High temporal and spatial resolution multiplexed wide-field Raman imaging system and method based on flat-top illumination

Abstract

A high-temporal-spatial-resolution multiplexed wide-field Raman imaging system and method based on flat-top illumination includes a light source module, a laser modulation module, a confocal excitation module, a wide-field excitation module based on flat-top illumination, a spectral module, a wide-field imaging module, and a spectral and image control module. The confocal excitation mode is used to obtain a high-signal-to-noise ratio Raman spectrum to determine the characteristic peaks of the Raman spectrum. The wide-field excitation mode based on flat-top illumination can excite the sample with a large field of view and uniform intensity, while simultaneously achieving rapid detection of a single Raman characteristic peak or monitoring of its dynamic changes with high spatiotemporal resolution. It can also capture wide-field hyperspectral images covering the entire spectral range. Multiplexed wide-field imaging enables fully spatiotemporally synchronized monitoring and comparison of the dynamic changes of multiple characteristic peaks. This system facilitates accurate quantitative or qualitative analysis of systems with spatially heterogeneous sample distribution, intensity fluctuations, or dynamic changes, and has broad application prospects in fields such as biomedicine and physical chemistry.

Description

Technical Field

[0001] The present invention relates to the fields of optical microscopic imaging technology and spectral imaging technology, and in particular to a high temporal and spatial resolution multiplexed wide-field Raman imaging system and method based on flat-top illumination. Background Art

[0002] As a label-free molecular fingerprint spectrum, Raman spectroscopy is mainly used to achieve qualitative or quantitative analysis of the molecular structure and composition of substances. Generally, traditional Raman spectroscopy data is one-dimensional, and it is difficult to distinguish the differences and dynamic changes in chemical information in different regions of the sample space. Raman spectroscopy imaging technology combines Raman spectroscopy technology with optical imaging technology, and can simultaneously obtain hyperspectral images containing sample spatial information and spectral information, thereby achieving qualitative, quantitative and positioning analysis of substance molecules. However, the classic step-by-step scanning imaging method, such as point scanning or line scanning imaging method, has a scanning time of at least seconds or even slower for samples in larger areas. At the same time, due to the time delay and imaging error of the step-by-step scanning method, the imaging process has serious spatiotemporal asynchrony, and it is impossible to study the dynamic change process with a time resolution of most milliseconds.

[0003] Fortunately, widefield imaging methods can address these challenges. This method directly acquires large-field, single-wavelength imaging information within a single camera exposure (milliseconds), exhibiting spatiotemporal synchronization and high spatial and temporal resolution. This approach is highly suitable for dynamic systems where only characteristic Raman peaks need to be studied. For static or relatively stable samples, this method not only enables rapid detection of characteristic peaks but also allows for the acquisition of hyperspectral images in a very short time using a wavelength scanning device, while maintaining imaging speeds faster than most scanning imaging techniques.

[0004] However, in conventional wide-field Raman imaging systems, a single imaging detector cannot achieve temporal and spatial synchronous dynamic imaging comparison and analysis of multiple characteristic peaks, which limits the possibility of simultaneous analysis of multiple species. Secondly, the excitation light source of a wide-field Raman imaging system is usually a laser with a Gaussian intensity distribution. Its uneven excitation intensity will cause the source of intensity information in the wide-field Raman intensity image to be uncertain, which is not conducive to the study of systems with spatial distribution heterogeneity, intensity fluctuations or dynamic changes. At the same time, uneven excitation intensity will produce field-dependent image quality and spectral signal-to-noise ratio, reduce the effective imaging field of view, and hinder high-throughput, high-quality wide-field Raman imaging. Summary of the Invention

[0005] The present invention aims to address the aforementioned problems in the prior art by providing a high-temporal-spatial-resolution multiplexed wide-field Raman imaging system and method based on flat-top illumination. A confocal excitation mode is used to obtain a high signal-to-noise ratio Raman spectrum, thereby determining the Raman spectral characteristic peaks of the sample. A wide-field excitation mode based on flat-top illumination can excite the sample with a large field of view and uniform intensity, while simultaneously enabling rapid detection of a single Raman characteristic peak or monitoring of its dynamic changes with high spatiotemporal resolution. It can also capture wide-field hyperspectral images covering the entire spectral range. Furthermore, the multiplexed wide-field imaging method enables dynamic monitoring and comparison of multiple characteristic peaks with complete spatiotemporal synchronization. The present invention addresses the problems of slow imaging speed and spatiotemporal asynchrony in scanning imaging technology, as well as the field-dependent image quality and spectral signal-to-noise ratio, as well as the limited effective field of view, caused by uneven laser intensity excitation. This facilitates accurate quantitative or qualitative analysis of systems with spatially heterogeneous sample distribution, intensity fluctuations, or dynamic changes, and has broad application prospects in fields such as biomedicine and physical chemistry.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention relates to a high temporal and spatial resolution dual-channel wide-field Raman imaging system based on flat-top illumination, comprising a light source module, a laser modulation module, a confocal excitation module, a wide-field excitation module based on flat-top illumination, a spectrum module, a wide-field imaging module, and a spectrum and image control module;

[0008] The light source module is used to select lasers of different wavelengths;

[0009] The laser modulation module is used to achieve modulation of laser line width, power, beam quality and beam size;

[0010] The confocal excitation module is used to excite the sample to generate Raman scattering in a confocal manner;

[0011] The wide-field excitation module based on flat-top illumination is used to excite the sample with uniform intensity in a wide-field manner to generate Raman scattering;

[0012] The spectral module is used to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio;

[0013] The wide-field imaging module is used to realize single-wavelength or high-spectral wide-field Raman imaging and multiplexed spatiotemporal synchronous wide-field Raman imaging;

[0014] The spectrum and image control module is used to control the grating conversion and detector image acquisition parameter settings of the spectrum module, and to control the wavelength selection and scanning, multi-channel detector image acquisition and synchronous triggering of the wide-field imaging module.

[0015] Preferably, the light source module comprises two parallel, equi-height placed first and second high-quality single-mode lasers, and the laser wavelengths include but are not limited to 532 nm and 660 nm.

[0016] Preferably, in the laser modulation module, a first laser line filter and a first reflector are placed after the first high-quality single-mode laser; and a second laser line filter and a dichroic mirror are placed after the second high-quality single-mode laser.

[0017] Preferably, in the laser modulation module, the laser light emitted by the first or second high-quality single-mode laser passes through a dichroic mirror and then sequentially passes through an adjustable polarization attenuator, a spatial filter, a variable magnification and zoom beam expander and a second reflector.

[0018] Preferably, the adjustable polarization attenuator comprises a rotatable mount, an achromatic or zero-order half-wave plate, a laser or broadband polarization beam splitter cube, a light block or a light trap.

[0019] Preferably, the spatial filter includes an aspheric lens or an objective lens, a high damage threshold precision pinhole, a first lens, and a first adjustable aperture.

[0020] Preferably, the variable magnification and zoom beam expander is an achromatic variable magnification and zoom beam expander with a sliding focus or a rotating focus mechanism.

[0021] Preferably, the confocal excitation module transmits lasers of different wavelengths through a first flip mirror, a second lens, a third lens, a third mirror, a second flip mirror, a Raman filter and an objective lens to a sample plane to excite the sample to generate Raman scattering.

[0022] Preferably, the Raman filter comprises a 532nm or 660nm edge filter.

[0023] Preferably, the objective lens is an air immersion, water immersion or oil immersion objective lens.

[0024] Preferably, the wide-field excitation module based on flat-top illumination includes a fourth reflector, a beam shaper, a fourth lens, a fifth lens, a sixth lens, a Raman filter and an objective lens.

[0025] Preferably, the beam shaper is an achromatic beam shaper capable of adjusting laser divergence.

[0026] Preferably, the spectral module includes a reflective mirror block of a microscope turret, a seventh lens, an eighth lens, an adjustable slit, a multi-grating spectrometer and a detector.

[0027] Preferably, the multi-grating spectrometer includes but is not limited to gratings of 300 gr / mm, 1200 gr / mm and 1800 gr / mm.

[0028] Preferably, the detector is a CCD.

[0029] Preferably, the wide-field imaging module includes a space of a microscope turret, a beam splitter, a first wide-field imaging channel, a fifth reflecting mirror, and a second wide-field imaging channel.

[0030] Preferably, the beam splitter is a beam splitter with a 50:50 ratio or other light splitting ratio.

[0031] Preferably, the first wide-field imaging channel includes a first imaging lens, a first wavelength scanning device, and a first detector which are placed in sequence.

[0032] Preferably, the first wavelength scanning device is a liquid crystal tunable filter.

[0033] Preferably, the first detector is an EMCCD or sCMOS.

[0034] Preferably, the second wide-field imaging channel includes a second imaging lens, a second wavelength scanning device, and a second detector which are placed in sequence.

[0035] Preferably, the second wavelength scanning device is an angle-tunable filter.

[0036] Preferably, the second detector is an EMCCD or sCMOS.

[0037] A high temporal and spatial resolution multiplexed wide-field Raman imaging method based on flat-top illumination comprises the following steps:

[0038] (1) Selecting lasers of different wavelengths through the light source module, the laser wavelengths including but not limited to 532nm and 660nm;

[0039] (2) using the adjustable polarization attenuator to adjust the power of lasers of different wavelengths;

[0040] (3) Using the zoom beam expander to adjust the diameter of lasers of different wavelengths;

[0041] (4) When the confocal excitation module is used, the first flip mirror and the second flip mirror are cut into the optical path, and the variable magnification and zoom beam expander is appropriately adjusted so that the laser beam diameter can completely fill the clear aperture of the objective lens, so as to generate a minimum focus spot to excite the sample to produce Raman scattering;

[0042] (5) After the Raman scattering signal is obtained using the confocal excitation module, the reflector of the microscope turret needs to be cut into the optical path, and the Raman signal enters the spectral module to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio, which is used to determine the characteristic peak of the Raman spectrum;

[0043] (6) When using the wide-field excitation module based on flat-top illumination, the first flip mirror and the second flip mirror are cut out of the optical path, and the variable-magnification and zoom beam expander is used to set specific laser beam diameters for lasers of different wavelengths to match the beam shaping requirements of the beam shaper and obtain a flat-top beam. The flat-top beam is focused on or near the rear focal plane of the objective lens, and a large field of view and uniform intensity excitation of the sample is achieved on the sample plane through the objective lens;

[0044] Preferably, the diameter of the 532nm and 660nm specific laser beams is between 6mm and 7mm;

[0045] Preferably, the wide field excitation module based on flat top illumination can generate a maximum of about 110×110 μm on the sample plane. 2 Effective field of view, including but not limited to 110×110μm 2 ;

[0046] (7) After obtaining the Raman scattering signal using the wide-field excitation module based on flat-top illumination, it is necessary to cut the idle position of the microscope turret into the optical path, and the Raman signal enters the first or second wide-field imaging channel to quickly obtain a characteristic peak image or a hyperspectral image;

[0047] (8) After obtaining the Raman scattering signal using the wide-field excitation module based on flat-top illumination, it is necessary to cut the idle space of the microscope turret into the optical path, and the Raman signal simultaneously enters the first and second wide-field imaging channels to realize multi-channel wide-field Raman imaging.

[0048] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0049] (1) The present invention uses confocal Raman spectroscopy technology to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio, which can determine more accurate Raman spectral characteristic peaks;

[0050] (2) The high spatiotemporal resolution wide-field Raman imaging technology of the present invention has a millisecond-level imaging time resolution, which can achieve high-throughput, high spatiotemporal resolution dynamic imaging of a single Raman characteristic peak or rapid hyperspectral imaging of the entire spectral range;

[0051] (3) The multiplexed wide-field Raman imaging technology of the present invention can realize the temporal and spatial synchronous dynamic imaging comparison and analysis of multiple characteristic peaks, as well as background subtraction of complex systems for multi-species analysis;

[0052] (4) The wide-field Raman imaging technology based on flat-top illumination of the present invention can obtain field-independent image quality and spectral signal-to-noise ratio, improve the effective imaging field of view, and enhance the imaging throughput and quality of the wide-field Raman imaging system. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 4 is a block diagram of a high spatiotemporal resolution multiplexed wide-field Raman imaging system based on flat-top illumination in an embodiment of the present invention;

[0054] Figure 2 Schematic diagram of the optical path structure of a high spatiotemporal resolution multiplexed wide-field Raman imaging system based on flat-top illumination in an embodiment of the present invention;

[0055] Figure 3 for Figure 2 Schematic diagram of the disassembled confocal excitation mode;

[0056] Figure 4 for Figure 2 Schematic diagram of the structure of the wide-field excitation mode based on flat-top illumination disassembled in [4];

[0057] Figure 5 Diagram showing the implementation steps of the high spatiotemporal resolution multiplexed wide-field Raman imaging method based on flat-top illumination. DETAILED DESCRIPTION

[0058] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0059] like Figure 1 As shown, a high temporal and spatial resolution multiplexed wide-field Raman imaging system based on flat-top illumination includes a light source module, a laser modulation module, a confocal excitation module, a spectral module, a wide-field excitation module based on flat-top illumination, a wide-field imaging module, and a spectral and image control module;

[0060] The light source module is used to provide lasers of different wavelengths;

[0061] The laser modulation module is connected to the laser modulation module and is used to realize the modulation of laser line width, power, beam quality and beam size;

[0062] The confocal excitation module is connected to the laser modulation module and is used to excite the sample to generate Raman scattering in a confocal manner;

[0063] The wide-field excitation module based on flat-top illumination is connected to the laser modulation module and is used to excite the sample to generate Raman scattering in a wide-field manner with a light beam of uniform intensity distribution;

[0064] The spectral module is connected to the confocal excitation module to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio;

[0065] The wide-field imaging module is connected to a wide-field excitation module based on flat-top illumination to realize single-wavelength or high-spectral wide-field Raman imaging and multiplexed spatiotemporal synchronous wide-field Raman imaging;

[0066] The spectrum and image control module is connected to the spectrum module and the wide-field imaging module, and is used to control the grating conversion and detector image acquisition of the spectrum module, as well as the wavelength selection and scanning, multi-channel detector image acquisition and synchronous triggering of the wide-field imaging module.

[0067] The light source module includes a first laser and a second laser with different wavelengths, and the laser wavelengths include 532nm and 660nm.

[0068] The laser modulation module includes a first laser line filter, a first reflector, and a second laser line filter, a dichroic mirror, an adjustable polarization attenuator, a spatial filter, a variable magnification and zoom beam expander assembly, and a second reflector, which are sequentially arranged behind the first laser.

[0069] The adjustable polarization attenuator includes a rotating half-wave plate, a polarization beam splitter cube and a light block arranged in sequence; the rotating half-wave plate includes an adjustable rotating mount and a half-wave plate; the adjustable polarization attenuator is used to continuously change the ratio of S-polarized light and P-polarized light of the incident linearly polarized laser. After reflection, the S-polarized light is absorbed by the light block, thereby achieving continuously adjustable attenuation of the P-polarized light.

[0070] The spatial filter is used to improve the quality of the laser beam and increase the laser ellipticity, and includes an aspheric lens or objective lens, a precision pinhole, a first lens and an adjustable iris arranged in sequence; the adjustable iris is used to eliminate high-order diffraction; the variable magnification and zoom beam expander assembly includes a variable magnification and zoom beam expander and an adjustable iris; the variable magnification and zoom beam expander is used to adjust the laser beam size and laser collimation; the adjustable iris is used to further eliminate high-order diffraction.

[0071] The confocal excitation module includes a first flip reflector, a second lens, a third lens, a third reflector, a second flip reflector, a Raman filter and an objective lens which are arranged in sequence.

[0072] The wide-field excitation module based on flat-top illumination includes a fourth reflector, a beam shaper, a fourth lens, a fifth lens, a sixth lens, a Raman filter and an objective lens, which are arranged in sequence; the beam shaper is used to convert laser light into a flat-top beam.

[0073] The spectrum module includes a reflector block of a microscope turret, a seventh lens, an eighth lens, an adjustable slit, a multi-grating spectrometer and a detector which are arranged in sequence; the detector is a CCD camera.

[0074] The wide-field imaging module includes a gap on the microscope turret, a beam splitter, a first wide-field imaging channel, a fifth reflector, and a second wide-field imaging channel; the first wide-field imaging channel includes a first imaging lens, a first wavelength scanning device, and a first detector placed in sequence; the second wide-field imaging channel includes a second imaging lens, a second wavelength scanning device, and a second detector placed in sequence; the first or second wavelength scanning device includes a liquid crystal tunable filter and an angle tunable filter; the first or second detector includes an EMCCD camera or an sCMOS camera.

[0075] Specifically, if Figure 2 As shown, a high-temporal-spatial-resolution multiplexed wide-field Raman imaging system based on flat-top illumination is shown. When laser 1 is used, the laser light emitted by laser 1 is reflected by laser line filter 2, reflector 3, and dichroic mirror 6 into the system's main optical path. When laser 4 is used, the laser light emitted by laser 4 is transmitted through laser line filter 5 and dichroic mirror 6 into the system's main optical path. Lasers of different wavelengths pass through an adjustable polarization attenuator consisting of a rotating achromatic half-wave plate 7, a broadband polarization beam splitter cube 8, and a light barrier 9 to adjust the laser power. A spatial filter consisting of an aspheric lens or objective lens 10, a precision pinhole 11, a lens 12, and an adjustable aperture 13 improves the laser beam quality, increases the laser ellipticity, and eliminates higher-order diffraction. The laser beam diameter and collimation are then adjusted by a variable-magnification and zoom beam expander 14. An adjustable aperture 15 further eliminates higher-order diffraction. The laser light then passes through a reflector 16 and enters the subsequent optical path. At this point, a flip mirror 17 can be used to split the excitation light path into two.

[0076] If the flip mirror 17 is cut into the light path, the laser passes through the lens 18 and the lens 19 to adjust the laser beam diameter and extend the propagation distance of the laser beam. The laser is reflected by the mirror 20 to the flip mirror 21 that is also cut into the light path. Then the collimated laser is reflected by the Raman filter 22 to the back focal plane of the objective lens 23. The objective lens 23 generates a confocal point light spot on the sample plane to stimulate the sample to produce Raman scattering. In this process, the sample three-dimensional adjustment platform 24 is needed to focus and select the area of ​​interest of the sample.

[0077] If the flip mirror 17 and the flip mirror 21 are cut out of the light path, the laser will be reflected by the mirror 25 and enter the beam shaper 26, converting the laser into a flat-top beam. The flat-top beam then passes through lenses 27 and 28 to further expand the beam diameter, improve the uniformity of the flat-top beam, and extend the propagation distance of the flat-top beam. The flat-top beam is then focused by lens 29 onto the back focal plane or near the back focal plane of the objective lens 23. The objective lens 23 generates a collimated or defocused uniform intensity large light spot on the sample plane to excite the sample to produce Raman scattering. In this process, the sample three-dimensional adjustment platform 24 is required to focus and select the area of ​​interest of the sample.

[0078] The Raman scattering signals obtained under the above different paths are collected by the objective lens 23 and then filtered out Rayleigh scattering and stray light by the Raman filter 22. At this time, the Raman scattering signal will pass through the microscope turret, which can divide the collection light path into two paths.

[0079] If the reflector block 30 of the microscope turret is cut into the optical path, the Raman scattering signal is reflected by the reflector and then relayed by lenses 31 and 32 to extend the propagation of the Raman scattering signal. The Raman scattering signal is then focused onto an adjustable slit 34 by lens 33 to control spectral resolution and block stray light. The filtered Raman scattering signal then enters the spectrometer 35 and CCD 36 to collect a one-dimensional Raman spectrum.

[0080] If the gap 37 in the microscope turret is cut into the optical path, the Raman scattering signal propagates in the infinity-corrected optical path composed of lens 38 and lens 39, and then can be divided into equal-proportion Raman scattering signals after passing through a 50:50 beam splitter. Half of the Raman scattering signal enters the first wide-field imaging channel composed of an imaging lens 40, a liquid crystal tunable filter 42, and an EMCCD 43, and the other half of the Raman scattering signal enters the second wide-field imaging channel composed of an angle-tunable filter 45, an imaging lens 46, and an EMCCD 47 through a reflector 44. Either the first or the second wide-field imaging channel can achieve wide-field Raman imaging.

[0081] Finally, during actual operation, the computer 48 can be used to control the spectrometer 35, CCD 36, liquid crystal tunable filter 42, EMCCD 43, angle tunable filter 45, and EMCCD 47, thereby realizing the acquisition and control settings of the spectrum and image.

[0082] Figure 3 for Figure 2 A schematic diagram of the disassembled confocal excitation mode structure in a high-temporal-spatial-resolution multiplexed wide-field Raman imaging system based on flat-top illumination. In confocal excitation mode, the light source module selects lasers of different wavelengths. The laser modulation module then modulates the laser intensity, beam quality, and beam diameter. The variable-magnification and zoom beam expander 14 then appropriately adjusts the laser beam diameter to ensure that the laser in the confocal excitation module completely fills the aperture of the objective lens 23, achieving a minimum focused spot to excite the sample and generate Raman scattering. The Raman scattering signal then enters the spectral module to obtain a high-signal-to-noise ratio one-dimensional Raman spectrum for determining the characteristic peaks of the Raman spectrum. During this process, the spectral and image control module is used to control the grating conversion and CCD image acquisition parameter settings of the spectral module.

[0083] Figure 4 for Figure 2A schematic diagram of the disassembled widefield excitation mode structure based on flat-top illumination in a high-temporal-spatial-resolution multiplexed widefield Raman imaging system based on flat-top illumination. In widefield excitation mode, the light source module selects lasers of different wavelengths, and the laser modulation module modulates the laser intensity, beam quality, and beam diameter. The variable-magnification beam expander 14 adjusts the specific beam diameter for each wavelength to match the beam shaping requirements of the beam shaper 26 and produce a flat-top beam. The widefield excitation module based on flat-top illumination then excites the sample over a large field of view with uniform intensity and obtains a Raman scattering signal. The Raman scattering signal then enters the widefield imaging module. Using only a single widefield imaging channel, high-throughput, high-temporal-spatial-resolution dynamic imaging of a single Raman characteristic peak or rapid hyperspectral imaging across the entire spectral range can be achieved. Using two widefield imaging channels simultaneously enables simultaneous temporal-spatial dynamic imaging, comparison, and analysis of multiple characteristic peaks, as well as background subtraction for complex systems involving multi-species analysis. In this process, the spectral and image control module controls wavelength selection and scanning, multi-channel detector image acquisition, and synchronous triggering.

[0084] like Figure 5 As shown, an imaging method of an ultra-fast large-field super-resolution fluorescence microscopy imaging system comprises the following steps:

[0085] (1) Selecting lasers of different wavelengths through the light source module, the laser wavelengths including but not limited to 532nm and 660nm;

[0086] (2) using the adjustable polarization attenuator to adjust the power of lasers of different wavelengths;

[0087] (3) Using the zoom beam expander to adjust the diameter of lasers of different wavelengths;

[0088] (4) When using the confocal excitation mode, the flip mirror 17 and the flip mirror 21 are cut into the optical path, and the zoom beam expander 14 is appropriately adjusted so that the laser beam diameter can completely fill the clear aperture of the objective lens 23, so as to generate a minimum focus spot to excite the sample to produce Raman scattering;

[0089] (5) After obtaining the Raman scattering signal using the confocal excitation mode, the reflector block 30 of the microscope turret is cut into the optical path, and the Raman scattering signal enters the spectral module to achieve one-dimensional Raman spectrum acquisition with a high signal-to-noise ratio;

[0090] (6) When the wide-field excitation mode based on flat-top illumination is used, the flip mirror 17 and the flip mirror 21 are cut out of the optical path, and the variable-magnification and zoom beam expander 14 is used to set the specific laser beam diameters for lasers of different wavelengths to match the beam shaping requirements of the beam shaper 26 and obtain a flat-top beam. The flat-top beam is focused on the rear focal plane of the objective lens 23 and then on the sample plane. Then, the wide-field excitation module based on flat-top illumination is used to achieve large-field-of-view, uniform-intensity excitation of the sample and obtain Raman scattering signals;

[0091] (7) After obtaining the Raman scattering signal using the wide-field excitation module based on flat-top illumination, the gap 37 of the microscope turret cuts into the optical path, and the Raman scattering signal enters the wide-field imaging module to quickly obtain a characteristic peak image or a hyperspectral image, or to achieve multi-channel wide-field Raman imaging;

[0092] (8) In steps (5) and (7), the computer 48 is used to realize the acquisition and control of the spectrum and image, mainly used to control the grating conversion and CCD image acquisition parameter setting of the spectrum module, or to control the selection and scanning of wavelengths, multi-channel detector image acquisition and synchronous triggering;

[0093] In summary, the present invention provides a high-temporal-spatial-resolution multiplexed wide-field Raman imaging system and method based on flat-top illumination. The method utilizes a confocal Raman spectroscopy optical path to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio, and can determine more accurate Raman spectral characteristic peaks. The method utilizes high-temporal-spatial-resolution wide-field Raman imaging technology with millisecond-level imaging time resolution to achieve high-throughput, high-temporal-spatial-resolution dynamic imaging of a single Raman characteristic peak or rapid hyperspectral imaging over the entire spectral range. The method utilizes multiplexed wide-field Raman imaging technology to achieve temporal-spatial-synchronous dynamic imaging comparison and analysis of multiple characteristic peaks, as well as background subtraction for complex systems for multi-species analysis. The method utilizes wide-field Raman imaging technology based on flat-top illumination to obtain field-independent image quality and spectral signal-to-noise ratio, thereby increasing the effective imaging field of view and improving the imaging throughput and quality of the wide-field Raman imaging system.

[0094] It should be noted that the above-described specific embodiment merely further illustrates a preferred embodiment of a high-temporal-spatial-resolution multiplexed wide-field Raman imaging system and method based on flat-top illumination, but the present invention is not limited to this embodiment. Any simple modifications, equivalent modifications, and variations made by a person of ordinary skill in the art to the optical paths, optical elements, or optical path switching methods of the above-described embodiments based on the technical essence of the present invention fall within the scope of protection of the technical solution of the present invention. Furthermore, although certain specific terms are used in this specification, these terms are for convenience only and do not constitute any limitation to the present invention.

Claims

1. A high temporal and spatial resolution multiplexed wide-field Raman imaging system based on flat-top illumination, characterized by: It includes light source module, laser modulation module, confocal excitation module, wide-field excitation module based on flat-top illumination, spectrum module, wide-field imaging module and spectrum and image control module; The light source module is used to provide lasers of different wavelengths; The laser modulation module is connected to the laser modulation module and is used to realize the modulation of laser line width, power, beam quality and beam size; The confocal excitation module is connected to the laser modulation module and is used to excite the sample to generate Raman scattering in a confocal manner; The wide-field excitation module based on flat-top illumination is connected to the laser modulation module and is used to excite the sample to generate Raman scattering in a wide-field manner with a light beam of uniform intensity distribution; The spectral module is connected to the confocal excitation module to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio; The wide-field imaging module is connected to a wide-field excitation module based on flat-top illumination to realize single-wavelength or high-spectral wide-field Raman imaging and multiplexed spatiotemporal synchronous wide-field Raman imaging; The spectrum and image control module is connected to the spectrum module and the wide-field imaging module, and is used to control the grating conversion and detector image acquisition of the spectrum module, as well as the wavelength selection and scanning, multi-channel detector image acquisition and synchronous triggering of the wide-field imaging module; The confocal excitation module includes a first flip reflector, a second lens, a third lens, a third reflector, a second flip reflector, a Raman filter and an objective lens, which are arranged in sequence; The wide-field excitation module based on flat-top illumination includes a fourth reflector, a beam shaper, a fourth lens, a fifth lens, a sixth lens, a Raman filter, and an objective lens, which are arranged in sequence; the beam shaper is used to convert laser light into a flat-top beam; The first flip reflector and the second flip reflector are used to switch the light path into the confocal excitation module or the wide-field excitation module based on flat-top illumination; when the first flip reflector and the second flip reflector are cut in, the light path enters the confocal excitation module; when the first flip reflector and the second flip reflector are cut out, the light path enters the wide-field excitation module based on flat-top illumination; The spectrum module includes a reflector block of a microscope turret, a seventh lens, an eighth lens, an adjustable slit, a multi-grating spectrometer and a detector arranged in sequence; the detector is a CCD camera; The wide-field imaging module includes a gap on the microscope turret, a beam splitter, a first wide-field imaging channel, a fifth reflector, and a second wide-field imaging channel; the first wide-field imaging channel includes a first imaging lens, a first wavelength scanning device, and a first detector placed in sequence; the second wide-field imaging channel includes a second wavelength scanning device, a second imaging lens, and a second detector placed in sequence; the first wavelength scanning device includes a liquid crystal tunable filter, and the second wavelength scanning device includes an angle tunable filter; the first detector includes an EMCCD camera or an sCMOS camera, and the second detector includes an EMCCD camera or an sCMOS camera.

2. The high spatiotemporal resolution multiplexed wide-field Raman imaging system based on flat-top illumination according to claim 1, characterized in that: The light source module includes a first laser and a second laser with different wavelengths, and the laser wavelengths include 532 nm and 660 nm.

3. The high spatiotemporal resolution multiplexed wide-field Raman imaging system based on flat-top illumination according to claim 2, characterized in that: The laser modulation module includes a first laser line filter, a first reflector, and a second laser line filter, a dichroic mirror, an adjustable polarization attenuator, a spatial filter, a variable magnification and zoom beam expander assembly, and a second reflector, which are sequentially arranged behind the first laser.

4. The high spatiotemporal resolution multiplexed wide-field Raman imaging system based on flat-top illumination according to claim 3, characterized in that: The adjustable polarization attenuator includes a rotating half-wave plate, a polarization beam splitter cube and a light block arranged in sequence; the rotating half-wave plate includes an adjustable rotating mount and a half-wave plate; the adjustable polarization attenuator is used to continuously change the ratio of S-polarized light and P-polarized light of the incident linearly polarized laser. After reflection, the S-polarized light is absorbed by the light block, thereby achieving continuously adjustable attenuation of the P-polarized light.

5. The high spatiotemporal resolution multiplexed wide-field Raman imaging system based on flat-top illumination according to claim 4, characterized in that: The spatial filter is used to improve the quality of the laser beam and increase the laser ellipticity, and includes an aspheric lens or objective lens, a precision pinhole, a first lens and an adjustable iris arranged in sequence; the adjustable iris is used to eliminate high-order diffraction; the variable magnification and zoom beam expander assembly includes a variable magnification and zoom beam expander and an adjustable iris; the variable magnification and zoom beam expander is used to adjust the laser beam size and laser collimation; the adjustable iris is used to further eliminate high-order diffraction.

6. A high temporal and spatial resolution multiplexed wide-field Raman imaging method based on flat-top illumination, characterized in that: The following steps are involved: (1) Use the light source module to select lasers with different wavelengths, including 532 nm and 660 nm; (2) Using an adjustable polarization attenuator to adjust the power of lasers of different wavelengths; (3) Use a variable magnification and zoom beam expander to adjust the laser diameter of different wavelengths; (4) When using the confocal excitation module, the first flip mirror and the second flip mirror are cut into the optical path, and the zoom beam expander is appropriately adjusted so that the laser beam diameter can completely fill the aperture of the objective lens, which is used to generate the minimum focus spot to excite the sample to produce Raman scattering; (5) After obtaining the Raman scattering signal using the confocal excitation module, the reflector block of the microscope turret is cut into the optical path, and the Raman signal enters the spectral module to obtain a one-dimensional Raman spectrum with a high signal-to-noise ratio, which is used to determine the characteristic peaks of the Raman spectrum; (6) When using a wide-field excitation module based on flat-top illumination, the first flip mirror and the second flip mirror are cut out of the optical path, and a variable-magnification zoom beam expander is used to set the specific laser beam diameter for different wavelength lasers to match the beam shaping requirements of the beam shaper and obtain a flat-top beam. The flat-top beam is focused to the rear focal plane of the objective lens or near the rear focal plane, and a large field of view and uniform intensity excitation of the sample is achieved on the sample plane through the objective lens; (7) After obtaining the Raman scattering signal using a wide-field excitation module based on flat-top illumination, the gap of the microscope turret is cut into the optical path, and the Raman signal enters a single wide-field imaging channel to quickly obtain a characteristic peak image or a hyperspectral image, which is used to achieve high-throughput, high-temporal-resolution dynamic imaging of a single characteristic peak or hyperspectral wide-field imaging of the entire spectral range; (8) After obtaining the Raman scattering signal using a wide-field excitation module based on flat-top illumination, the gap of the microscope turret is cut into the optical path, and the Raman signal enters two wide-field imaging channels at the same time, which is used to achieve temporal and spatial synchronous dynamic imaging comparison and analysis of multiple characteristic peaks.

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