Microscopic imaging system and method

Abstract

The present application is applicable to the field of optical microscope imaging technology, and provides a microscopic imaging system and method, which includes: an illumination optical path module, a detection optical path module, an imaging processing module and a stage, wherein the illumination optical path module is used to incident a laser beam into an observation sample on the stage at a preset incident angle; the detection optical path module is used to collect the fluorescence signal refracted by the observation sample, separate and transform the multiple wavelength beams in the fluorescence signal, and obtain the spectrum information of the multiple wavelength beams in the fluorescence signal; perform photoelectric signal conversion on the spectrum information of the multiple wavelength beams in the fluorescence signal to obtain a captured image of the observation sample; the imaging processing module is used to perform deconvolution reconstruction on the captured image to obtain a reconstructed image of the observation sample. Through this microscopic imaging system, it is possible to distinguish multiple different wavelength beams emitted by fluorescent groups in the observation sample in one imaging, thereby making the microscopic imaging process simpler to operate and faster in imaging speed.

Description

Technical Field

[0001] The present application belongs to the field of optical microscope imaging technology, and in particular, relates to a microscopic imaging system and method. Background Art

[0002] At present, total internal reflection multi-fluorescence microscopy is an optical imaging technology that uses the evanescent wave generated during total internal reflection to illuminate the observed sample. In total internal reflection, the illumination beam enters the cover glass at an angle greater than the critical angle, and an evanescent wave is generated on one side of the observed sample along the direction perpendicular to the interface. Since its intensity decays exponentially with the penetration distance, the evanescent wave can only excite the fluorescent groups in the thin layer of the observed sample close to the cover glass to emit fluorescence, while the fluorescent groups deeper in the observed sample cannot be excited. Multicolor fluorescence microscopy plays a key role in biological research. It can simultaneously label multiple proteins or organelles and study multiple targets or features, thereby providing a more comprehensive cell image. In order to better achieve multicolor, by utilizing the high dispersion properties, a high dispersion device is used to distinguish light of different wavelengths, so that light beams of different wavelengths can be imaged at different depths after passing through the device.

[0003] However, the existing total internal reflection technology faces great challenges in achieving multi-color fluorescence microscopy. Since the excitation and emission wavelengths of the fluorescent groups in the observed samples often overlap, spectral crossover is prone to occur during multi-color fluorescence microscopy, resulting in interference and distortion of the fluorescence signal. In addition, the existing multi-color fluorescence microscopy process usually requires the use of multiple filters and light sources, making the imaging operation complicated and the imaging speed slow. Summary of the invention

[0004] The embodiments of the present application provide a microscopic imaging system and method, which can solve the problems of complex operation and slow imaging speed in the existing multi-color fluorescence microscopic imaging of the observed sample using total internal reflection, due to the overlap of the excitation and emission wavelengths of the fluorescent groups, which easily causes spectral cross-talk and fluorescence signal distortion. Multiple filters and multiple light sources are usually used to excite the fluorescent groups in the observed sample and perform imaging.

[0005] In a first aspect, an embodiment of the present application provides a microscopic imaging system, the system comprising: an illumination optical path module, a detection optical path module, an imaging processing module and an object stage, wherein:

[0006] The illumination light path module forms an illumination light path with the stage, and is used to make the laser beam incident on the observation sample on the stage at a preset incident angle; wherein the preset incident angle is determined by the refractive index of the cover glass on the observation sample; the observation sample is a material or structure used for experimental observation after being fluorescently labeled;

[0007] The detection optical path module forms a detection optical path with the stage, and is used to collect the fluorescence signal refracted by the observed sample, separate and transform the multiple wavelength light beams in the fluorescence signal, and obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal; and perform photoelectric signal conversion on the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain a captured image of the observed sample; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and spectrum capture; the spectrum information is used to characterize the characteristics of the observed sample at different angles;

[0008] The imaging processing module establishes a communication connection with the detection optical path module, and is used to perform deconvolution reconstruction on the captured image to obtain a reconstructed image of the observed sample.

[0009] In a possible implementation of the first aspect, the detection optical path module includes a collection unit, a processing unit, and a detection unit, and the detection optical path includes a first detection optical path and a second detection optical path; wherein,

[0010] The collection unit forms the first detection light path with the stage, and is used to collect the fluorescence signal refracted by the observation sample;

[0011] The processing unit, the collection unit, and the detection unit form a second detection optical path, which is used to separate and transform the multiple wavelength light beams in the fluorescence signal to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal;

[0012] The detection unit is used to convert the spectrum information of multiple wavelength light beams in the fluorescence signal into photoelectric signals to obtain the captured image of the observed sample.

[0013] In a possible implementation manner of the first aspect, the processing unit includes a Fourier transform component, a high dispersion device, and a microlens array component; wherein,

[0014] The Fourier transform component is used to perform Fourier transform on the fluorescence signal to obtain frequency domain information of multiple wavelength light beams in the fluorescence signal;

[0015] The high-dispersion device is used to perform dispersion separation on the fluorescence signal to obtain spectral information of multiple wavelength light beams in the fluorescence signal;

[0016] The microlens array assembly is used to capture the frequency domain information and the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal;

[0017] The second detection light path passes through the Fourier transform component, the high-dispersion device, the microlens array component in sequence from the acquisition unit to the detection unit, or the second detection light path passes through the high-dispersion device, the Fourier transform component, the microlens array component in sequence from the acquisition unit to the detection unit.

[0018] In a possible implementation of the first aspect, the acquisition unit includes an objective lens and a dichroic mirror, and the first detection light path passes through the objective lens, the dichroic mirror and the processing unit in sequence from the stage, wherein:

[0019] The objective lens is used to collect the fluorescence signal refracted by the observation sample;

[0020] The dichroic mirror is used to reflect the fluorescent signal passing through the objective lens to the processing unit.

[0021] In a possible implementation of the first aspect, the illumination optical path module includes a laser, a collimating beam expansion system, and an incident device, and the illumination optical path sequentially passes through the collimating beam expansion system, the incident device, and the stage from the laser, wherein:

[0022] The laser is used to emit the laser beam;

[0023] The collimation and beam expansion system is used to collimate and expand the laser beam emitted by the laser to form a parallel beam;

[0024] The incident device is used to cause the parallel light beam to be incident on the observation sample at the preset incident angle.

[0025] In a possible implementation manner of the first aspect, an attenuation unit is further provided on the illumination light path, wherein:

[0026] The attenuation unit is located between the laser and the collimating beam expanding system, and is used to perform attenuation processing on the laser beam, and make the attenuated laser beam incident on the collimating beam expanding system.

[0027] In a second aspect, an embodiment of the present application provides a microscopic imaging method, which is applied to a microscopic imaging system as described in any one of the above, and the method comprises:

[0028] Collecting a fluorescent signal refracted by an observation sample, wherein the observation sample is a material or structure that is fluorescently labeled and used for experimental observation;

[0029] Separating and transforming the multiple wavelength beams in the fluorescence signal to obtain spectrum information of the multiple wavelength beams in the fluorescence signal; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and information capture; the spectrum information is used to characterize the characteristics of the observed sample at different angles;

[0030] Performing photoelectric signal conversion on the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain a captured image of the observed sample;

[0031] Deconvolution reconstruction is performed on the captured image to obtain a reconstructed image of the observed sample.

[0032] In a possible implementation manner of the second aspect, the performing separation and transformation processing on the multiple wavelength light beams in the fluorescence signal to obtain spectrum information of the multiple wavelength light beams in the fluorescence signal includes:

[0033] Performing Fourier transform on the fluorescence signal through a Fourier transform component to obtain frequency domain information of multiple wavelength light beams in the fluorescence signal;

[0034] Performing dispersion separation on the fluorescence signal through a high dispersion device to obtain spectral information of multiple wavelength light beams in the fluorescence signal;

[0035] The frequency domain information and spectrum information of the multiple wavelength light beams in the fluorescence signal are captured by a microlens array component to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal.

[0036] In a possible implementation manner of the second aspect, before collecting the fluorescent signal refracted by the observation sample, the method includes:

[0037] The laser beam emitted by the laser is incident on the collimation and beam expansion system, and a parallel beam is obtained after being collimated and expanded by the collimation and beam expansion system;

[0038] The parallel light beam is used to excite the observation sample at a preset incident angle through an incident device to obtain the fluorescence signal refracted by the observation sample; wherein the preset incident angle is determined by the refractive index of the cover glass on the observation sample.

[0039] In a possible implementation manner of the second aspect, collecting and observing the fluorescent signal refracted by the sample includes:

[0040] The fluorescent signal refracted by the observed sample is incident on an objective lens, and is collected by the objective lens and reflected by a dichroic mirror, so that the Fourier transform component obtains the fluorescent signal.

[0041] Compared with the prior art, the embodiments of the present invention have the following beneficial effects:

[0042] The embodiment of the present application provides a microscopic imaging system, which includes: an illumination optical path module, a detection optical path module, an imaging processing module and an object stage, wherein the illumination optical path module forms an illumination optical path with the object stage, and is used to make a laser beam incident on an observation sample on the object stage at a preset incident angle; wherein the preset incident angle is determined by the refractive index of a cover glass on the observation sample; the observation sample is a material or structure used for experimental observation after being fluorescently labeled; the detection optical path module forms a detection optical path with the object stage, and is used to collect a fluorescent signal refracted by the observation sample, perform separation and transformation processing on multiple wavelength beams in the fluorescent signal, and obtain spectrum information of the multiple wavelength beams in the fluorescent signal; and perform photoelectric signal conversion on the spectrum information of the multiple wavelength beams in the fluorescent signal, and obtain a captured image of the observation sample; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and spectrum capture; the spectrum information is used to characterize the characteristics of the observation sample at different angles; the imaging processing module establishes a communication connection with the detection optical path module, and is used to perform deconvolution reconstruction on the captured image, and obtain a reconstructed image of the observation sample. The microscopic imaging system makes the laser beam incident on the observation sample on the stage at a preset incident angle so that the observation sample refracts a fluorescent signal, and performs Fourier transform, dispersion separation, spectrum capture and other separation and transformation processing and photoelectric signal conversion on the multiple wavelength beams in the fluorescent signal to image the captured image of the observation sample. Finally, the captured image is deconvoluted and reconstructed to obtain a reconstructed image of the observation sample. The microscopic imaging system can distinguish multiple different wavelength beams emitted by the fluorescent group in the observation sample in one imaging, thereby making the microscopic imaging process simpler to operate and faster in imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 is a structural schematic diagram of a microscopic imaging system provided in one embodiment of the present application;

[0045] Figure 2 It is a schematic flow chart of a microscopic imaging method provided in one embodiment of the present application;

[0046] Figure 3 It is a schematic diagram of a simulation process of a microscopic imaging system provided in one embodiment of the present application.

[0047] In the figure, 10-microscope imaging system, 20-illumination optical path module, 21-laser, 22-collimation and beam expansion system, 30-detection optical path module, 31-acquisition unit, 311-objective lens, 312-dichroic mirror, 32-processing unit, 321-Fourier transform component, 322-high dispersion device, 323-microlens array component, 33-detection unit, 40-imaging processing module, 50-stage. DETAILED DESCRIPTION

[0048] In the following description, specific details such as specific system structures, technologies, etc. are provided for the purpose of illustration rather than limitation, so as to provide a thorough understanding of the embodiments of the present application. However, it should be clear to those skilled in the art that the present application may also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to prevent unnecessary details from obstructing the description of the present application.

[0049] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or combinations thereof.

[0050] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0051] As used in the specification and appended claims of this application, the term "if" can be interpreted as "when" or "uponce" or "in response to determining" or "in response to detecting", depending on the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "uponce it is determined" or "in response to determining" or "uponce [described condition or event] is detected" or "in response to detecting [described condition or event]", depending on the context.

[0052] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0053] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0054] See also Figure 1 , Figure 1 Schematic diagram of a microscopic imaging system provided in one embodiment of the present application. Figure 1 In the embodiment, the microscopic imaging system 10 includes: an illumination optical path module 20, a detection optical path module 30, an imaging processing module 40 and an object stage 50, wherein:

[0055] The illumination optical path module 20 forms an illumination optical path with the stage 50, and is used to make the laser beam incident on the observation sample on the stage 50 at a preset incident angle; wherein the preset incident angle is determined by the refractive index of the cover glass on the observation sample; the observation sample is a material or structure used for experimental observation after being fluorescently labeled;

[0056] The detection optical path module 30 forms a detection optical path with the stage 50, and is used to collect the fluorescence signal refracted by the observation sample, separate and transform the multiple wavelength beams in the fluorescence signal, and obtain the spectrum information of the multiple wavelength beams in the fluorescence signal; and perform photoelectric signal conversion on the spectrum information of the multiple wavelength beams in the fluorescence signal to obtain a captured image of the observation sample; wherein the separation and transformation processing includes at least one of the following: Fourier transformation, dispersion separation, and spectrum capture; the spectrum information is used to characterize the characteristics of the observation sample at different angles;

[0057] The imaging processing module 40 establishes a communication connection with the detection optical path module 30 and is used to perform deconvolution reconstruction on the captured image to obtain a reconstructed image of the observed sample.

[0058] like Figure 1 As shown, the microscopic imaging system 10 is a system for performing high-precision imaging of experimental observation materials or structures (i.e., observation samples) that have been fluorescently labeled. The microscopic imaging system 10 achieves detailed observation and analysis of the observation samples through the illumination optical path module 20, the detection optical path module 30, and the imaging processing module 40.

[0059] It should be noted that the microscopic imaging system 10 utilizes the property of total internal reflection so that the laser beam emitted by the illumination optical path module 20 only excites the fluorescent groups in the thin layer of the observation sample close to the cover glass to emit fluorescence. Therefore, the microscopic imaging system 10 images the observation sample in the thin layer.

[0060] The observation sample is a material or structure used for experimental observation after being fluorescently labeled. Among them, fluorescent labeling is a process that causes the sample to emit a fluorescent signal under the irradiation of light of a specific wavelength. The cover glass is a thin sheet covering the observation sample when observing the observation sample under a microscope. The stage 50 is a platform for supporting the observation sample.

[0061] Among them, the illumination light path module 20 is a module for generating and guiding a laser beam, and the illumination light path module 20 is used to irradiate the laser beam to the observation sample on the stage 50 at a preset incident angle. The preset incident angle is determined according to the refractive index of the cover glass on the observation sample to ensure that the laser beam can excite the fluorescent group in the thin layer of the observation sample to emit a fluorescent signal. It should be noted that when the laser beam is incident on the cover glass on the observation sample at an incident angle greater than the critical angle, an evanescent wave is generated on one side of the observation sample in a direction perpendicular to the interface. Since the intensity of the evanescent wave decays exponentially with the penetration distance, the evanescent wave can only excite the fluorescent group in a thin layer of the observation sample (i.e., the thickness of one wavelength) close to the cover glass to emit a fluorescent signal, while the fluorescent group in the deeper layer of the observation sample cannot be excited. Therefore, in this embodiment, the preset incident angle refers to an incident angle greater than the critical angle, and the critical angle is determined by the refractive index of the cover glass, that is, the preset incident angle is determined by the refractive index of the cover glass on the observation sample. Among them, let the critical angle be ,but , is the refractive index of the high refractive index medium, is the refractive index of the low-refractive-index medium.

[0062] The detection optical path module 30 is used to collect the fluorescence signal refracted by the observed sample, and to separate and transform the multiple wavelength light beams in the fluorescence signal (i.e., Fourier transform, dispersion separation, and spectrum capture) to obtain the spectrum information of the fluorescence signal, and to convert the spectrum information in the fluorescence signal into a photoelectric signal, thereby obtaining a captured image of the observed sample. Among them, Fourier transform is a transformation method for converting a signal from a spatial domain to a frequency domain. In a microscopic imaging system, Fourier transform can be used to analyze the frequency components of the fluorescence signal, that is, the distribution of light beams of different wavelengths in the signal, thereby obtaining the frequency domain information of the fluorescence signal. Dispersion separation is a method for dispersing a light beam into different paths according to the wavelength of light. In the microscopic imaging process, dispersion separation can focus light beams of different wavelengths to different axial positions through optical elements such as high dispersion devices, thereby obtaining the spectrum information of the fluorescence signal. Spectrum capture is used to capture the frequency domain information and spectrum information of the fluorescence signal to obtain the spectrum information of light beams of different wavelengths in the fluorescence signal, and convert it into a photoelectric signal to obtain the characteristics of the observed sample at different angles, thereby obtaining a captured image of the observed sample. It can be understood that by separating and transforming the multiple wavelength light beams in the fluorescence signal, it is possible to gain a deeper understanding of the characteristics of the observed sample at different angles, thereby improving the resolution, sensitivity and accuracy of the imaging system.

[0063] The imaging processing module 40 is used to receive the captured image provided by the detection optical path module 30, and perform deconvolution reconstruction on the captured image to eliminate blur and distortion and obtain a clearer reconstructed image of the observed sample. The deconvolution algorithm may adopt the Richardson-Lucy deconvolution algorithm.

[0064] It can be understood that the embodiment of the present application provides a microscopic imaging system, which includes: an illumination light path module 20, a detection light path module 30, an imaging processing module 40 and a stage 50, wherein the illumination light path module 20 forms an illumination light path with the stage 50, which is used to make the laser beam incident on the observation sample on the stage 50 at a preset incident angle; wherein the preset incident angle is determined by the refractive index of the cover glass on the observation sample; the observation sample is a material or structure for experimental observation after being fluorescently labeled; the detection light path module 30 forms a detection light path with the stage 50, which is used to collect The fluorescence signal refracted by the observation sample is separated and transformed to obtain the spectrum information of the multiple wavelength beams in the fluorescence signal; and the spectrum information of the multiple wavelength beams in the fluorescence signal is converted into a photoelectric signal to obtain a captured image of the observation sample; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and spectrum capture; the spectrum information is used to characterize the characteristics of the observation sample at different angles; the imaging processing module 40 establishes a communication connection with the detection optical path module 30, and is used to perform deconvolution reconstruction on the captured image to obtain a reconstructed image of the observation sample. The microscopic imaging system 10 images the captured image of the observation sample by incidenting a laser beam into the observation sample on the stage at a preset incident angle so that the observation sample refracts the fluorescence signal, and performs separation and transformation processing such as Fourier transform, dispersion separation, and spectrum capture and photoelectric signal conversion on the multiple wavelength beams in the fluorescence signal, and finally, deconvolution reconstruction is performed on the captured image to obtain a reconstructed image of the observation sample. The microscopic imaging system 10 can distinguish a plurality of light beams of different wavelengths emitted by fluorescent groups in the observed sample in one imaging, thereby making the microscopic imaging process simpler to operate and faster in imaging speed.

[0065] In a possible implementation, the detection optical path module 30 includes a collection unit 31, a processing unit 32 and a detection unit 33, and the detection optical path includes a first detection optical path and a second detection optical path; wherein,

[0066] The collection unit 31 forms a first detection light path with the stage 50, and is used to collect the fluorescence signal refracted by the observation sample;

[0067] The processing unit 32 forms a second detection optical path with the collection unit 31 and the detection unit 33, and is used to separate and transform the multiple wavelength light beams in the fluorescence signal to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal;

[0068] The detection unit 33 is used to convert the spectrum information of multiple wavelength light beams in the fluorescence signal into photoelectric signals to obtain a captured image of the observed sample.

[0069] like Figure 1As shown, the detection optical path module 30 includes a collection unit 31, a processing unit 32 and a detection unit 33. The collection unit 31 is a device for collecting the fluorescence signal refracted by the observed sample. The processing unit 32 is a device for performing Fourier transform, dispersion separation and spectrum capture on the fluorescence signal to obtain spectrum information. The detection unit 33 is a device for performing photoelectric signal conversion on the spectrum information of the fluorescence signal to obtain a captured image.

[0070] The detection optical path is the transmission path of the fluorescence signal from the stage 50 to the detection unit 33. In this embodiment, the detection optical path includes a first detection optical path and a second detection optical path, wherein the first detection optical path is the transmission path of the fluorescence signal from the stage 50 to the collection unit 31; the second detection optical path is the transmission path of the fluorescence signal from the collection unit 31 through the processing unit 32 to the detection unit 33.

[0071] It can be understood that the detection optical path module 30 realizes the collection, processing and signal conversion of the fluorescence signal through the collection unit 31, the processing unit 32 and the detection unit 33, and provides the necessary image capture data for the imaging processing module of the system.

[0072] Further, the detection unit 33 is a charge coupled device CCD camera or a complementary metal oxide semiconductor CMOS camera. Among them, CCD camera and CMOS camera are two commonly used image sensors. CCD camera captures images through a series of closely arranged photosensitive elements (such as photodiodes). CMOS camera captures images by integrating signal processing circuits next to the photosensitive elements.

[0073] In a possible implementation, the processing unit 32 includes a Fourier transform component 321, a high dispersion device 322, and a microlens array component 323; wherein,

[0074] A Fourier transform component 321 is used to perform Fourier transform on the fluorescence signal to obtain frequency domain information of multiple wavelength light beams in the fluorescence signal;

[0075] A high dispersion device 322 is used to perform dispersion separation on the fluorescence signal to obtain spectrum information of multiple wavelength light beams in the fluorescence signal;

[0076] The microlens array assembly 323 is used to capture the frequency domain information and spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal;

[0077] The second detection light path passes through the Fourier transform component 321, the high dispersion device 322, the microlens array component 323 from the collection unit 31 to the detection unit 33, or the second detection light path passes through the high dispersion device 322, the Fourier transform component 321, the microlens array component 323 from the collection unit 31 to the detection unit 33.

[0078] like Figure 1 As shown, the processing unit 32 is a device for performing Fourier transform, dispersion separation and spectrum capture on the fluorescence signal to obtain spectrum information. The processing unit 32 includes a Fourier transform component 321, a high dispersion device 322 and a microlens array component 323.

[0079] The Fourier transform component 321 is a confocal system (i.e., a 4f system) composed of two lenses with equal focal lengths. The Fourier transform component 321 converts the fluorescence signal from the spatial domain to the frequency domain by performing Fourier transform on the fluorescence signal, thereby obtaining frequency domain information of multiple wavelength beams in the fluorescence signal, which helps to detect the characteristics of the observed sample at different angles. In this embodiment, the Fourier transform component 321 can be a 4f system composed of a tube lens and a Fourier lens.

[0080] The high dispersion device 322 is an optical element that can disperse light into different paths according to its wavelength. In this embodiment, the fluorescence signal is dispersed and separated by the high dispersion device 322, and light beams of different wavelengths are focused to different axial positions, thereby obtaining the spectral information of multiple wavelength light beams in the fluorescence signal, that is, the intensity and distribution of light beams of different wavelengths.

[0081] The microlens array assembly 323 is an array composed of many tiny lenses, each of which corresponds to a specific frequency range and can focus the incident light. The microlens array assembly 323 is used to capture the frequency domain information and spectral information of the fluorescence signal after Fourier transformation and dispersion separation.

[0082] The second detection optical path is the transmission path of the fluorescence signal from the collection unit 31 to the detection unit 33. In this embodiment, the second detection optical path can be from the collection unit 31 through the Fourier transform component 321, the high dispersion device 322, the microlens array component 323 to the detection unit 33; it can also be from the collection unit 31 through the high dispersion device 322, the Fourier transform component 321, the microlens array component 323 to the detection unit 33. That is, the high dispersion device 322 can be arranged before the Fourier transform component 321, or it can be close to the surface of the microlens array component 323, that is, in this embodiment, the processing order of Fourier transform and dispersion separation of the fluorescence signal is not limited.

[0083] It can be understood that the processing unit 32 realizes the conversion of the fluorescence signal from the spatial domain to the frequency domain and the dispersion separation processing through the coordinated work of the Fourier transform component 321, the high dispersion device 322 and the microlens array component 323, and captures the frequency domain information and spectral information of the fluorescence signal through the microlens array, providing the necessary data and information for the subsequent imaging process of the system.

[0084] In a possible implementation, the acquisition unit 31 includes an objective lens 311 and a dichroic mirror 312, and the first detection light path passes through the objective lens 311, the dichroic mirror 312 and the processing unit 32 from the stage 50 in sequence, wherein:

[0085] Objective lens 311, used for collecting the fluorescence signal refracted by the observation sample;

[0086] The dichroic mirror 312 is used to reflect the fluorescent signal passing through the objective lens to the processing unit 32 .

[0087] like Figure 1 As shown, the acquisition unit 31 is a device for collecting the fluorescence signal refracted by the observed sample. The acquisition unit 31 includes an objective lens 311 and a dichroic mirror 312. Among them, the objective lens 311 is used to collect the fluorescence signal emitted by the observed sample after being excited by the laser beam. The objective lens 311 has a high magnification and a high numerical aperture, which can capture the weak fluorescence signal emitted by the observed sample and focus it into the subsequent detection light path. The dichroic mirror 312 is a spectroscopic element with special optical properties. The dichroic mirror 312 can separate the light beam into different paths according to the wavelength of the light beam to reflect the fluorescence signal emitted by the observed sample and guide it to the processing unit 32.

[0088] The first detection light path is a transmission path of the fluorescence signal from the stage 50 to the processing unit 32. In this embodiment, the first detection light path is from the stage 50 through the objective lens 311, the dichroic mirror 312 to the processing unit 32 in sequence.

[0089] It can be understood that the acquisition unit 31 achieves efficient capture and guidance of the fluorescent signals emitted by the observed sample through the coordinated work of the objective lens 311 and the dichroic mirror 312, so that these fluorescent signals are transmitted to the processing unit 32 for further processing and analysis to obtain the structure and characteristics of the observed sample.

[0090] In a possible implementation, the illumination optical path module 20 includes a laser 21, a collimating beam expansion system 22, and an incident device (not shown in the figure), and the illumination optical path sequentially passes through the collimating beam expansion system 22, the incident device, and the stage 50 from the laser 21, wherein:

[0091] A laser 21, used for emitting a laser beam;

[0092] The collimation and beam expansion system 22 is used to collimate and expand the laser beam emitted by the laser 21 to form a parallel beam;

[0093] The incident device is used to make the parallel light beam incident on the observation sample at a preset incident angle.

[0094] like Figure 1 As shown, the illumination optical path module 20 includes a laser 21 and a collimating and beam expanding system 22. Among them, the laser 21 is the core light source of the illumination optical path module, which is used to emit a laser beam. The collimating and beam expanding system 22 is a complex optical component, which is usually composed of a pair of convex lenses with different focal lengths. The function of the collimating and beam expanding system 22 is to collimate and expand the laser beam to form parallel light with uniform intensity, so that the parallel light can be uniformly and stably excited when it is irradiated on the observation sample at a preset incident angle. Among them, collimation is to adjust the laser beam into a parallel beam to ensure that the laser beam maintains directionality during transmission; beam expansion is to increase the diameter of the laser beam to obtain a larger observation area. The incident device is a device for guiding parallel light to the observation sample at a preset incident angle. In this embodiment, the incident device can be a prism type or an objective lens type.

[0095] The illumination light path is the transmission path of the laser beam from the laser 21 to the stage 50. In this embodiment, the illumination light path is from the laser 21 through the collimating beam expansion system 22, the incident device to the stage 50 in sequence.

[0096] It can be understood that the illumination optical path module 20 achieves high-quality illumination of the observed sample through the coordinated work of the laser 21, the collimation and expansion system 22 and the incident device. The laser beam is collimated and expanded to provide the system with stable and uniform lighting conditions, thereby helping to improve the imaging quality of the system and enabling the system to more accurately capture the structure and characteristics of the observed sample.

[0097] In a possible implementation, an attenuation unit (not shown in the figure) is further provided on the illumination light path, wherein:

[0098] The attenuation unit is located between the laser and the collimating beam expansion system, and is used to attenuate the laser beam and direct the attenuated laser beam into the collimating beam expansion system 22 .

[0099] It should be noted that the attenuation unit is a device for attenuating the laser beam emitted by the laser 21. Since the laser beam generated by the laser usually has a high energy density, directly irradiating the observed sample will cause damage to the sample or produce unnecessary thermal effects. Therefore, before the laser beam enters the collimating and expanding system 22, the laser beam is attenuated by the attenuation unit to reduce the energy density of the laser beam to ensure the stability and safety of the illumination optical path. In this embodiment, the attenuation unit can be an attenuation plate, which is an optical element that can absorb or reflect part of the energy of the laser beam. By adjusting the thickness, material or surface coating of the attenuation plate, the laser beam can be attenuated to different degrees. In this embodiment, the illumination optical path is from the laser 21 through the attenuation unit, the collimating and expanding system 22, the incident device to the stage 50 in sequence.

[0100] It can be understood that by providing an attenuation unit on the illumination light path, the energy density of the laser beam can be precisely controlled to ensure the stability and safety of the illumination light path, thereby obtaining a higher quality imaging image.

[0101] Specifically, Figure 1 As shown, the imaging process of the microscopic imaging system 10 is as follows: the laser beam emitted by the laser 21 in the illumination optical path module 20 is incident on the collimation and expansion system 22 composed of two lenses through the attenuation unit, and is collimated and expanded by the collimation and expansion system 22 to form parallel light with uniform intensity. The parallel light is incident on the observation sample on the stage 50 at a preset incident angle through the incident device. Then, the fluorescence signal excited by the observation sample is collected by the objective lens 311 in the detection optical path module, and is reflected into the Fourier transform component 321 through the dichroic mirror 312, and the fluorescence signal is Fourier transformed, and the back focal plane (i.e., the spectrum plane) of the Fourier lens in the Fourier transform component 321 is modulated by the high dispersion device 322 and the microlens array component 323, and the detection unit 33 (such as an EMCCD camera) placed on the back focal plane of the microlens array component 323 is imaged. Finally, the captured image is deconvoluted and reconstructed in the imaging processing module 40 to obtain a reconstructed image of the observation sample.

[0102] A microscopic imaging system corresponding to the above embodiment, Figure 2 A schematic diagram of a microscopic imaging method provided by an embodiment of the present application is shown. Figure 2 , the microscopic imaging method is applied to any of the microscopic imaging systems described above, and the microscopic imaging method comprises:

[0103] S11, collecting a fluorescent signal refracted by an observation sample, wherein the observation sample is a material or structure that is fluorescently labeled and used for experimental observation;

[0104] S12, performing separation and transformation processing on multiple wavelength beams in the fluorescence signal to obtain spectrum information of the multiple wavelength beams in the fluorescence signal; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and information capture; the spectrum information is used to characterize the characteristics of the observed sample at different angles;

[0105] S13, performing photoelectric signal conversion on the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain a captured image of the observed sample;

[0106] S14. Perform deconvolution reconstruction on the captured image to obtain a reconstructed image of the observed sample.

[0107] It should be noted that the microscopic imaging system 10 utilizes the property of total internal reflection so that the laser beam emitted by the illumination optical path module 20 only excites the fluorescent groups in the thin layer of the observation sample close to the cover glass to emit fluorescence. Therefore, the microscopic imaging system 10 images the observation sample in the thin layer.

[0108] Specifically, first, the fluorescence signal refracted by the observed sample is collected; second, the fluorescence signal is Fourier transformed to analyze its frequency components to obtain the frequency domain information of the fluorescence signal; the fluorescence signal is dispersed and separated to focus light beams of different wavelengths to different axial positions to obtain the spectral information of the fluorescence signal; the frequency domain information and spectral information of the fluorescence signal are captured to obtain the spectrum information of light beams of different wavelengths in the fluorescence signal; then, the spectrum information of light beams of different wavelengths in the fluorescence signal is converted into photoelectric signals to obtain the characteristics of the observed sample at different angles, thereby obtaining a captured image of the observed sample; finally, the captured image is deconvolved and reconstructed to eliminate blur and distortion to obtain a clearer reconstructed image of the observed sample.

[0109] It can be understood that the microscopic imaging method has a simple structure. In order to separate the different wavelength information of the multi-color observation sample, the excited fluorescence signal is imaged at different depths by Fourier transform, dispersion separation, etc., so that the different wavelength information of the same plane is converted into depth information. The information of the observation sample can be captured at the same time by only one imaging. At the same time, it is also easier to build a microscopic imaging system, avoiding the need to segment the fluorescence emitted by multiple fluorophores of different wavelengths because the camera cannot distinguish the wavelength of light. Therefore, the complexity of the system will not increase with the increase of colors in the observation sample, and more colors can be distinguished at one time.

[0110] It can be understood that the embodiment of the present application provides a microscopic imaging method, including: collecting fluorescent signals refracted by an observed sample, wherein the observed sample is a material or structure used for experimental observation after being fluorescently labeled; performing separation and transformation processing on multiple wavelength beams in the fluorescent signal to obtain spectrum information of multiple wavelength beams in the fluorescent signal; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and information capture; the spectrum information is used to characterize the characteristics of the observed sample at different angles; performing photoelectric signal conversion on the spectrum information of multiple wavelength beams in the fluorescent signal to obtain a captured image of the observed sample; performing deconvolution reconstruction on the captured image to obtain a reconstructed image of the observed sample. Through this microscopic imaging method, it is possible to distinguish multiple different wavelength beams emitted by fluorescent groups in the observed sample in one imaging, thereby making the microscopic imaging process simpler to operate and faster in imaging speed.

[0111] In a possible implementation, multiple wavelength light beams in the fluorescence signal are separated and transformed to obtain spectrum information of the multiple wavelength light beams in the fluorescence signal, including:

[0112] Performing Fourier transform on the fluorescence signal through the Fourier transform component 321 to obtain frequency domain information of multiple wavelength light beams in the fluorescence signal;

[0113] The fluorescence signal is dispersed and separated by a high dispersion device 322 to obtain spectrum information of multiple wavelength light beams in the fluorescence signal;

[0114] The frequency domain information and spectrum information of the multiple wavelength light beams in the fluorescence signal are captured by the microlens array assembly 323 to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal.

[0115] It should be noted that the Fourier transform component 321 is a confocal system (i.e., a 4f system) composed of two lenses with equal focal lengths. The Fourier transform component 321 converts the fluorescence signal from the spatial domain to the frequency domain by performing Fourier transform on the fluorescence signal, thereby obtaining frequency domain information of multiple wavelength beams in the fluorescence signal, which helps to detect the characteristics of the observed sample at different angles. In this embodiment, the Fourier transform component 321 can be a 4f system composed of a tube lens and a Fourier lens.

[0116] The high dispersion device 322 is an optical element that can disperse light into different paths according to its wavelength. In this embodiment, the fluorescence signal is dispersed and separated by the high dispersion device 322, and light beams of different wavelengths are focused to different axial positions, thereby obtaining the spectral information of multiple wavelength light beams in the fluorescence signal, that is, the intensity and distribution of light beams of different wavelengths.

[0117] The microlens array assembly 323 is an array composed of many tiny lenses, each of which corresponds to a specific frequency range and can focus the incident light. The microlens array assembly 323 is used to capture the frequency domain information and spectral information of the fluorescence signal after Fourier transformation and dispersion separation.

[0118] It can be understood that, in this embodiment, the separation and transformation process of the fluorescence signal includes methods such as Fourier transform, dispersion separation and spectrum capture, through which the spectrum information of multiple wavelength light beams in the fluorescence signal is obtained. The spectrum information shows the distribution characteristics of the fluorescence signal at different frequencies and wavelengths, providing support for subsequent signal analysis, processing and imaging.

[0119] In a possible implementation, before collecting the fluorescent signal refracted by the observation sample, the microscopic imaging method includes:

[0120] The laser beam emitted by the laser 21 is incident on the collimating and beam expanding system 22, and a parallel beam is obtained after being collimated and expanded by the collimating and beam expanding system 22;

[0121] The incident device excites the observed sample with a parallel light beam at a preset incident angle to obtain a fluorescent signal refracted by the observed sample; wherein the preset incident angle is determined by the refractive index of the cover glass on the observed sample.

[0122] It should be noted that the laser 21 is the core light source of the illumination optical path module, which is used to emit a laser beam. The collimation and beam expansion system 22 is a complex optical component, usually composed of a pair of convex lenses with different focal lengths. The function of the collimation and beam expansion system 22 is to collimate and expand the laser beam to form parallel light with uniform intensity, so that the parallel light can be uniformly and stably excited when it is irradiated on the observation sample at a preset incident angle. Among them, collimation is to adjust the laser beam into a parallel beam to ensure that the laser beam maintains directionality during transmission; beam expansion is to increase the diameter of the laser beam to obtain a larger observation area. The incident device is a device for guiding parallel light into the observation sample at a preset incident angle. In this embodiment, the incident device can be a prism type or an objective lens type.

[0123] The preset incident angle is determined according to the refractive index of the cover glass on the observed sample to ensure that the laser beam can excite the fluorescent groups in the thin layer of the observed sample to emit a fluorescent signal. In this microscopic imaging method, through the total internal reflection property, the exciter 21 only excites the fluorescent groups in the thin layer of the observed sample close to the cover glass to emit fluorescence, making the light path for excitation simpler.

[0124] Specifically, first, the laser beam emitted by the laser 21 is incident on the collimating and expanding system 22, and a parallel beam is obtained after the collimation and expansion processing of the collimating and expanding system 22; then, the parallel beam is used to excite the observed sample at a preset incident angle through the incident device to obtain a fluorescence signal refracted by the observed sample.

[0125] It can be understood that high-quality illumination of the observed sample is achieved through the coordinated work of the laser 21, the collimation and expansion system 22 and the incident device. The laser beam is collimated and expanded to provide the system with stable and uniform lighting conditions, thereby helping to improve the imaging quality of the system and enabling the system to more accurately capture the structure and characteristics of the observed sample.

[0126] It should be noted that since the laser beam generated by the laser usually has a high energy density, directly irradiating the observed sample will cause damage to the sample or produce unnecessary thermal effects. Therefore, before the laser beam enters the collimation and expansion system 22, it is necessary to attenuate the laser beam through an attenuation unit to reduce the energy density of the laser beam to ensure the stability and safety of the illumination optical path.

[0127] In a possible implementation, collecting and observing a fluorescent signal refracted by a sample includes:

[0128] The fluorescent signal refracted by the observed sample is incident on the objective lens 311 , and is collected by the objective lens 311 and reflected by the dichroic mirror 312 , so that the Fourier transform component 321 obtains the fluorescent signal.

[0129] It should be noted that the objective lens 311 is used to collect the fluorescence signal emitted by the observed sample after being excited by the laser beam. The objective lens 311 has a high magnification and a high numerical aperture, and can capture the weak fluorescence signal emitted by the observed sample and focus it into the subsequent detection light path. The dichroic mirror 312 is a spectroscopic element with special optical properties. The dichroic mirror 312 can separate the light beam into different paths according to the wavelength of the light beam to reflect the fluorescence signal emitted by the observed sample and guide it to the processing unit 32.

[0130] Specifically, the fluorescence signal excited by the observed sample is collected by the objective lens 311 and then reflected by the dichroic mirror 312 into the Fourier transform component 321, thereby achieving efficient collection of the fluorescence signal.

[0131] Furthermore, in this embodiment, the microscopic imaging system is simulated by MATLAB software. Figure 3 As shown, Figure 3 1 is a schematic diagram of a microscopic imaging system simulation process provided by an embodiment of the present application. The specific process of simulating the microscopic imaging system is as follows: first, a point spread function of a simulation system corresponding to the microscopic imaging system is generated. Figure 3 As shown in (3-a), Figure 3 (3-a) is a schematic diagram of the point spread function of different wavelengths under total internal reflection excitation. Figure 3 In (3-a), points of different colors represent different wavelengths. Since, after total internal reflection, light of different wavelengths will appear at different positions along the direction of dispersion after passing through the microscopic imaging system, the wavelength Transformed into the axial position Z, therefore, different colors (i.e. different wavelengths) can be distinguished according to different positional relationships. Figure 3 As shown in (3-b), Figure 3 (3-b) in FIG. 1 is a schematic diagram of an artificially set two-dimensional multi-color sample. The artificially set two-dimensional multi-color sample is simulated in the simulation system. Then, the simulation system simulates and obtains the captured image, such as Figure 3 As shown in (3-c), Figure 3 (3-c) in the figure is the captured image obtained by simulating the simulation system. Finally, the captured image obtained by simulating the simulation system is reconstructed by Richardson-Lucy deconvolution with the point spread function of the simulation system to obtain a total internal reflection multi-color reconstructed image, as shown in Figure 3 As shown in (3-d), Figure 3 (3-d) in the figure is the reconstructed image obtained by simulating the simulation system.

[0132] It should be noted that the point spread function is the light intensity distribution generated when an optical system images a point light source, which describes the imaging characteristics of the optical system. Ideally, a point light source should be imaged as a point, but due to diffraction, aberrations and other reasons of the optical system, the actual imaging will produce a more diffuse spot on the focal plane. The point spread function describes the shape and intensity distribution of this diffusion. In the imaging process, the point spread function is used to restore the real image structure under the premise of a known degree of imaging blur. For example, in fluorescence microscopy, the point spread function can be used to correct the image to make the sample structure clearer.

[0133] Specifically, the process of generating the point spread function of the simulation system corresponding to the microscopic imaging system is as follows: in the MATLAB software, first, set the relevant parameters of the FLFM (light field fluorescence microscope) system, such as the numerical aperture (NA) of the objective lens, the magnification, and the optical layout of the microscope (such as the structure and arrangement of the microlens array, etc.); secondly, calculate the performance parameters of the system, such as the lateral and longitudinal resolutions, etc.; finally, generate the three-dimensional point spread function of the wide-field imaging system, and Fourier transform it to the position where the microlens array component is placed.

[0134] Figure 3The two-dimensional multicolor sample provided in (3-b) contains multiple two-dimensional light intensity distribution matrices, which represent the luminous intensity of samples of different colors at different positions. In MATLAB software, based on the light intensity data of the two-dimensional multicolor sample, by simulating the optical properties of the light field microscope, the point spread function of the imaging system and the dispersion effect, a "photo" of the sample taken by a virtual camera can be generated, that is, Figure 3 The captured image obtained by simulating the simulation system provided in (3-c).

[0135] It can be understood that by simulating the microscopic imaging system in MATLAB software, the feasibility of the optical path design in the microscopic imaging system can be verified, the parameters of related components can be optimized, and the microscopic imaging system can also be evaluated and improved through the simulation to improve the imaging quality and system performance.

[0136] It should be noted that the above-mentioned microscopic imaging method embodiment and the system embodiment of the present application are based on the same concept. Their specific functions and technical effects can be found in the system embodiment section and will not be repeated here.

[0137] It should be understood that the size of the serial numbers of the steps in the above embodiments does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0138] If the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the present application implements all or part of the processes in the above-mentioned embodiment method, which can be completed by instructing the relevant hardware through a computer program. The computer program can be stored in a computer-readable storage medium. When the computer program is executed by the processor, the steps of the above-mentioned method embodiments can be implemented. Among them, the computer program includes computer program code, and the computer program code can be in source code form, object code form, executable file or some intermediate form. The computer-readable medium may at least include: any entity or device that can carry the computer program code to the camera / terminal device, recording medium, computer memory, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), electric carrier signal, telecommunication signal and software distribution medium. For example, a USB flash drive, a mobile hard disk, a magnetic disk or an optical disk. In some jurisdictions, according to legislation and patent practice, computer-readable media cannot be electric carrier signals and telecommunication signals.

[0139] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0140] Those of ordinary skill in the art will appreciate that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Professional and technical personnel can use different methods to implement the described functions for each specific application, but such implementation should not be considered to be beyond the scope of this application.

[0141] In the embodiments provided in the present application, it should be understood that the disclosed devices / network equipment and methods can be implemented in other ways. For example, the device / network equipment embodiments described above are only schematic. For example, the division of modules or units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms.

[0142] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0143] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. A microscopic imaging system, characterized in that: The system comprises: an illumination optical path module, a detection optical path module, an imaging processing module and an object stage, wherein: The illumination light path module forms an illumination light path with the stage, and is used to make the laser beam incident on the observation sample on the stage at a preset incident angle; wherein the preset incident angle is determined by the refractive index of the cover glass on the observation sample; the observation sample is a material or structure used for experimental observation after being fluorescently labeled; The detection optical path module forms a detection optical path with the stage, and is used to collect the fluorescence signal refracted by the observed sample, separate and transform the multiple wavelength light beams in the fluorescence signal, and obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal; and perform photoelectric signal conversion on the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain a captured image of the observed sample; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and spectrum capture; the spectrum information is used to characterize the characteristics of the observed sample at different angles; The imaging processing module establishes a communication connection with the detection optical path module, and is used to perform deconvolution reconstruction on the captured image to obtain a reconstructed image of the observed sample; wherein, The detection optical path module includes a collection unit, a processing unit and a detection unit, and the detection optical path includes a first detection optical path and a second detection optical path; wherein, The collection unit forms the first detection light path with the stage, and is used to collect the fluorescence signal refracted by the observation sample; The processing unit, the collection unit, and the detection unit form a second detection optical path, which is used to separate and transform the multiple wavelength light beams in the fluorescence signal to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal; The detection unit is used to convert the spectrum information of the multiple wavelength light beams in the fluorescence signal into a photoelectric signal to obtain the captured image of the observed sample; and The processing unit includes a Fourier transform component, a high dispersion device and a microlens array component; wherein, The Fourier transform component is used to perform Fourier transform on the fluorescence signal to obtain frequency domain information of multiple wavelength light beams in the fluorescence signal; The high-dispersion device is used to perform dispersion separation on the fluorescence signal to obtain spectral information of multiple wavelength light beams in the fluorescence signal; The microlens array assembly is used to capture the frequency domain information and the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal; The second detection light path passes through the Fourier transform component, the high-dispersion device, the microlens array component in sequence from the acquisition unit to the detection unit, or the second detection light path passes through the high-dispersion device, the Fourier transform component, the microlens array component in sequence from the acquisition unit to the detection unit.

2. The microscopic imaging system according to claim 1, characterized in that: The acquisition unit includes an objective lens and a dichroic mirror, and the first detection light path passes through the objective lens, the dichroic mirror and the processing unit in sequence from the stage, wherein: The objective lens is used to collect the fluorescence signal refracted by the observation sample; The dichroic mirror is used to reflect the fluorescent signal passing through the objective lens to the processing unit.

3. The microscopic imaging system according to claim 1, characterized in that: The illumination optical path module includes a laser, a collimating beam expansion system and an incident device, and the illumination optical path sequentially passes through the collimating beam expansion system, the incident device and the stage from the laser, wherein: The laser is used to emit the laser beam; The collimation and beam expansion system is used to collimate and expand the laser beam emitted by the laser to form a parallel beam; The incident device is used to cause the parallel light beam to be incident on the observation sample at the preset incident angle.

4. The microscopic imaging system according to claim 3, characterized in that: An attenuation unit is also provided on the illumination light path, wherein: The attenuation unit is located between the laser and the collimating beam expanding system, and is used to perform attenuation processing on the laser beam, and make the attenuated laser beam incident on the collimating beam expanding system.

5. A microscopic imaging method, characterized in that: Applied to the microscopic imaging system according to any one of claims 1 to 4, the method comprises: Collecting a fluorescent signal refracted by an observation sample, wherein the observation sample is a material or structure that is fluorescently labeled and used for experimental observation; Separating and transforming the multiple wavelength beams in the fluorescence signal to obtain spectrum information of the multiple wavelength beams in the fluorescence signal; wherein the separation and transformation processing includes at least one of the following: Fourier transform, dispersion separation, and information capture; the spectrum information is used to characterize the characteristics of the observed sample at different angles; Performing photoelectric signal conversion on the spectrum information of the multiple wavelength light beams in the fluorescence signal to obtain a captured image of the observed sample; Performing deconvolution reconstruction on the captured image to obtain a reconstructed image of the observed sample; wherein, The step of performing separation and transformation processing on the multiple wavelength light beams in the fluorescence signal to obtain spectrum information of the multiple wavelength light beams in the fluorescence signal includes: Performing Fourier transform on the fluorescence signal through a Fourier transform component to obtain frequency domain information of multiple wavelength light beams in the fluorescence signal; Performing dispersion separation on the fluorescence signal through a high dispersion device to obtain spectral information of multiple wavelength light beams in the fluorescence signal; The frequency domain information and spectrum information of the multiple wavelength light beams in the fluorescence signal are captured by a microlens array component to obtain the spectrum information of the multiple wavelength light beams in the fluorescence signal.

6. The microscopic imaging method according to claim 5, characterized in that: Before collecting and observing the fluorescence signal refracted by the sample, the method includes: The laser beam emitted by the laser is incident on the collimation and beam expansion system, and a parallel beam is obtained after being collimated and expanded by the collimation and beam expansion system; The parallel light beam is used to excite the observation sample at a preset incident angle through an incident device to obtain the fluorescence signal refracted by the observation sample; wherein the preset incident angle is determined by the refractive index of the cover glass on the observation sample.

7. The microscopic imaging method according to claim 6, characterized in that: The collecting and observing the fluorescence signal refracted by the sample comprises: The fluorescent signal refracted by the observed sample is incident on an objective lens, and is collected by the objective lens and reflected by a dichroic mirror, so that the Fourier transform component obtains the fluorescent signal.

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