Surface-enhanced raman imaging system applied to single-cell metabolic detection

Abstract

The application provides a surface enhanced Raman imaging system applied to single cell metabolism detection, comprising a laser emission module, a microscopic Raman generation and imaging module, a Raman collection module and a central control module; the laser emission module generates a laser beam and is coupled to the microscopic Raman generation and imaging module; the laser beam emitted by the laser emission module is sequentially reflected by a Raman dichroic band edge filter, transmitted by an imaging dichroic band edge filter and reflected by a second reflector of the Raman generation and imaging module, and then focused by a microscope objective to a sample to be detected; backscattering Raman spectrum signals generated by the sample to be detected are converted into parallel light by the microscope objective, reflected by the second reflector, transmitted by the imaging dichroic band edge filter and the Raman dichroic band edge filter, and then output to the Raman collection module for spectral spectroscopy and detection; the central control module is connected with the laser emission module, the microscopic Raman generation and imaging module and the Raman collection module and performs data interaction.

Description

Technical Field

[0001] This invention relates to the field of optical detection, and more particularly to a surface-enhanced Raman imaging system for single-cell metabolic detection. Background Technology

[0002] Single cells are the basic unit of function and evolution of cellular life on Earth. Based on this characteristic, single-cell metabolic assays overcome the limitations of traditional population-based cell assays, which rely on averaging. These assays can precisely analyze the metabolic activity, substance transport, and energy metabolism characteristics of individual cells. They not only reveal cellular heterogeneity but also capture metabolic differences among rare cells, stem cells, and tumor microenvironment cells, providing crucial microscopic metabolic evidence for cell differentiation mechanisms, disease pathogenesis, tumor drug resistance research, and drug target screening.

[0003] Current single-cell detection primarily focuses on morphological sorting techniques, such as traditional flow cytometry, spectroscopic flow cytometry, and imaging flow cytometry. These techniques mainly achieve precise, high-throughput sorting of single cells through fluorescence and surface imaging. However, for extremely weak single-cell metabolic signals, these characterization methods are limited by the fundamental problem of extremely low signal-to-noise ratio. Mass cytometry uses metal isotope-labeled antibodies to atomize cells and then delivers them to a plasma torch to ionize the metal labels. Time-of-flight mass spectrometry is then used to detect the ion mass, thereby enabling the detection of weak single-cell metabolic signals. However, this technology cannot be applied to live-cell detection, and the instruments are expensive.

[0004] Raman spectroscopy requires no sample labeling, preserves cell viability, and possesses strong fingerprint characteristics, enabling rapid acquisition of chemical information from biological samples under near-physiological conditions. However, ordinary spontaneous Raman scattering signals are inherently very weak, and when directly detecting trace metabolites within single cells, the signal-to-noise ratio remains insufficient, making it difficult to distinguish characteristic signals from low-concentration metabolites. Compared to conventional Raman spectroscopy, surface-enhanced Raman spectroscopy (SERS) leverages the localized surface plasmon resonance effect of precious metal nanoparticles such as gold and silver. This enhances the Raman scattering signal of target molecules adsorbed on their surface or in the near-field region by millions of times, achieving single-molecule detection sensitivity and meeting the detection application needs in the field of single-cell molecular metabolism.

[0005] Currently, single-cell metabolic detection based on surface-enhanced Raman scattering (SERS) technology is mainly carried out using self-built micro-Raman spectroscopy systems. During the detection process, sample positioning, signal acquisition, and other operations mostly require manual execution, resulting in extremely limited automation. This not only leads to poor detection efficiency, but errors introduced by human operation also affect the repeatability and accuracy of the detection results. Meanwhile, relatively mature commercially available general-purpose micro-Raman spectroscopy systems typically face problems such as mismatch between system performance and detection requirements, inflexible system parameter adjustment, and mismatch between test spectrum acquisition planning and actual application needs, making them unable to effectively conduct spectroscopic detection in the field of single-cell metabolism.

[0006] Therefore, the present invention addresses the above-mentioned problems by providing a surface-enhanced Raman imaging system for single-cell metabolic detection. Summary of the Invention

[0007] This invention provides a surface-enhanced Raman imaging system for single-cell metabolism detection, enabling automatic detection of weak spectra of single-cell metabolism, improving the efficiency of single-cell metabolism detection, and enhancing the repeatability and accuracy of detection results.

[0008] This invention provides a surface-enhanced Raman imaging system for single-cell metabolism detection, comprising a laser emission module, a micro Raman generation and imaging module, a Raman collection module, and a central control module; The laser emitting module includes a laser, a first shutter, a narrowband filter, a plano-concave lens, a plano-convex lens, and a first reflecting mirror arranged in sequence. The laser generates a laser beam, the first shutter controls the rapid opening and closing of the laser beam, the narrowband filter allows light of a set wavelength band of the laser beam to pass through, the plano-concave lens and the plano-convex lens together adjust the diameter of the emitted laser beam, and the first reflecting mirror reflects and couples the laser beam to the micro Raman generation and imaging module. The micro Raman generation and imaging module includes at least a Raman dichroic band-edge filter, an imaging dichroic band-edge filter, a second reflecting mirror, and a microscope objective. The laser beam emitted by the laser emitting module is reflected sequentially by the Raman dichroic band-edge filter, transmitted by the imaging dichroic band-edge filter, and reflected by the second reflecting mirror. It is then focused onto the sample under test by the microscope objective. The backscattered Raman spectral signal generated by the sample under test is collected by the microscope objective and converted into parallel light. It is then reflected sequentially by the second reflecting mirror, transmitted by the imaging dichroic band-edge filter, and transmitted by the Raman dichroic band-edge filter before being output to the Raman collection module. The Raman collection module includes a third reflecting mirror, a long-pass filter, a first lens, and a spectrometer. The Raman spectral signal output by the micro Raman generation and imaging module is reflected by the third reflecting mirror and transmitted through the long-pass filter, and then focused by the first lens into the slit of the spectrometer. The spectrometer then performs spectral dispersion and detection. The central control module includes a controller and a host computer. The controller is connected to the laser, the first shutter, the spectrometer, and the host computer and interacts with them for data exchange.

[0009] Preferably, the micro Raman generation and imaging module further includes a white LED light source, a second shutter, a second lens, a fourth reflector, and an imaging camera. The white LED light source and the second shutter are positioned below the sample to be tested. The white LED light source provides an illumination beam for imaging the sample. The second shutter controls the rapid opening and closing of the illumination beam. After the illumination beam illuminates the sample through the second shutter, it is collected by the microscope objective and output as parallel light. Subsequently, it is reflected sequentially by the second reflector, reflected by the imaging dichroic bandgap filter, focused by the second lens, and reflected by the fourth reflector to the imaging camera for imaging. The controller is connected to the white LED light source, the second shutter, and the imaging camera and performs data interaction.

[0010] Preferably, the microscope objective is mounted on a piezoelectric displacement stage, and the controller is connected to the piezoelectric displacement stage and controls the piezoelectric displacement stage to move the microscope objective vertically to adjust the focusing height.

[0011] Preferably, the sample to be tested is placed on a two-dimensional motion platform, and the controller is connected to the two-dimensional motion platform and controls the two-dimensional motion platform to move the sample to be tested on the plane to adjust the focusing position on the sample to be tested.

[0012] Preferably, the plano-concave lens is a negative focal length lens, with the concave surface of the plano-concave lens facing the incident laser beam; the plano-convex lens is a positive focal length lens, with the plane of the plano-convex lens facing the incident laser beam, and the distance between the plano-concave lens and the plano-convex lens is the sum of their working focal lengths.

[0013] Preferably, the ratio of the positive focal length of the plano-convex lens to the negative focal length of the plano-concave lens is equal to the ratio of the diameter of the entrance pupil of the microscope objective to the diameter of the laser beam emitted by the laser.

[0014] Preferably, both the plano-concave lens and the plano-convex lens have an anti-reflection coating for the incident laser band on their surfaces.

[0015] Preferably, the first reflector is positioned at a 45° angle to the incident laser beam, and the Raman dichroic banded filter is positioned at a 45° angle to the incident laser beam.

[0016] Preferably, the spectrometer includes a slit, an internal mirror, a first concave mirror, a reflective grating, a second concave mirror, and an internal camera. After the Raman spectral signal is transmitted through the slit, it is reflected by the internal mirror to the first concave mirror, collimated by the mirror into parallel light, and incident on the surface of the reflective grating to complete the spectral dispersion. The dispersed beam is reflected again to the second concave mirror, and after being reflected and focused by the mirror, it is projected onto the focal plane of the internal camera.

[0017] Preferably, the focal length of the first lens matches the focal length of the equivalent optical path corresponding to the NA factor of the spectrometer.

[0018] Compared with the prior art, the technical solutions of the embodiments of the present invention have beneficial effects.

[0019] For example, the surface-enhanced Raman imaging system for single-cell metabolism detection provided by this invention includes a laser emission module, a micro-Raman generation and imaging module, a Raman collection module, and a central control module. A monochromatic laser emitted from a laser source is adjusted and controlled by the laser emission module and the micro-Raman generation and imaging module, and then focused onto a single-cell droplet sample fused with metal nanoparticles, exciting the sample to generate a surface-enhanced Raman scattering signal. This Raman signal is transmitted to the Raman collection module, completing the conversion and output of the photoelectric signal. Through the collaborative work between the modules, the entire single-cell metabolism detection is automated, improving the efficiency of single-cell metabolism detection while also enhancing the repeatability and accuracy of the detection results.

[0020] For example, a two-dimensional motion platform carrying the sample to be tested is set up, and a piezoelectric displacement stage carrying the microscope objective is set up. The controller drives the two-dimensional motion platform to achieve precise displacement of the single-cell sample, and drives the piezoelectric displacement stage to achieve automatic focusing of the microscope objective. In conjunction with microscopic imaging, automatic positioning and identification of single cells, automatic image focusing of metal nanoparticles, and automatic planning and acquisition of spectral detection areas are achieved. The whole process does not require manual adjustment and can meet the detection needs of single-cell samples of different sizes and types. It not only reduces the error caused by manual operation, but also significantly shortens the detection time. Attached Figure Description

[0021] Figure 1 This is an optical path diagram of the surface-enhanced Raman imaging system for single-cell metabolism detection according to the present invention; Figure 2 This is an automated Z-axis focusing image of metal nanoparticles surrounding a single cell in a droplet, as presented in this invention. Figure 3This is the image recognition result and path planning diagram of a single cell in a droplet according to the present invention. Detailed Implementation

[0022] To make the objectives, features, and beneficial effects of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described below are merely for explaining the present invention and are not intended to limit the present invention. Furthermore, the same or similar reference numerals may be used in the drawings to refer to the same or similar elements in different embodiments, and descriptions of the same or similar elements in different embodiments, as well as descriptions of prior art elements, features, effects, etc., may be omitted.

[0023] Reference Figure 1 This invention provides a surface-enhanced Raman imaging system for single-cell metabolic detection.

[0024] Specifically, the surface-enhanced Raman imaging system for single-cell metabolism detection provided in this embodiment of the invention includes a laser emission module 11, a micro Raman generation and imaging module 12, a Raman collection module 13, and a central control module 14. The laser emitting module 11 includes a laser 111, a first shutter 112, a narrowband filter 113, a plano-concave lens 114, a plano-convex lens 115, and a first reflector 116 arranged in sequence. The laser 111 generates a laser beam, the first shutter 112 controls the rapid opening and closing of the laser beam, the narrowband filter 113 allows light of a set wavelength band of the laser beam to pass through, the plano-concave lens 114 and the plano-convex lens 115 are combined to adjust the diameter of the emitted laser beam, and the first reflector 116 reflects and couples the laser beam to the micro Raman generation and imaging module 12. The micro Raman generation and imaging module 12 includes at least a Raman dichroic band-edge filter 121, an imaging dichroic band-edge filter 122, a second reflecting mirror 123, and a microscope objective 125. The laser beam emitted from the laser emitting module 11 is reflected by the Raman dichroic band-edge filter 121, transmitted by the imaging dichroic band-edge filter 122, and reflected by the second reflecting mirror 123, and then focused by the microscope objective 125 onto the sample 126 to be tested. The backscattered Raman spectral signal generated by the sample 126 to be tested is collected by the microscope objective 125 and converted into parallel light, and then reflected by the second reflecting mirror 123, transmitted by the imaging dichroic band-edge filter 122, and transmitted by the Raman dichroic band-edge filter 121, and then output to the Raman collection module 13. The Raman collection module 13 includes a third reflecting mirror 131, a long-pass filter 132, a first lens 133, and a spectrometer 134. The Raman spectral signal output by the micro Raman generation and imaging module 12 is reflected by the third reflecting mirror 131 and transmitted through the long-pass filter 132, and then focused by the first lens 133 into the slit 134-1 of the spectrometer 134. The spectrometer 134 then performs spectral dispersion and detection. The central control module 14 includes a controller 141 and a host computer 142. The controller 141 is connected to the laser 111, the first shutter 112, the spectrometer 134 and the host computer 142 respectively and performs data interaction.

[0025] In some embodiments, the micro Raman generation and imaging module 12 further includes a white LED light source 129, a second shutter 128, a second lens 1210, a fourth reflector 1211, and an imaging camera 1212. The white LED light source 129 and the second shutter 128 are disposed below the sample 126 to be tested. The white LED light source 129 provides an illumination beam for imaging the sample 126 to be tested. The second shutter 128 controls the rapid opening and closing of the illumination beam. After the illumination beam is irradiated onto the sample 126 to be tested by the second shutter 128, it is collected by the microscope objective 125 and output as parallel light. Subsequently, it is reflected sequentially by the second reflector 123, reflected by the imaging dichroic bandgap filter 122, focused by the second lens 1210, and reflected by the fourth reflector 1211 to the imaging camera 1212 for imaging. The controller 141 is connected to the white LED light source 129, the second shutter 128, and the imaging camera 1212 and performs data interaction.

[0026] Specifically, during single-cell image acquisition, a white LED light source 129 provides illumination for the overall imaging optical path, and the imaging contrast is enhanced by transmissive illumination. The white LED light source 129 has a focusing lens on its end face, so that the emitted light beam is distributed in parallel.

[0027] Specifically, the second lens 1210 is selected from a matching tube lens or a cemented doublet achromatic lens to improve image quality.

[0028] Specifically, the laser emission module 11 is the basis for generating efficient Raman signals. The laser 111 provides a pump source for the generation of Raman signals and has the characteristics of narrow linewidth, miniaturization, high power and wavelength stability. The first shutter 112 is used for the rapid opening and closing of the laser beam to meet the rapid switching requirements of spectral detection and white light imaging. The narrowband filter 113 is used to purify the spectral properties of the laser output 111 and isolate the interference from other bands of the laser.

[0029] In some embodiments, the plano-concave lens 114 is a negative focal length lens, with the concave surface of the plano-concave lens 114 facing the incident laser beam; the plano-convex lens 115 is a positive focal length lens, with the plane of the plano-convex lens 115 facing the incident laser beam, and the distance between the plano-concave lens 114 and the plano-convex lens 115 is the sum of their working focal lengths.

[0030] In some embodiments, the ratio of the positive focal length of the plano-convex lens 115 to the negative focal length of the plano-concave lens 114 is equal to the ratio of the diameter of the entrance pupil of the microscope objective 125 to the diameter of the laser beam emitted by the laser 111.

[0031] Specifically, the plano-concave lens 114 and the plano-convex lens 115 are combined to form a Galilean telescope system, so that the diameter of the emitted laser beam is equal to the size of the entrance pupil of the microscope objective 125, thereby improving the beam coupling efficiency of the microscope objective 125 and further improving the quality of the imaging spot.

[0032] In some embodiments, the surfaces of the plano-concave lens 114 and the plano-convex lens 115 are provided with an anti-reflection coating for the incident laser band to reduce the power loss of the emitted laser beam.

[0033] In some embodiments, the first reflector 116 is set at a 45° angle to the incident laser beam, and the Raman dichroic band-edge filter 121 is set at a 45° angle to the incident laser beam; when the incident beam forms a 45° angle with the surface of the Raman dichroic band-edge filter 121, it has a high laser reflectivity, thereby avoiding power loss when the pump laser is coupled into the micro Raman system.

[0034] The working process of the laser emitting module 11 is as follows: the laser beam output by the laser 111 passes through the shutter 112 and the narrowband filter 113, and is then vertically incident on the plano-concave lens 114 and the plano-convex lens 115. The center of the laser beam is incident on the reflector 116 and then achieves 45° mirror reflection.

[0035] In some embodiments, the sample to be tested 126 is placed on a two-dimensional motion platform 127, and the controller 141 is connected to the two-dimensional motion platform 127 and controls the two-dimensional motion platform 127 to move the sample to be tested 126 on the plane to adjust the focusing position on the sample to be tested 126.

[0036] Specifically, the two-dimensional motion platform 127 carries the sample 126 to be tested and realizes large-scale, high-precision, and rapid mechanical scanning in the two-dimensional XY plane, thereby providing hardware support for the effective range and efficiency of the single-cell target to be tested.

[0037] Specifically, the two-dimensional motion platform 127 is an existing position movement control platform. Its structure is not limited here, as long as it meets the usage requirements, and will not be described in detail.

[0038] In some embodiments, the microscope objective 125 is disposed on the piezoelectric displacement stage 124, and the controller 141 is connected to the piezoelectric displacement stage 124 and controls the piezoelectric displacement stage 124 to drive the microscope objective 125 to move vertically to adjust the focusing height.

[0039] Specifically, the piezoelectric displacement stage 124 is equipped with a microscope objective 125 to achieve real-time dynamic focusing of the microscope objective 125. This addresses the problem of inconsistent Z-axis height distribution between metabolite molecules in single-cell micro-regions and metal nanoparticles in the sample under test, enabling high-performance real-time Raman signal acquisition in micro-regions.

[0040] Specifically, the piezoelectric displacement stage 124 adopts an existing nanometer-level high-precision piezoelectric displacement control platform. Its structure is not limited here, as long as it meets the usage requirements, and will not be described in detail.

[0041] Specifically, the microscope objective 125 uses an ultra-long working distance plan achromatic infinity objective, which can provide a larger working space for the sample and facilitate the actual operation of the sample.

[0042] In some embodiments, the spectrometer 134 includes a slit 134-1, an internal mirror 134-2, a first concave mirror 134-3, a reflective grating 134-4, a second concave mirror 134-5, and an internal camera 134-6. After the Raman spectral signal is transmitted through the slit 134-1, it is reflected by the internal mirror 134-2 to the first concave mirror 134-3, collimated by it into parallel light, and incident on the surface of the reflective grating 134-4 to complete the spectral dispersion. The dispersed beam is reflected again to the second concave mirror 134-5, and after being reflected and focused by it, it is projected onto the focal plane of the internal camera 134-6.

[0043] Specifically, after the Raman signal is redirected by the third reflecting mirror 131, it is incident perpendicularly onto the long-pass filter 132 and focused by the first lens 133 into the slit 134-1 of the spectrometer 134 to achieve spectral dispersion and detection. The power of the incident laser beam is significantly attenuated by the long-pass filter 132, thereby ensuring that the laser power incident on the Raman spectrometer does not exceed the safety threshold of the internal camera 134-6 of the spectrometer 134, and minimizing interference with the true Raman spectral intensity.

[0044] Specifically, the spectrometer 134 performs high-sensitivity spectral dispersion and data acquisition on the incident Raman signal. The internal camera 134-6 uses a high-sensitivity low-temperature CCD camera for spectral detection. To match the different spectral band ranges of the Raman characteristic peaks of the actual sample 126, a spectrometer with a rotatable reflection grating 134-4 is selected. By rotating the angle of the internal reflection grating 134-4, the Raman spectral band range can be adjusted, thereby meeting the adjustment requirements of the actual spectral coverage band according to the Raman spectral band usage requirements of different samples.

[0045] In some embodiments, the focal length of the first lens 133 is matched with the focal length of the equivalent optical path corresponding to the NA (Numerical Aperture) factor of the spectrometer 134. That is, the focal length of the first lens 133 is the same as the focal length of the equivalent optical path calculated by the spectrometer 134 based on the NA value and the aperture D, so as to obtain better Raman beam acquisition efficiency.

[0046] Specifically, the first lens 133 is a plano-convex lens with an anti-reflection coating on its surface.

[0047] Specifically, the central control module 14 realizes the signal connection and control of the laser emission module 11, the micro Raman generation and imaging module 12 and the Raman collection module 13, and completes the final data graphic display and storage through software.

[0048] Specifically, the host computer 142 is equipped with image recognition software, which realizes functions such as intelligent recognition of the single cell image to be tested within the detection field of view, intelligent planning of Raman spectroscopy micro-region path, and automatic signal acquisition. The image recognition software adopts existing image recognition software, which only needs to meet the functional requirements, and will not be described in detail here.

[0049] Figure 2 This is an automated Z-axis focusing image of metal nanoparticles surrounding a single cell in a droplet, as presented in this invention. Figure 3 This is the image recognition result and path planning diagram of a single cell in a droplet according to the present invention.

[0050] See Figure 2 and Figure 3 , Figure 2 To determine the optimal nano-metal particles near a single cell by using a particle sharpness algorithm to screen out the surface images with the most ideal focusing effect on the particle surface in the images captured by the imaging camera 1212, and to carry out Raman spectral path planning and spectral acquisition work at this spatial height layer. Figure 3 The study demonstrates image recognition results of single cells in droplets, as well as an automated planning scheme and acquisition process for the Raman spectroscopy detection region required for single-cell metabolic analysis, thereby improving the efficiency of single-cell metabolic detection and enhancing the repeatability and accuracy of the detection results.

[0051] The surface-enhanced Raman imaging system for single-cell metabolism detection provided by this invention includes a laser emission module 11, a micro-Raman generation and imaging module 12, a Raman collection module 13, and a central control module 14. Through the collaborative operation of these modules, an automated detection process for single-cell metabolism is achieved: a monochromatic laser emitted by the laser source 111, after being adjusted and controlled by the laser emission module 11 and the micro-Raman generation and imaging module 12, is focused onto the single-cell sample, exciting the sample to generate a Raman scattering signal; this Raman signal is transmitted to the Raman collection module 13 to complete the photoelectric signal processing. The conversion and output module 12 is the core component of the single-cell metabolic surface-enhanced Raman imaging optical system. It provides hardware support for the efficient generation of micro Raman spectral signals and the acquisition of single-cell bright-field images, ensuring the acquisition of single-cell images within the micro-area of ​​the measured sample, automatic centering of single cells, and automatic focusing of nano-metal particles. The central control module 14, in conjunction with the above three modules, realizes the functions of intelligent recognition of the single-cell image to be tested within the detection field of view, intelligent planning of the Raman spectral micro-area path, and automatic signal acquisition.

[0052] The central control module 14 can control the shutter closure in the spectral acquisition and imaging optical paths according to preset parameters, avoiding the influence of the imaging light source on the spectral acquisition quality and the influence of laser intensity on the imaging quality. The controller 141 drives the two-dimensional motion platform 127 to achieve precise displacement of single-cell samples, and drives the piezoelectric displacement stage 124 to achieve automatic focusing of the microscope objective 125. In conjunction with microscopic imaging, it realizes automatic positioning and identification of single cells, automatic image focusing of metal nanoparticles, and automatic planning and acquisition of spectral detection areas. The whole process does not require manual adjustment, which can meet the detection needs of single-cell samples of different sizes and types, reducing the errors caused by manual operation and significantly shortening the detection time.

[0053] Although specific embodiments have been described above, these embodiments are not intended to limit the scope of the invention, even when only a single embodiment is described with respect to a particular feature. The feature examples provided in this disclosure are intended to be illustrative and not limiting, unless otherwise stated. In practice, one or more technical features of the dependent claims may be combined with the technical features of the independent claims as needed and where technically feasible, and may be derived from the technical features of the respective independent claims in any suitable manner rather than solely by the specific combinations listed in the claims.

[0054] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A surface-enhanced Raman imaging system for single-cell metabolic detection, characterized in that, It includes a laser emission module, a micro Raman generation and imaging module, a Raman collection module, and a central control module; The laser emitting module includes a laser, a first shutter, a narrowband filter, a plano-concave lens, a plano-convex lens, and a first reflecting mirror arranged in sequence. The laser generates a laser beam, the first shutter controls the rapid opening and closing of the laser beam, the narrowband filter allows light of a set wavelength band of the laser beam to pass through, the plano-concave lens and the plano-convex lens together adjust the diameter of the emitted laser beam, and the first reflecting mirror reflects and couples the laser beam to the micro Raman generation and imaging module. The micro Raman generation and imaging module includes at least a Raman dichroic band-edge filter, an imaging dichroic band-edge filter, a second reflecting mirror, and a microscope objective. The laser beam emitted by the laser emitting module is reflected sequentially by the Raman dichroic band-edge filter, transmitted by the imaging dichroic band-edge filter, and reflected by the second reflecting mirror. It is then focused onto the sample under test by the microscope objective. The backscattered Raman spectral signal generated by the sample under test is collected by the microscope objective and converted into parallel light. It is then reflected sequentially by the second reflecting mirror, transmitted by the imaging dichroic band-edge filter, and transmitted by the Raman dichroic band-edge filter before being output to the Raman collection module. The Raman collection module includes a third reflecting mirror, a long-pass filter, a first lens, and a spectrometer. The Raman spectral signal output by the micro Raman generation and imaging module is reflected by the third reflecting mirror and transmitted through the long-pass filter, and then focused by the first lens into the slit of the spectrometer. The spectrometer then performs spectral dispersion and detection. The central control module includes a controller and a host computer. The controller is connected to the laser, the first shutter, the spectrometer, and the host computer and interacts with them for data exchange.

2. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The micro-Raman generation and imaging module further includes a white LED light source, a second shutter, a second lens, a fourth mirror, and an imaging camera. The white LED light source and the second shutter are positioned below the sample to be tested. The white LED light source provides an illumination beam for imaging the sample. The second shutter controls the rapid opening and closing of the illumination beam. After the illumination beam illuminates the sample through the second shutter, it is collected by the microscope objective and output as parallel light. Subsequently, it is reflected sequentially by the second mirror, reflected by the imaging dichroic bandgap filter, focused by the second lens, and reflected by the fourth mirror to the imaging camera for imaging. The controller is connected to the white LED light source, the second shutter, and the imaging camera and performs data interaction.

3. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The microscope objective is mounted on a piezoelectric displacement stage, and the controller is connected to the piezoelectric displacement stage and controls the piezoelectric displacement stage to move the microscope objective vertically to adjust the focusing height.

4. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The sample to be tested is placed on a two-dimensional motion platform. The controller is connected to the two-dimensional motion platform and controls the two-dimensional motion platform to move the sample to be tested on the plane to adjust the focusing position on the sample.

5. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The plano-concave lens is a negative focal length lens, with its concave surface facing the incident laser beam; the plano-convex lens is a positive focal length lens, with its flat surface facing the incident laser beam, and the distance between the plano-concave lens and the plano-convex lens is the sum of their working focal lengths.

6. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 5, characterized in that, The ratio of the positive focal length of the plano-convex lens to the negative focal length of the plano-concave lens is equal to the ratio of the diameter of the entrance pupil of the microscope objective to the diameter of the laser beam emitted by the laser.

7. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 5, characterized in that, Both the plano-concave lens and the plano-convex lens have an anti-reflection coating on their surfaces for the incident laser band.

8. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The first reflector is set at a 45° angle to the incident laser beam, and the Raman dichroic banded filter is set at a 45° angle to the incident laser beam.

9. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The spectrometer includes a slit, an internal mirror, a first concave mirror, a reflective grating, a second concave mirror, and an internal camera. The Raman spectral signal is transmitted through the slit and reflected by the internal mirror to the first concave mirror, where it is collimated into parallel light and incident on the surface of the reflective grating to complete the spectral dispersion. The dispersed beam is then reflected again to the second concave mirror, where it is reflected and focused before being projected onto the focal plane of the internal camera.

10. The surface-enhanced Raman imaging system for single-cell metabolic detection according to claim 1, characterized in that, The focal length of the first lens matches the focal length of the equivalent optical path corresponding to the NA factor of the spectrometer.

Images