Single-pixel fluorescent Raman bimodal imaging system and method

Abstract

The invention discloses a single-pixel fluorescent Raman bimodal imaging system and method, and belongs to the technical field of optical imaging. Comprising a light source module, a coding structured light module, a microscopic imaging module, a sample control module and a signal collection module which are sequentially arranged along a light path, the light source module is used for generating laser with required wavelength; the encodable structured light module converts the received laser into structured light; the microscopic imaging module amplifies the structured light, projects the amplified structured light to a sample plane, and collects fluorescence, elastic scattering and inelastic scattering generated after the sample is excited; the signal collection module amplifies, separates and collects the received fluorescence signals and scattering signals, converts the light signals into electric signals, and transmits the electric signals to the control and calculation module for storage and subsequent processing. According to the invention, the problem that the existing imaging system cannot obtain time-space consistent multi-modal images under the same excitation source and cannot realize image fusion is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical imaging, and in particular relates to a single-pixel fluorescence Raman dual-modality imaging system and method. Background Art

[0002] Bioimaging is an important research method to understand the structure of biological tissues and to clarify the various physiological functions of organisms. It uses optical or electron microscopes to directly obtain microscopic structural images of biological cells and tissues, and analyzes the obtained images to understand the various physiological processes of biological cells. At present, the commonly used bioimaging technologies are fluorescence imaging and Raman imaging. Fluorescence imaging uses the changes in the fluorescence characteristics of organisms to obtain optical images, and Raman microscopy is a non-labeled material analysis method. Due to the lack of endogenous fluorescent materials in many biological structures and biological processes, it is difficult to use the intrinsic fluorescence of biological samples for imaging and detection. Therefore, in most cases, it is necessary to introduce exogenous fluorescent materials as contrast agents to calibrate specific cells or tissue organs, which can realize the positioning and morphological analysis of imaged cells in tissues. However, there are great challenges for the fluorescent labeling of various lipids, sugars and other substances in tissue cells. The simultaneous analysis of multiple biological macromolecules in cells requires solving the problems of optical path design, the difficulty of simultaneous labeling of multiple fluorescent molecules, and the high research cost. Raman imaging technology can perform non-labeled specific imaging of biological samples and avoid interference from aqueous solutions. However, traditional Raman imaging has problems such as slow imaging speed in point scanning, field curvature and photon scattering in line scanning, and low imaging quality in wide-field illumination.

[0003] Based on the problems existing in the above-mentioned fluorescence imaging and Raman imaging, the prior art combines the two detection modes of fluorescence and Raman to obtain a fluorescence Raman dual-modal imaging system, uses transmission fluorescence imaging technology as the positioning system of imaging cells in tissues, and obtains the molecular fingerprint spectrum of imaging cells through Raman scattering technology, making full use of their respective advantages for complementarity and synergy, so that the detection sensitivity and accuracy are greatly improved. However, in the existing fluorescence / Raman wide-field imaging, different devices need to be used to obtain them separately, such as scientific research-grade CCD as a sensor for image acquisition, and the imaging field of view is perpendicular to the CCD axis. Therefore, when the Raman / fluorescence image comes from different array detectors that are not coaxial, the image parameters such as image field of view, imaging angle, resolution, number of pixels, etc. are all different, that is, spatial mismatch. On the other hand, different imaging devices have different clocks, so there may be synchronization difficulties between the two in time. It can be seen that it is difficult to obtain images of different modalities at the same time on the same machine, resulting in time and space mismatches between the images, which brings difficulties to image fusion, image analysis, and target positioning. Summary of the invention

[0004] Technical issues to be solved:

[0005] In order to avoid the shortcomings of the prior art, the present invention provides a single-pixel fluorescence Raman dual-modality imaging system and method, which fuses fluorescence imaging and Raman imaging, and then combines single-pixel imaging technology to perform fluorescence Raman dual imaging, that is, the field of view, resolution, number of pixels and other parameters of single-pixel images obtained using different detectors under the same excitation source are completely matched, thereby achieving error-free image fusion. The present invention solves the problem that the existing imaging system cannot obtain multi-modality images that are consistent in time and space under the same excitation source and achieve image fusion.

[0006] The technical solution of the present invention is: a single-pixel fluorescence Raman dual-modal imaging system, comprising a light source module, a coded structured light module, a microscopic imaging module, a sample control module, and a signal collection module arranged in sequence along the optical path, wherein the signal collection module transmits the collected fluorescence signal and Raman signal to the control and calculation module, and obtains a two-dimensional or three-dimensional image after processing;

[0007] The light source module is used to generate laser light of a required wavelength;

[0008] The encodable structured light module converts the received laser into structured light and projects it to the microscopic imaging module;

[0009] The microscopic imaging module amplifies the structured light and projects it onto the sample plane, while collecting the fluorescence, Rayleigh scattered light and Raman scattered light generated by the sample after being excited;

[0010] The sample control module is used to adjust and fix the position of the sample, and place the sample at the position of the imaging field of view;

[0011] The signal collection module amplifies, separates, and collects the received fluorescence signals and scattered signals, and then converts the optical signals into electrical signals, which are then transmitted to the control and calculation module for storage and subsequent processing.

[0012] A further technical solution of the present invention is that the light source module is a laser with different wavelengths, a continuous wave laser or a multi-wavelength LED light source.

[0013] A further technical solution of the present invention is: the codable structured light module generates structured light through a digital micromirror device, a spatial light modulator or a physical grating; the digital micromirror device generates structured light by loading a binary grating pattern on the digital micromirror device, and generating structured light in the focal plane of the objective lens by projection or imaging; the spatial light modulator generates structured light by combining filtering and utilizing the interference of two coherent light beams of ±1 order diffraction to generate structured light in the focal plane of the objective lens.

[0014] A further technical solution of the present invention is: a beam shaping unit is arranged between the continuous wave laser and the digital micromirror device, including a first filter, a first lens and a second lens arranged in sequence along the optical path to expand the light source emitted by the laser; the first filter uses a bandpass filter to filter out stray light and retain the excitation light of the imaging object; the first lens and the second lens are both convex lenses with focal lengths of 50mm and 150mm respectively, forming a 4f system for beam expansion; the first filter is placed on the front focus of the first lens, and the coded structured light plane is located on the back focus of the second lens.

[0015] A further technical solution of the present invention is: the microscopic imaging module inputs structured light into the sample to excite fluorescence signals and Raman scattering signals, including a collimator, a first dichroic mirror, and a microscope objective lens arranged in sequence along the optical path; the first dichroic mirror transmits the excitation signal to the signal collection module, and refracts the light within the wavelength range of the excitation light of the imaging object to the focal plane of the microscope objective lens through the first dichroic mirror; the fluorescence, Rayleigh scattered light and Raman scattered light generated by the excited sample are collected by the microscope objective lens, and the Rayleigh scattered light is filtered out by the first dichroic mirror to obtain a fluorescence-Raman signal.

[0016] A further technical solution of the present invention is: the sample control module is a multi-dimensional rotating table, which can adjust any position and angle of the sample in space.

[0017] A further technical solution of the present invention is: the signal collection module includes a panoramic photosynthesizer, a second dichroic mirror, a fluorescence signal collection unit and a Raman signal collection unit; after the output light of the microscopic imaging module passes through the panoramic photosynthesizer, the fluorescence-Raman signal is separated by the second dichroic mirror and transmitted to the fluorescence signal collection unit and the Raman signal collection unit respectively; wherein the fluorescence signal of the reflected fluorescence signal collection unit passes through the second filter and the third lens in sequence and is collected by the first single-point detector; the Raman signal transmitted to the Raman signal collection unit passes through the third filter and the tube lens in sequence and is collected by the second single-point detector; the first single-point detector collection and the second single-point detector collection both send the collected data to the control and calculation module through the collection card.

[0018] A further technical solution of the present invention is: the second dichroic mirror is selected according to the wavelength range of the fluorescence signal to be reflected and the Raman signal to be transmitted; the central wavelengths of the second filter and the third filter match the wavelengths of the fluorescence or Raman signal to be separated, the bandpass width of the second filter only allows the fluorescence signal to pass through and blocks light of other wavelengths, and the bandpass width of the third filter only allows the Raman signal to pass through and blocks light of other wavelengths.

[0019] A further technical solution of the present invention is: the control and calculation module includes a control unit and a calculation unit, the light source module, the codable structured light module, the microscopic imaging module, the sample control module, and the signal collection module are controlled by the control unit, and the collected photoelectric signal is restored into a two-dimensional or three-dimensional image by the calculation unit. The computational imaging algorithm used is a corresponding algorithm for matching the structured light, the structured light is Fourier basis light, and the corresponding algorithm is an inverse Fourier algorithm.

[0020] An imaging method of a single-pixel fluorescence Raman dual-modality imaging system, the specific steps are as follows:

[0021] According to the wavelength band of light excited by the sample, select the filter to collect fluorescence signal and Raman signal;

[0022] A light source module, a codable structured light module, a microscopic imaging module, a sample control module, and a signal collection module are arranged along the light path and are connected to the control and calculation modules respectively;

[0023] Fluorescently label the samples before imaging;

[0024] The outgoing laser of the light source module is passed through the coded structured light module to obtain structured light with bright and dark stripes; the structured light is projected onto the sample through the microscopic imaging module, and the microscopic imaging module collects the fluorescence, Rayleigh scattered light and Raman scattered light generated by the sample excitation, and after filtering out the Rayleigh scattered light, it is emitted to the signal collection module to collect the fluorescence signal and Raman signal respectively, and the collected signals are sent to the control and calculation module for storage and subsequent processing;

[0025] The control and calculation module processes the fluorescence signal to obtain a fluorescence image, acquires the fluorescence localization of the cells to be tested in the tissue and the morphology and biochemical information of specific organelles; and processes the Raman signal to obtain a Raman spectrum image of the cells to be tested with fluorescence localization in the sample tissue.

[0026] Beneficial Effects

[0027] The beneficial effects of the present invention are as follows: the present invention combines a Raman microscopy imaging system based on structured light illumination with a fluorescence microscopy imaging system into dual-modal single-pixel imaging, providing synergistic advantages over any single modality; a panoramic photosynthesizer is used in the fusion process of the two imaging systems to ensure that the fluorescence-Raman signal is collected in one space while obtaining a large field of view, and combined with single-pixel imaging technology, the image fusion problem of the existing fluorescence-Raman imaging system is solved.

[0028] The present invention uses single-pixel imaging to solve the problem of fluorescence-Raman dual-modal image fusion, without the need for complex scanning or the use of complex multi-array detectors. The field of view, resolution, number of pixels and other parameters of single-pixel images obtained using different detectors are completely matched, achieving error-free image fusion. Since the fluorescence and Raman processes usually occur within picoseconds to tens of nanoseconds, they can be regarded as occurring simultaneously on a macro scale. The addition of a panoramic photosynthesizer allows the fluorescence signal and the Raman signal to be collected by the detector at the same time. Therefore, single-pixel imaging can achieve the use of the same excitation source to obtain a multi-modal image that is consistent in time and space, solving the image fusion problem from the principle of image acquisition, facilitating precise co-localization, and bringing great convenience to image fusion and image analysis. On the other hand, using two single-point detectors instead of scientific research-grade CCDs as sensors for image acquisition is more cost-effective.

[0029] Preferably, the present invention uses a single-point detector combined with a spatial coding strategy to replace the planar array detector as a weak light signal detection unit. The spatial coding strategy can use a random coding pattern, a Fourier coding pattern, and a Hadamard pattern. The complex image acquisition process can be simplified into a series of single-point measurements, reducing the complexity of data acquisition and the amount of data processed. When these patterns are irradiated onto the sample, the structural information of the sample will be encoded into the phase and amplitude of the light wave, and a computational reconstruction algorithm (such as compressed sensing technology) is used to reconstruct the image of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a structural diagram of a single-pixel fluorescence Raman dual-modality imaging system according to an embodiment of the present invention;

[0031] Figure 2 This is a working principle diagram of a single-pixel fluorescence Raman dual-modality imaging system according to an embodiment of the present invention.

[0032] Explanation of the accompanying drawings: 1. Laser source, 2. First filter, 3. First lens, 4. Second lens, 5. Digital micromirror array, 6. First dichroic mirror, 7. Microscope objective, 8. Three-dimensional rotating sample stage, 9. Panoramic photosynthesizer, 10. Second dichroic mirror, 11. Second filter, 12. Third lens, 13. First single-point detector, 14. Third filter, 15. Tube lens, 16. Second single-point detector acquisition, 17. Acquisition card, 18. Computer. DETAILED DESCRIPTION

[0033] The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0034] Based on the problem that the existing imaging system cannot obtain multi-modal images with consistent time and space under the same excitation source and realize image fusion, the present invention provides a single-pixel fluorescence Raman dual-modal imaging system, comprising a light source module, a codable structured light module, a microscopic imaging module, a sample control module, and a signal collection module arranged in sequence along the optical path, wherein the signal collection module transmits the respectively collected fluorescence signals and Raman signals to the control and calculation module, and obtains a two-dimensional or three-dimensional image after processing; the light source module is used to generate laser light of required wavelength; the codable structured light module converts the received laser light into structured light and projects it to the microscopic imaging module; the microscopic imaging module amplifies the structured light and projects it to the sample plane, and at the same time collects the fluorescence, Rayleigh scattered light and Raman scattered light generated by the sample after being excited; the sample control module is used to adjust the sample and fix the position of the sample, and place the sample at the position of the imaging field of view; the signal collection module amplifies, separates and collects the received fluorescence signal and scattered signal, and then converts the optical signal into an electrical signal, and transmits it to the control and calculation module for storage and subsequent processing.

[0035] Specifically, the light source module is a laser with different wavelengths, a continuous wave laser or a multi-wavelength LED light source.

[0036] Specifically, the codable structured light module generates structured light through a digital micromirror device, a spatial light modulator or a physical grating; the digital micromirror device generates structured light by loading a binary grating pattern on the digital micromirror device, and generating structured light in the focal plane of the objective lens by projection or imaging; the spatial light modulator generates structured light by combining filtering and utilizing the interference of two coherent light beams of ±1 order diffraction to generate structured light in the focal plane of the objective lens.

[0037] Specifically, a beam shaping unit is arranged between the continuous wave laser and the digital micromirror device, including a first filter, a first lens and a second lens arranged in sequence along the optical path to expand the light source emitted by the laser. According to the emission wavelength of the required laser, the first filter uses a bandpass filter to filter out stray light and retain the excitation light of the imaging object. The first lens and the second lens are convex lenses with focal lengths of 50mm and 150mm respectively to form a 4f system for beam expansion, and the diameter of the light beam is adjusted so that the excitation light can completely cover the spatial light modulator. The focal length of the first lens is different from the focal length of the second lens. The first filter is placed at the front focus of the first lens, and the coded structured light plane is located at the back focus of the second lens. The focal length used by the lens is determined according to the range of the imaging object.

[0038] Specifically, the microscopic imaging module includes a collimator, a first dichroic mirror, and a microscope objective lens, which are arranged in sequence along the optical path, and are used to input structured light into the sample to excite fluorescence signals and Raman scattering signals. According to needs, the first dichroic mirror is used to transmit the excited signals to the fluorescence imaging module or the Raman imaging module, and the light within the wavelength range of the excitation light of the imaging object is refracted to the focal plane of the microscope objective lens through the first dichroic mirror; the fluorescence, Rayleigh scattered light and Raman scattered light generated by the excited sample are collected by the microscope objective lens, and the Rayleigh scattered light is filtered out by the first dichroic mirror to obtain a fluorescence-Raman signal.

[0039] Specifically, the sample control module is a multi-dimensional rotating table, which can adjust any position and angle of the sample in space.

[0040] Specifically, the signal collection module includes a panoramic photosynthesizer, a second dichroic mirror, a fluorescence signal collection unit and a Raman signal collection unit. After the output light of the microscopic imaging module passes through the panoramic photosynthesizer, the fluorescence-Raman signal is separated by the second dichroic mirror and transmitted to the fluorescence signal collection unit and the Raman signal collection unit respectively, wherein the fluorescence signal reflected from the fluorescence signal collection unit passes through the second filter and the third lens in sequence and is collected by the first single-point detector, and the Raman signal transmitted to the Raman signal collection unit passes through the third filter and the tube lens in sequence and is collected by the second single-point detector; the first single-point detector collection and the second single-point detector collection both send the collected data to the control and computing module through the collection card.

[0041] A single-point detector combined with a spatial coding strategy is used to replace the array detector as a weak light signal detection unit. The spatial coding strategy can use random coding patterns, Fourier coding patterns, and Hadamard patterns. The complex image acquisition process can be simplified into a series of single-point measurements, reducing the complexity of data acquisition and the amount of data processed. When these patterns are irradiated onto the sample, the structural information of the sample will be encoded into the phase and amplitude of the light wave, and a computational reconstruction algorithm (such as compressed sensing technology) is used to reconstruct the image of the sample.

[0042] Specifically, the dichroic mirror needs to be optimized for a specific wavelength to reflect or transmit light of a specific wavelength. For fluorescence signals and Raman signals, the fluorescence signal is usually in a shorter wavelength range (such as the ultraviolet to visible light region), while the Raman signal is in a longer wavelength range (such as the near-infrared region). Therefore, the second dichroic mirror needs to be selected according to these wavelength ranges. The center wavelength of the second filter and the third filter should match the wavelength of the fluorescence or Raman signal to be separated. The passband width of the filter needs to be narrow enough to allow only specific signals to pass while blocking light of other wavelengths. There should be enough depth in the cutoff area to prevent unwanted wavelengths from passing through. The focal length of the third lens and the tube lens needs to be selected according to the optical path design of the system to ensure that the light coming out of the filter can be correctly focused on the detector. The focal length of the lens is usually matched with the focal length of the objective lens to maintain the conjugate relationship of the system.

[0043] Specifically, the control and calculation module includes a control unit and a calculation unit. The control unit controls the light source module, the codable structured light module, the microscopic imaging module, the sample control module, and the signal collection module. The calculation unit restores the collected photoelectric signal into a two-dimensional or three-dimensional image. The computational imaging algorithm used is a corresponding algorithm that matches the structured light. The structured light is Fourier basis light, and the corresponding algorithm is an inverse Fourier algorithm.

[0044] The present invention provides an imaging method of a single-pixel fluorescence Raman dual-modality imaging system, and the specific steps are as follows:

[0045] Step 1: Select the filter to collect fluorescence and Raman signals according to the wavelength of light excited by the sample;

[0046] Step 2: Arrange the light source module, the coded structured light module, the microscopic imaging module, the sample control module, and the signal collection module along the light path, and connect them to the control and calculation modules respectively;

[0047] Step 3: The imaging object needs to be fluorescently labeled before imaging;

[0048] Use biological internal fluorescent groups or some exogenous fluorescent groups for calibration to confirm the exact location of the imaging target;

[0049] Step 4: The outgoing laser of the light source module is passed through the coded structured light module to obtain structured light with bright and dark stripes; the structured light is projected onto the sample through the microscopic imaging module, and the microscopic imaging module collects the fluorescence, Rayleigh scattered light and Raman scattered light generated by the sample excitation, and after filtering out the Rayleigh scattered light, it is emitted to the signal collection module to collect the fluorescence signal and Raman signal respectively, and the collected signals are sent to the control and calculation module for storage and subsequent processing;

[0050] Step 5: The control and calculation module processes the fluorescence signal to obtain a fluorescence image, acquires the fluorescence localization of the cells to be tested in the tissue and the morphology and biochemical information of specific organelles; and processes the Raman signal to obtain a Raman spectrum image of the cells to be tested with fluorescence localization in the sample tissue.

[0051] The above technical solution is further described below with reference to the accompanying drawings and examples:

[0052] Embodiment 1 takes a continuous wave laser, a digital micromirror array, and a computer as examples to specifically illustrate the invention. The hardware process is shown in the accompanying drawings.

[0053] Reference Figure 1 , Figure 2 As shown, this embodiment is based on a fluorescence Raman dual-modality single-pixel imaging system device, including a light source module, a structured light illumination module, a microscopic imaging module, a sample control module, a signal collection module, and a control and calculation module.

[0054] Laser 1 is a continuous light semiconductor laser with an excitation wavelength of 532nm; the continuous wave laser light source enters the beam shaping unit composed of multiple lenses at the rear end, and the LBTEK large-aperture fixed-magnification laser beam expander used is an optical system based on a telescope structure that can expand (or reduce) the diameter of the parallel incident light spot to a certain magnification, and then project it onto the DMD surface after beam expansion.

[0055] The model of the digital micromirror array modulator 5 (DMD) is the V-6501VIS spatial light modulator of the German ViALUX company. It is composed of 1920x1080 7.56um digital micromirrors, which can realize the modulation of light in the range of 400-2200nm, and generate structured light with bright and dark stripes after reflection by the DMD. In this embodiment, the modulation matrix adopts the Fourier matrix, and the projection objective receives the structured light stripes through the dichroic mirror, and projects it onto the sample plane after amplifying it at a certain magnification. The projection objective used is the Olympus plan achromatic objective lens, which has a standard RMS thread and adopts an infinity-corrected plan achromatic design. The plan achromatic objective lens effectively corrects the field curvature and distortion, and at the same time corrects the aberration of two wavelengths in the visible spectrum, so it has good field flatness and small spherical aberration and chromatic aberration; the dichroic mirror used is LBTEK brand, which can separate the light beam according to the wavelength. The long-wave dichroic mirror can transmit the light beam higher than the starting wavelength and reflect the light beam lower than the starting wavelength, and has the characteristics of high transmittance and high reflection. The cut-off wavelength can be selected as 550nm, and the average transmittance and average reflectance within the specified band can reach more than 90%.

[0056] After the sample is excited, fluorescence, elastic scattering and inelastic scattering are generated. The sample can be a polystyrene molecule with a fluorescent marker or a biological sample with a fluorescent marker. The signal of the system is collected by the microscope objective lens, and the Rayleigh scattered light is first filtered out by a dichroic mirror, and then the fluorescence and Raman signals of the sample are collected by a panoramic photosynthesizer. The panoramic photosynthesizer is a customized optical device for collecting light signals in a large area. After the fluorescence signal reflected by the dichroic mirror is selected by a narrow-band filter, the selected narrow-band filter is an ultra-narrow linewidth tunable filter BVF-300CL: the central wavelength is adjustable, the minimum filter linewidth can reach 30pm (3.7GHz), and the filter slope can reach 1500dB / nm. It is then collected by a large numerical aperture collection lens and received by a high-sensitivity single-point optical signal detector. The selected single-point detector is the S1227 Series silicon photodiode. The photosensitivity of this series of silicon photodiodes can reach 0.36-0.43A / W at typical wavelengths, ensuring high photoelectric conversion efficiency. It can capture light signals that are weaker than the human eye. It also has the characteristics of a wide wavelength range, covering a wide band from ultraviolet light to near-infrared light (usually 190nm to 1100nm). After converting the optical signal into an electrical signal, it is transmitted to the control and computing module for storage and subsequent processing. It can be accurately collected and processed in a multi-channel data acquisition device to obtain a fluorescent image, realize the fluorescence localization of the cells to be tested in the tissue, and obtain specific organelle morphology and biochemical information;

[0057] The Raman signal of the sample is transmitted through the dichroic mirror, and then collected by the tube lens with a focal length of 180mm matched with the objective lens used. After the wavelength is selected by the wavelength tunable filter, it is received by the single-point light signal detector. The detector converts the light signal into an image signal, and after processing, the Raman spectrum image of the fluorescently located cells to be tested in the sample tissue is obtained. The multi-channel data acquisition card model used in the multi-channel data acquisition device is NI-6361, and the maximum number of single-ended analog input channels is 16.

[0058] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A single-pixel fluorescence Raman dual-modality imaging system, characterized in that: It includes a light source module, a coded structured light module, a microscopic imaging module, a sample control module, and a signal collection module which are sequentially arranged along the light path. The signal collection module transmits the collected fluorescence signals and Raman signals to the control and calculation module, and obtains a two-dimensional or three-dimensional image after processing. The light source module is used to generate laser light of a required wavelength; The encodable structured light module converts the received laser into structured light and projects it to the microscopic imaging module; The microscopic imaging module amplifies the structured light and projects it onto the sample plane, while collecting the fluorescence, Rayleigh scattered light and Raman scattered light generated by the sample after being excited; The sample control module is used to adjust and fix the position of the sample, and place the sample at the position of the imaging field of view; The signal collection module amplifies, separates, and collects the received fluorescence signals and scattered signals, and then converts the optical signals into electrical signals, which are then transmitted to the control and calculation module for storage and subsequent processing.

2. A single-pixel fluorescence Raman dual-modality imaging system according to claim 1, characterized in that: The light source module is a laser with different wavelengths, a continuous wave laser or a multi-wavelength LED light source.

3. A single-pixel fluorescence Raman dual-modality imaging system according to claim 2, characterized in that: The codable structured light module generates structured light through a digital micromirror device, a spatial light modulator or a physical grating; the digital micromirror device generates structured light by loading a binary grating pattern on the digital micromirror device and generating structured light in the focal plane of the objective lens by projection or imaging; the spatial light modulator generates structured light by combining filtering and utilizing the interference of two coherent light beams of ±1 order diffraction to generate structured light in the focal plane of the objective lens.

4. The single-pixel fluorescence Raman dual-modality imaging system according to claim 1, characterized in that: A beam shaping unit is arranged between the continuous wave laser and the digital micromirror device, including a first filter, a first lens and a second lens arranged in sequence along the optical path to expand the light source emitted by the laser; the first filter uses a bandpass filter to filter out stray light and retain the excitation light of the imaging object; the first lens and the second lens are both convex lenses with focal lengths of 50 mm and 150 mm respectively, forming a 4f system for beam expansion; the first filter is placed on the front focus of the first lens, and the coded structured light plane is located on the back focus of the second lens.

5. A single-pixel fluorescence Raman dual-modality imaging system according to claim 4, characterized in that: The microscopic imaging module inputs structured light into the sample to excite the fluorescence signal and the Raman scattering signal, and includes a collimator, a first dichroic mirror, and a microscope objective lens arranged in sequence along the light path; the first dichroic mirror transmits the excited signal to the signal collection module, and refracts the light within the wavelength range of the excitation light of the imaging object to the focal plane of the microscope objective lens through the first dichroic mirror; The fluorescence, Rayleigh scattered light and Raman scattered light generated by the excited sample are collected by the microscope objective, and the Rayleigh scattered light is filtered out by the first dichroic mirror to obtain the fluorescence-Raman signal.

6. The single-pixel fluorescence Raman dual-modality imaging system according to claim 1, characterized in that: The sample control module is a multi-dimensional rotating table, which can adjust any position and angle of the sample in space.

7. The single-pixel fluorescence Raman dual-modality imaging system according to claim 1, characterized in that: The signal collection module comprises a panoramic photosynthesizer, a second dichroic mirror, a fluorescence signal collection unit and a Raman signal collection unit. After the outgoing light of the microscopic imaging module passes through the panoramic photosynthesizer, the fluorescence-Raman signal is separated by the second dichroic mirror and transmitted to the fluorescence signal collection unit and the Raman signal collection unit respectively. The fluorescence signal of the reflected fluorescence signal collection unit passes through the second filter and the third lens in sequence and is collected by the first single-point detector. The Raman signal transmitted to the Raman signal collection unit passes through the third filter and the tube lens in sequence and is collected by the second single-point detector. The first single-point detector and the second single-point detector both send the collected data to the control and calculation module through the acquisition card.

8. A single-pixel fluorescence Raman dual-modality imaging system according to claim 7, characterized in that: The second dichroic mirror is selected according to the wavelength range of the fluorescence signal to be reflected and the Raman signal to be transmitted; the central wavelengths of the second filter and the third filter match the wavelengths of the fluorescence or Raman signal to be separated, the bandpass width of the second filter only allows the fluorescence signal to pass through and blocks light of other wavelengths, and the bandpass width of the third filter only allows the Raman signal to pass through and blocks light of other wavelengths.

9. The single-pixel fluorescence Raman dual-modality imaging system according to claim 1, characterized in that: The control and calculation module includes a control unit and a calculation unit. The control unit controls the light source module, the codable structured light module, the microscopic imaging module, the sample control module, and the signal collection module. The calculation unit restores the collected photoelectric signal into a two-dimensional or three-dimensional image. The computational imaging algorithm used is a corresponding algorithm that matches the structured light. The structured light is Fourier basis light, and the corresponding algorithm is an inverse Fourier algorithm.

10. An imaging method of the single-pixel fluorescence Raman dual-modality imaging system according to any one of claims 1 to 9, characterized in that The specific steps are as follows: According to the wavelength band of light excited by the sample, select the filter to collect fluorescence signal and Raman signal; A light source module, a codable structured light module, a microscopic imaging module, a sample control module, and a signal collection module are arranged along the light path and are connected to the control and calculation modules respectively; Fluorescently label the samples before imaging; The laser emitted by the light source module passes through the coded structured light module to obtain structured light with bright and dark stripes; The structured light is projected onto the sample through the microscopic imaging module, and the microscopic imaging module collects the fluorescence, Rayleigh scattered light and Raman scattered light generated by the sample excitation. After filtering out the Rayleigh scattered light, it is transmitted to the signal collection module to collect the fluorescence signal and Raman signal respectively, and the collected signals are sent to the control and calculation module for storage and subsequent processing; The control and calculation module processes the fluorescence signal to obtain a fluorescence image, thereby obtaining the fluorescence localization of the cells to be tested in the tissue and the morphology and biochemical information of specific organelles; After processing the Raman signal, a Raman spectrum image of the fluorescently localized cells to be tested in the sample tissue is obtained.

Images