figure 10 As shown, it includes: a memory storing program code; and a processor connected to the memory, which, when executed by the processor, implements a line-scanning micro-Raman spectroscopy imaging method. For a detailed description of the method, please refer to the corresponding description in the above method embodiments, which will not be repeated here.

[0119] Example 4

[0120] A computer-readable storage medium, such as Figure 11 As shown, it stores program instructions, which, when executed by the processor, implement a line-scan micro-Raman spectroscopy imaging method. For a detailed description of the method, please refer to the corresponding description in the above method embodiments; it will not be repeated here.

[0121] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0122] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

[0123] The apparatus, electronic device, and non-volatile computer storage medium and method provided in the embodiments of this specification are corresponding. Therefore, the apparatus, electronic device, and non-volatile computer storage medium also have similar beneficial technical effects as the corresponding method. Since the beneficial technical effects of the method have been described in detail above, the beneficial technical effects of the corresponding apparatus, electronic device, and non-volatile computer storage medium will not be repeated here.

[0124] Those skilled in the art will also know that, besides implementing the controller in the form of purely computer-readable program code, the same functions can be achieved by logically programming the method steps, making the controller take the form of logic gates, switches, application-specific integrated circuits (ASICs), programmable logic controllers (PLCs), and embedded microcontrollers. Therefore, such a controller can be considered a hardware component, and the devices included within it for implementing various functions can also be considered structures within that hardware component. Alternatively, the devices for implementing various functions can be considered as both software units implementing the method and structures within a hardware component.

[0125] The systems, apparatuses, or units described in the above embodiments can be implemented by computer chips or physical entities, or by products with certain functions. For ease of description, the above apparatuses are described separately as various units based on their functions. Of course, when implementing one or more embodiments of this specification, the functions of each unit can be implemented in one or more software and / or hardware.

[0126] Those skilled in the art will understand that the embodiments of this specification can be provided as methods, systems, or computer program products. Therefore, the embodiments of this specification can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, the embodiments of this specification can take the form of a computer program product implemented on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0127] This specification is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this specification. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create a machine for implementing the flowchart illustrations and / or block diagrams. Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0128] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0129] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0130] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0131] This specification may be described in the general context of computer-executable instructions, such as program units, that are executed by a computer. Generally, program units include routines, programs, objects, components, data structures, etc., that perform a specific task or implement a specific abstract data type. This specification may also be practiced in distributed computing environments, where tasks are performed by remote processing devices connected via a communication network. In distributed computing environments, program units may reside in local and remote computer storage media, including storage devices.

[0132] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In particular, the system embodiments are basically similar to the method embodiments, so the description is relatively simple; relevant parts can be referred to the descriptions in the method embodiments.

[0133] The above description is merely an embodiment of this specification and is not intended to limit the scope of one or more embodiments of this specification. Various modifications and variations can be made to one or more embodiments of this specification by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of one or more embodiments of this specification should be included within the scope of the claims of one or more embodiments of this specification.

Claims

1. A line-scan microscopic Raman spectral imaging method, characterized by, The method comprises the following steps: rotating the sample to collect linear Raman signals of the sample in different directions, and obtaining Raman spectra of the sample in different directions; extracting spectral feature maps from the Raman spectra in different directions respectively; processing the spectral feature maps to obtain enhanced features corresponding to different directions; fusing the enhanced features to obtain a fused spectral feature map for qualitative or quantitative analysis.

2. A line-scan microscopic Raman spectral imaging method according to claim 1, wherein Before the step of rotating the sample to collect linear Raman signals of the sample in different directions, the method further comprises: taking a wide-field image of the sample to determine a region of interest, and then determining that the direction of the linear laser line at this time is the X direction.

3. A line-scan microscopic Raman spectral imaging method according to claim 2, wherein: The step of rotating the sample to collect linear Raman signals of the sample in different directions comprises: in the X direction, sequentially scanning and collecting linear Raman signals with the highest spatial resolution scale as a single-step step length. The sample is rotated by 90°, the direction of the linear laser line width is determined as the Y direction, the highest spatial resolution scale is used as a single step length, linear Raman signals are sequentially collected by sequentially scanning, and Raman spectra of the same sample in two orthogonal directions Z X ∈R B×C×L and Z Y ∈R B×C×L wherein B represents the moving size in the X direction or the Y direction, C represents the number of line scans, and L represents the spectral wavelength.

4. A line-scan microscopic Raman spectral imaging method according to claim 3, wherein The step of extracting spectral feature maps from the Raman spectra in different directions respectively comprises: extracting, by a feature extractor, an X direction spectral feature map Z X ∈R B / 2×C×L and a Y direction spectral feature map Z Y ∈R B / 2×C×L .

5. A line-scan micro-Raman spectral imaging method according to claim 4, wherein, The step of processing the spectral feature maps to obtain enhanced features corresponding to different directions comprises: The spectral feature map is processed through a cross-atlas relation model to obtain an enhanced feature map with atlas-related information in different directions 6. A line-scan micro-Raman spectral imaging method according to claim 5, wherein, The step of fusing the enhanced features to obtain a fused spectral feature map comprises: By concat(Z X ,Z Y )∈R B / 2×C×L get the fusion spectrum feature map in X direction and Y direction.

7. A line-scan microscopic Raman spectral imaging method according to claim 5, wherein The step of processing the spectral feature maps by using a cross-spectrum relationship model comprises: a Z-direction spectral feature map Z X and a Y-direction spectral feature map Z Y as input to the cross-spectral relationship model; X-direction spectral feature map Z X Z with Y-direction spectral characteristics Y Transformed into R B / 2×N The matrix; where N = C × L represents the number of spectral points in any displacement dimension; By performing matrix multiplication between the deformed X-direction spectral feature map Z X and the Y-direction spectral feature map Z Y , a cross-spectral relationship measurement matrix A ∈ R R×R is obtained, and the feature of each position in A is represented as follows: where i and j represent the feature map Z X and Z Y after deformation and respectively represent the feature values of the feature map Z X and Z Y at the i and j positions. applying a softmax layer to normalize the cross-spectrum relationship measurement matrix A to ensure that the value of each position in the cross-spectrum relationship measurement matrix is between 0 and 1, and the specific operation is as follows: In the atlas relationship measurement matrix A and the deformed feature matrix Z X The transpose between the matrix multiplication is carried out, the attention enhancement of the original feature map is realized, and its result is deformed into R B / 2×C×L ; The deformed result is multiplied by a scale parameter λ, and an element-level summation operation is performed on the original feature map to obtain a final enhanced feature map It is defined as shown below:

8. A line-scan micro-Raman spectral imaging system implementing the method according to any one of claims 1 to 7, characterized in that: The system comprises a laser light source, a laser shaping module, an excitation light modulation module, an objective lens, a turntable, a Raman signal collection module, and a controller. The turntable is used to place and rotate the sample. The laser light source is used to output excitation light to irradiate the sample. The laser shaping module is used to split and process the excitation light with high uniformity. The excitation light modulation module is used to enter the processed linear excitation light beam into the pupil of the objective lens, focus it on the sample through the objective lens, and excite Raman light. The Raman signal collection module is used to collect linear Raman signals before and after the sample is rotated to obtain Raman spectra of the sample in different directions. The controller is used to obtain Raman spectra of the sample in different directions, and process the Raman spectra to obtain a fused spectral feature map for qualitative or quantitative analysis.

9. A line-scan microscopic Raman spectral imaging system as claimed in claim 8, characterized in that: The laser shaping module comprises a micro-lens array or a DOE element, an expander, and a beam splitter cube. The laser beam emitted by the laser light source is expanded in beam diameter by the expander, then split by the beam splitter cube, and then processed with high uniformity by the micro-lens array or the DOE element.

10. A line-scan microscopic Raman spectral imaging system as claimed in claim 9, characterized in that: The excitation light modulation module comprises a Fourier lens, a first plane mirror, a notch filter, a second plane mirror, a first lens, a first beam splitter, a second beam splitter, and a third plane mirror arranged in sequence. The light beam processed by the micro-lens array or the DOE element forms parallel light through the Fourier lens. The parallel light is reflected by the first plane mirror to the notch filter, and then reflected by the notch filter to be sequentially focused on the back focal plane of the objective lens through the second plane mirror, the first lens, the first beam splitter, the second beam splitter, and the third plane mirror.

11. A line-scan microscopic Raman spectral imaging system according to claim 10, wherein: The bright field imaging module is further included for wide field shooting of the sample to clearly define the region of interest.

12. A line-scan micro-Raman spectral imaging system according to claim 11, wherein: The bright field imaging module comprises a white light camera and a white light source. The white light camera is used for imaging the light beam split by the first beam splitter, and the white light source is used for irradiating white light to the sample through the second beam splitter.

13. A line-scan microscopic Raman spectral imaging system according to claim 10, wherein: The Raman signal collection module comprises a fourth plane mirror, a second lens, a third lens, and a slit arranged in sequence. The excited Raman light enters the spectrometer through the objective lens, the third plane mirror, the second beam splitter, the first beam splitter, the first lens, the second plane mirror, the notch filter, the fourth plane mirror, the second lens, the third lens, and the slit in sequence.

14. An electronic device, comprising: The method comprises: a memory having program code stored thereon; a processor connected to the memory, and when the program code is executed by the processor, the method as claimed in any one of claims 1-7 is implemented.

15. A computer readable storage medium characterized by: a memory having program code stored thereon; a processor connected to the memory, and when the program code is executed by the processor, the method as claimed in any one of claims 1-7 is implemented.

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