Cell tissue topography microscopic imaging method and imaging device thereof

Abstract

The cell tissue morphology microscopic imaging method and imaging device solve the contradiction between the field of view and the resolution of commercial microscopes and the problem that only 2D information can be obtained, and belong to the field of optical microscopic imaging. The Fourier superposition illumination light beam is used to irradiate a 3D cell tissue sample to be measured in parallel, the diffraction light generated by the 3D cell tissue sample to be measured is collected, the diffraction light is divided into two light signals according to a set proportion, low-resolution three-dimensional image information is obtained according to one of the light signals, and two-dimensional image information is obtained according to the other light signal; three-dimensional reconstruction is carried out according to the obtained low-resolution three-dimensional image information and two-dimensional image information, and the cell tissue morphology image of the 3D cell tissue sample to be measured is obtained. The four-dimensional light field and the Fourier superposition technology are fused in the light path, and three-dimensional reconstruction is proposed, so that large-field-of-view, high-resolution cell morphology imaging is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a cell tissue morphology microscopic imaging method and an imaging device thereof, and belongs to the field of optical microscopic imaging. BACKGROUND

[0002] Three-dimensional microscopic imaging technology can provide three-dimensional information at the micro-nano scale, allowing researchers to gain a deep understanding of biological tissue and cell structure. This technology is widely used in many scientific fields such as biology, medicine, physics, and materials science.

[0003] Currently, microscopic three-dimensional imaging technology has made significant progress. Existing three-dimensional microscopic imaging techniques include confocal microscopy, multi-photon microscopy, light sheet microscopy, three-dimensional microscopic imaging based on structured light, and three-dimensional microscopic imaging based on light field, etc. High-resolution three-dimensional microscopic imaging can only be achieved by layer-by-layer scanning, which is low in efficiency and shallow in depth of field; while direct three-dimensional imaging is difficult to improve resolution due to structural limitations.

[0004] In biomedical research, some cells often need to be cultured in vitro in 3D, and their state needs to be observed during the culture process for dynamic process research. Currently, commercial microscopes are mainly used for observation. However, commercial microscopes have low resolution when the field of view is large, and small field of view when the resolution is high, and can only obtain 2D image information, which restricts the observation and research application of 3D cell culture process. SUMMARY

[0005] To solve the contradiction between the field of view and resolution of commercial microscopes and the problem of only obtaining 2D information, the present application provides a cell tissue morphology microscopic imaging method and an imaging device thereof.

[0006] The cell tissue morphology microscopic imaging method of the present application comprises:

[0007] The Fourier superposition illumination beam is used to irradiate the 3D cell tissue sample to be measured in parallel, and the diffraction light generated by the 3D cell tissue sample to be measured is collected. The diffraction light is divided into two light signals according to a set proportion, the low-resolution three-dimensional image information is obtained according to one of the light signals, and the two-dimensional image information is obtained according to the other light signal.

[0008] The low-resolution three-dimensional image information and the two-dimensional image information are used to perform three-dimensional reconstruction to obtain the cell tissue morphology image of the 3D cell tissue sample to be measured.

[0009] As a preferred embodiment, the method for obtaining the cell tissue morphology image of the 3D cell tissue sample to be measured by three-dimensional reconstruction according to the obtained low-resolution three-dimensional image information and two-dimensional image information comprises:

[0010] Two models are constructed, the first model is a mathematical model for simulating the Fourier ptychographic process under a virtual aperture, and the first model takes a three-dimensional point cloud as input and outputs a spectrum and a phase map under different apertures, and the second model is a mathematical model for simulating an optical sensor, and the input is a three-dimensional point cloud, and the output is a three-dimensional image;

[0011] The three-dimensional point cloud of different focal lengths is constructed by using the obtained low-resolution three-dimensional image information, and is used as the initial input of the two models; the three-dimensional point cloud is used as an optimization variable, and a regularization term and a constraint term conforming to prior knowledge are added, the outputs of the two models are compared with the obtained low-resolution three-dimensional image information and two-dimensional image information, and the three-dimensional point cloud is optimized, and the cell tissue morphology image of the 3D cell tissue sample to be measured is obtained according to the optimized three-dimensional point cloud.

[0012] As preferred, the method for adopting the Fourier ptychographic illumination light beam is as follows:

[0013] The light sources at different positions are time-sharingly lighted up in a mutually overlapping and redundant manner, and then the diffraction light with high-frequency and low-frequency information is separately collected.

[0014] The application further provides an imaging device for the cell tissue morphology microscopic imaging method.

[0015] The light source module is used for parallelly irradiating the 3D cell tissue sample to be measured by adopting the Fourier ptychographic illumination light beam.

[0016] The acquisition module is used for collecting the diffraction light generated by the 3D cell tissue sample to be measured, dividing the diffraction light into two light signals according to a set proportion, obtaining low-resolution three-dimensional image information according to one of the light signals, and obtaining two-dimensional image information according to the other light signal.

[0017] The three-dimensional reconstruction module is used for performing three-dimensional reconstruction according to the obtained low-resolution three-dimensional image information and two-dimensional image information, and obtaining the cell tissue morphology image of the 3D cell tissue sample to be measured.

[0018] As preferred, the light source module comprises a two-dimensional LED array and an eyepiece lens group.

[0019] The two-dimensional LED array adopts Fourier ptychographic illumination, and the LED light of the two-dimensional LED array is parallelly irradiated to the 3D cell tissue sample to be measured after passing through the eyepiece lens group.

[0020] As preferred, the light sources at different positions in the two-dimensional LED array are time-sharingly lighted up in a mutually overlapping and redundant manner by the light source control system, so that the acquisition module separately collects the diffraction light with high-frequency and low-frequency information.

[0021] As preferred, the eyepiece lens group is a single lens, a multi-lens group or a compound eye lens group.

[0022] As preferred, the acquisition module comprises a microscopic objective, a beam splitter prism, a two-dimensional image acquisition module and an optical sensor;

[0023] The microscopic objective collects the diffraction light generated by the 3D cell tissue sample to be measured, and the diffraction light is incident to the beam splitter prism, which is divided into two light signals in a set proportion, one of which enters the optical sensor, and the other enters the two-dimensional image acquisition module;

[0024] The optical sensor acquires low-resolution three-dimensional image information according to one of the light signals, and the two-dimensional image acquisition module acquires two-dimensional image information according to the other light signal.

[0025] As preferred, the optical sensor comprises a No. 1 tube lens group, a microlens array and a No. 1 camera;

[0026] One of the light signals is sequentially passed through the No. 1 tube lens group and the microlens array, and is collected by the No. 1 camera, and the image collected by the No. 1 camera is low-resolution three-dimensional image information.

[0027] As preferred, the two-dimensional image acquisition module comprises a No. 2 tube lens group and a No. 2 camera;

[0028] The other light signal is collected by the No. 2 camera through the No. 2 tube lens group, and the image collected by the No. 2 camera is two-dimensional image information.

[0029] The present application has the advantages that the present application uses Fourier ptychographic illumination to improve the effective numerical aperture value of the resolution, has a large field of view and high-resolution cell morphology imaging; uses light field microscopic imaging technology to acquire spatial position and angle information, realizes quantitative three-dimensional imaging, and provides an algorithm for three-dimensional reconstruction, the reconstruction method of the present application is efficient, has low requirements for acquisition, and saves computing resources. The present application does not need a precise and complex motion system and a motion control system, and has the characteristics of low cost and simple structure. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a schematic diagram of the principle of the cell tissue morphology microscopic imaging device;

[0031] Figure 2 It is a schematic diagram of the principle comparison of traditional illumination and Fourier ptychographic illumination of the present application;

[0032] Figure 3 It is a schematic diagram of the imaging principle of a traditional microscopic system;

[0033] Figure 4 It is a schematic diagram of the three-dimensional imaging principle of the present application. DETAILED DESCRIPTION

[0034] With reference to the drawings and specific embodiments, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0035] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0036] The present application will be further described below in combination with the drawings and specific embodiments, but is not limited by the embodiments.

[0037] Specific embodiment one, a cell tissue topography microscopic imaging method, comprising:

[0038] The Fourier superposition illumination light beam is used to irradiate the 3D cell tissue sample to be measured in parallel, the diffraction light generated by the 3D cell tissue sample to be measured is collected, the diffraction light is divided into two light signals according to a set proportion, low-resolution three-dimensional image information is obtained according to one of the light signals, and two-dimensional image information is obtained according to the other light signal.

[0039] Three-dimensional reconstruction is performed according to the obtained low-resolution three-dimensional image information and two-dimensional image information, and a cell tissue topography image of the 3D cell tissue sample to be measured is obtained.

[0040] The traditional microscope uses a light beam with a certain aperture angle to illuminate the sample, and the microscopic objective lens collects the high-frequency and low-frequency information of the sample at the same time. The two produce aliasing and the low-frequency intensity is much greater than the high-frequency intensity, which leads to the inability to distinguish the high-frequency detail information of the sample. The Fourier superposition illumination provided in the present embodiment illuminates the sample at different positions at different time points, separates the collection of high-frequency and low-frequency information, and uses the provided high-frequency and low-frequency low-resolution pictures to perform three-dimensional reconstruction.

[0041] Traditional microscopy systems can only obtain object position intensity information in the camera plane and have a small imaging depth, and cannot meet the needs of 3D imaging application scenarios; or can only realize 3D reconstruction through multi-layer splicing in a layer scanning manner, but this method requires a precise and expensive focusing and motion system. The light field imaging method reduces the 7-dimensional parameters (x, y, z, θ, φ, λ, t) in the traditional light field to 4 dimensions (s, w, u, v) by adding a set of microlens arrays at the focal plane position of the tube lens group, where x, y and z represent three-dimensional spatial coordinates, θ represents the elevation angle of the light in the spatial coordinate system, φ represents the projection angle of the light in the spatial coordinate system, λ represents the wavelength, and t represents the time; s represents the horizontal coordinate of the defined position plane in the double-plane space, w represents the vertical coordinate of the defined position plane in the double-plane space, u represents the horizontal coordinate of the defined direction plane in the double-plane space, and v represents the vertical coordinate of the defined direction plane in the double-plane space; (s, t, u, v) contains different depth sample information of the object space. The space formed by the plane where the microlens array is located and the detection plane can not only obtain the position intensity information of the imaging plane, but also obtain the angle information, so that the refocusing algorithm based on the Fourier slice theorem is used to reconstruct the images at different positions in the space to generate three-dimensional images with quantitative depth.

[0042] The present embodiment combines the resolution-enhanced Fourier superposition illumination technology and the 4-dimensional light field technology capable of obtaining quantitative depth information in the optical path, so that quantitative three-dimensional imaging function under a large field of view and high resolution can be realized without the need for a complex motion mechanism.

[0043] The main function of Fourier superposition-based microscopic imaging is to improve the resolution of two-dimensional images, and the three-dimensional reconstruction method of the present embodiment simultaneously combines the phase, amplitude and three-dimensional image information of low resolution acquired by the light field sensor obtained by the Fourier superposition imaging method on this basis. After the acquisition of the original data of the sensor is completed, the corresponding algorithm is applied to the data for reconstruction and fusion. In order to fully utilize the fluorescence and light field image data of different depths to reconstruct more accurate three-dimensional cell communication signal visualization effect, in the preferred embodiment, the method for obtaining the cell tissue morphology image of the 3D cell tissue sample according to the obtained low-resolution three-dimensional image information and two-dimensional image information in the present embodiment includes:

[0044] Step 1, two models are constructed, the first model is a mathematical model for simulating the Fourier ptychographic process under a virtual aperture, the first model takes a three-dimensional point cloud as input and outputs a spectrum and a phase diagram under different apertures, and the second model is a mathematical model for simulating an optical sensor, the input is a three-dimensional point cloud, and the output is a three-dimensional image; the microlens array 7 in the light field sensor has a larger size compared to a camera pixel, and a certain distance is left between the microlenses to avoid light aliasing, resulting in a lower resolution of three-dimensional imaging, so there is a certain gap between the three-dimensional point cloud generated in this step and the real three-dimensional structure;

[0045] Step 2, use the obtained low-resolution three-dimensional image information to construct three-dimensional point clouds of different focal lengths, and use them as initial inputs of the two models; the three-dimensional point cloud is used as an optimization variable, and a regular term and a constraint term conforming to prior knowledge are added to ensure that the calculated three-dimensional point cloud has visual characteristics such as smoothness, continuity and spatial correlation. The outputs of the two models are compared with the obtained low-resolution three-dimensional image information and two-dimensional image information, and the input three-dimensional point cloud is optimized, and the cell tissue morphology image of the 3D cell tissue sample to be measured is obtained according to the optimized three-dimensional point cloud. Due to the highly nonlinear and non-convex characteristics of the imaging model, it is difficult to use an optimization algorithm for fast global optimization directly, therefore, the initial three-dimensional point cloud of different focal lengths is constructed and optimized to avoid the nonlinear optimization from falling into a local extreme value with a large gap from the real three-dimensional point cloud. The cell tissue morphology image of the 3D cell tissue sample to be measured is obtained according to the optimized three-dimensional point cloud.

[0046] The method of the embodiment uses Fourier ptychographic illumination to improve the effective numerical aperture value of the resolution, has a large field of view and a high-resolution cell morphology imaging; uses light field microscopic imaging technology to obtain spatial position and angle information, realizes quantitative three-dimensional imaging, and provides an algorithm for three-dimensional reconstruction. The reconstruction method of the application is efficient, has low requirements for collection, and saves computing resources. The application does not need a precise and complex motion system and a motion control system, and has the characteristics of low cost and simple structure.

[0047] The embodiment also provides an imaging device based on the cell tissue morphology microscopic imaging method, which comprises:

[0048] A light source module is configured to irradiate the 3D cell tissue sample to be measured with a Fourier ptychographic illumination beam in parallel;

[0049] A collection module is configured to collect diffracted light generated by the 3D cell tissue sample to be measured, divide the diffracted light into two light signals according to a set proportion, obtain low-resolution three-dimensional image information according to one of the light signals, and obtain two-dimensional image information according to the other light signal;

[0050] The three-dimensional reconstruction module is configured to perform three-dimensional reconstruction according to the obtained low-resolution three-dimensional image information and two-dimensional image information, so as to obtain a cell tissue morphology image of the 3D cell tissue sample to be measured.

[0051] In the preferred embodiment, the light source module includes a two-dimensional LED array 1 and an eyepiece lens group 2.

[0052] The 3D cell tissue sample to be measured is placed in a culture dish 3, the two-dimensional LED array 1 adopts Fourier superposition illumination, preferably the LEDs at different positions in the two-dimensional LED array 1 are lit in a time-sharing manner by the light source control system in an interlaced and redundant manner, so that the collection module separately collects the diffracted light with high-frequency and low-frequency information, and the LED light of the two-dimensional LED array 1 is parallelly irradiated on the 3D cell tissue sample to be measured in the culture dish 3 after passing through the wide-angle eyepiece lens group 2, thereby providing a uniform plane wave.

[0053] The traditional microscope adopts a certain aperture angle light beam to illuminate the sample, and the microscope objective simultaneously collects the high-frequency and low-frequency information of the sample, the two produce aliasing and the low-frequency intensity is much greater than the high-frequency intensity, which leads to the inability to distinguish the high-frequency detail information of the sample, the Fourier superposition illumination provided by the embodiment separates the collection of the high-frequency and low-frequency information by lighting the LEDs at different positions at different time points to illuminate the sample, and the three-dimensional reconstruction method provided later reconstructs by using the provided high-frequency and low-frequency low-resolution pictures, Figure 2 The schematic diagram of the traditional illumination and the Fourier superposition illumination of the embodiment (the left side is the traditional illumination, and the right side is only one of the illuminations lit).

[0054] The collection module of the embodiment includes a microscope objective 4, a beam splitter prism 5, a two-dimensional image collection module and an optical sensor.

[0055] The microscope objective 4 collects the diffracted light generated by the 3D cell tissue sample to be measured, the diffracted light is incident on the beam splitter prism 5, the beam splitter prism 5 is divided into two light signals in a set ratio, which can be divided into two light signals in a 1:1 manner, or other ratios, one of the two light signals enters the optical sensor, and the other enters the two-dimensional image collection module.

[0056] The optical sensor acquires low-resolution three-dimensional image information according to the one light signal, and the two-dimensional image collection module acquires two-dimensional image information according to the other light signal.

[0057] In the preferred embodiment, the optical sensor of the embodiment includes a No. 1 tube lens group 6-1, a microlens array 7 and a No. 1 camera 8-1; the microlens array 7 is located at the focal plane position of the No. 1 tube lens group 6, and the No. 1 camera 8-1 is located at the focal plane position of the microlens array 7.

[0058] One of the two light signals is sequentially through the No. 1 tube lens group 6-1 and the microlens array 7, and is collected by the No. 1 camera 8-1. The image collected by the No. 1 camera 8-1 is a low-resolution three-dimensional image information. The No. 1 camera 8-1 will collect a series of low-resolution original images, and different low-resolution images correspond to different sub-spectrum regions of the sample. The image is transmitted to the upper computer, and the resolution is improved by using a three-dimensional reconstruction method.

[0059] In the preferred embodiment, the two-dimensional image acquisition module of the present embodiment includes a No. 2 tube lens group 6-2 and a No. 2 camera 8-2, and the No. 2 camera 8-2 is located at the focal plane position of the No. 2 tube lens group 6.

[0060] The other light signal is collected by the No. 2 camera 8-2 through the No. 2 tube lens group 6-2, and the image collected by the No. 2 camera 8-2 is a two-dimensional image.

[0061] In the traditional microscope, the system resolution where λ is the wavelength of the illumination light source, NA obj is the numerical aperture of the objective lens, and the resolution of the microscopic system is only determined by the objective lens used. When the illumination method of the present embodiment is used, the resolution of the microscopic system is where NA ill is the numerical aperture of the illumination system. As can be seen from the formula, the system resolution is greatly improved due to the introduction of the illumination aperture, thereby realizing high-resolution imaging on the basis of maintaining the field of view of the original microscope objective, and solving the problem of mutual contradiction between the field of view and the resolution of the traditional microscopic system.

[0062] As shown in Figure 3 , the traditional microscopic system can only obtain the position intensity information of the object in the camera plane and has a small imaging depth, and cannot meet the requirements of the application scenarios with 3D imaging requirements; or can only realize 3D reconstruction by multi-layer splicing through layer scanning, but this method requires a precise and expensive focusing and motion system. The light field imaging method reduces the 7-dimensional parameters (x, y, z, θ, φ, λ, t) in the traditional light field to 4-dimensional (s, w, u, v) by adding a set of microlens arrays at the focal plane position of the tube lens group. x, y, and z represent the three-dimensional space position coordinates, θ represents the elevation angle of the light ray in the space coordinate system, φ represents the projection angle of the light ray in the space coordinate system, λ represents the wavelength, and t represents the time; s represents the horizontal coordinate of the position plane defined in the double-plane space, w represents the vertical coordinate of the position plane defined in the double-plane space, u represents the horizontal coordinate of the direction plane defined in the double-plane space, and v represents the vertical coordinate of the direction plane defined in the double-plane space; (s, t, u, v) contains different depth sample information of the object space. The space formed by the plane where the microlens array 7 is located and the plane where the camera is located can not only obtain the position intensity information of the imaging surface, but also obtain the angle information, so that the refocusing algorithm based on the Fourier slice theorem is used to reconstruct the images at different positions in the space, and a three-dimensional image with quantitative depth is generated, as shown inFigure 4 as shown.

[0063] Based on the above cell tissue topography microscopic imaging device, the resolution of the Fourier superposition illumination technology is improved, and the 4-dimensional light field technology which can obtain quantitative depth information is fused with the optical path, so that the quantitative three-dimensional imaging function under the condition of large field of view and high resolution can be realized without complex motion mechanism.

[0064] The main function of the Fourier superposition-based microscopic imaging is to improve the resolution of the two-dimensional image, and the three-dimensional reconstruction method of the embodiment simultaneously combines the phase, amplitude and three-dimensional image information with lower resolution collected by the light field sensor obtained by the Fourier superposition imaging method on this basis. After the collection of the original data of the sensor is completed, the corresponding algorithm is applied to the data for reconstruction and fusion. In order to fully utilize the fluorescence and light field image data of different depths of field, and to reconstruct more accurate three-dimensional cell communication signal visualization effect, in the preferred embodiment, the three-dimensional reconstruction module of the embodiment is used to construct two models. The first model is a mathematical model simulating the Fourier superposition process under a virtual aperture, and the first model takes three-dimensional point cloud as input and takes frequency spectrum and phase diagram under different apertures as output. The second model is a mathematical model simulating the optical sensor, and the input is three-dimensional point cloud and the output is three-dimensional image. The microlens array 7 in the light field sensor has a larger size compared with the camera pixel, and a certain distance is left between the microlenses to avoid light aliasing, resulting in lower resolution of three-dimensional imaging. Therefore, there is a certain gap between the three-dimensional point cloud generated in this step and the real three-dimensional structure.

[0065] The three-dimensional reconstruction module of the embodiment is also used to construct three-dimensional point clouds of different focal lengths by using the obtained low-resolution three-dimensional image information, and the three-dimensional point clouds are used as initial inputs of the two models. The three-dimensional point cloud is used as an optimization variable, and a regular term and a constraint term conforming to prior knowledge are added to ensure that the calculated three-dimensional point cloud has visual characteristics such as smoothness, continuity and spatial correlation. The outputs of the two models are compared with the obtained low-resolution three-dimensional image information and two-dimensional image information, and the input three-dimensional point cloud is optimized. The cell tissue topography image of the 3D cell tissue sample to be measured is obtained according to the optimized three-dimensional point cloud. Due to the highly nonlinear and non-convex characteristics of the imaging model, it is difficult to use the optimization algorithm for fast global optimization directly, therefore, the three-dimensional point cloud of different focal lengths constructed is used as the initial three-dimensional point cloud for optimization, avoiding the nonlinear optimization from falling into a local extreme value with a large gap from the real three-dimensional point cloud. The cell tissue topography image of the 3D cell tissue sample to be measured is obtained according to the optimized three-dimensional point cloud.

[0066] The embodiment has the advantages of large field of view, high resolution, Fourier superposition illumination technique improving effective numerical aperture value in resolution, realizing quantitative three-dimensional imaging, obtaining spatial position and angle information, using algorithm for three-dimensional reconstruction, low specific cost and simple structure, not needing precise and complex motion system and motion control system, and high efficiency of the three-dimensional reconstruction module of the embodiment, low requirement for collection, and saving of calculation resources.

[0067] While the application has been described with reference to particular embodiments, it will be understood that the examples are merely there to illustrate the principles and applications of the present application. It will be understood that various modifications can be made to the example embodiments, and other arrangements can be devised without departing from the spirit and scope of the present application as defined by the appended claims. It will be understood that the features described with reference to separate embodiments can be used in combination with one another. It will be understood that the features described in relation to one example can be used in other examples.

Claims

1. A method for microscopic imaging of cell and tissue morphology, characterized in that, The method includes: The 3D cell tissue sample to be tested is illuminated in parallel by a Fourier layered illumination beam. The diffracted light generated by the 3D cell tissue sample to be tested is collected and the diffracted light is divided into two light signals according to a set ratio. Low-resolution three-dimensional image information is obtained from one light signal and two-dimensional image information is obtained from the other light signal. Based on the obtained low-resolution three-dimensional image information and two-dimensional image information, three-dimensional reconstruction is performed to obtain the cell tissue morphology image of the 3D cell tissue sample to be tested; The method for obtaining a 3D cell tissue morphology image of the 3D cell tissue sample to be tested by performing 3D reconstruction based on the obtained low-resolution 3D image information and 2D image information includes: Two models are constructed. The first model is a mathematical model that simulates the Fourier stacking process under a virtual aperture. The first model takes a three-dimensional point cloud as input and outputs the spectrum and phase map under different apertures. The second model is a mathematical model that simulates an optical sensor. The input is a three-dimensional point cloud and the output is a three-dimensional image. Three-dimensional point clouds with different focal lengths are constructed using the obtained low-resolution three-dimensional image information and used as the initial input of two models. The three-dimensional point clouds are used as variables to be optimized, and regularization terms and constraints that conform to prior knowledge are added. The outputs of the two models are compared with the obtained low-resolution three-dimensional image information and two-dimensional image information to optimize the input three-dimensional point clouds. The cell tissue morphology image of the 3D cell tissue sample to be tested is obtained based on the optimized three-dimensional point clouds. The method of using Fourier layered illumination beams is as follows: By using an overlapping and redundant method, light sources at different locations are illuminated in a time-sharing manner, thereby separately collecting diffracted light carrying high-frequency and low-frequency information.

2. The imaging device for the cell and tissue morphology microscopic imaging method according to claim 1, characterized in that, The device includes: The light source module is used to illuminate the 3D cell tissue sample under test in parallel using a Fourier layered illumination beam; The acquisition module is used to collect the diffracted light generated by the 3D cell tissue sample to be tested, and to divide the diffracted light into two optical signals according to a set ratio. Low-resolution three-dimensional image information is obtained from one optical signal, and two-dimensional image information is obtained from the other optical signal. The 3D reconstruction module is used to perform 3D reconstruction based on the obtained low-resolution 3D image information and 2D image information to obtain the cell tissue morphology image of the 3D cell tissue sample to be tested.

3. The imaging device according to claim 2, characterized in that, The light source module includes a two-dimensional LED array and an eyepiece lens group; The two-dimensional LED array uses Fourier layered illumination, and the LED light from the two-dimensional LED array is parallel to the 3D cell tissue sample to be tested after passing through the eyepiece lens group.

4. The imaging device according to claim 3, characterized in that, The light source control system illuminates LEDs at different positions in the two-dimensional LED array in a time-division manner with overlapping redundancy, so that the acquisition module can collect diffracted light with high-frequency and low-frequency information separately.

5. The imaging device according to claim 3, characterized in that, The eyepiece lens group can be a single lens, a multi-lens group, or a compound lens group.

6. The imaging device according to claim 2, characterized in that, The acquisition module includes a microscope objective, a beam splitter prism, a two-dimensional image acquisition module, and an optical sensor; The microscope objective collects the diffracted light generated by the 3D cell tissue sample to be tested. The diffracted light is incident on a beam splitter prism, which splits the light into two light signals according to a set ratio. One light signal enters the optical sensor, and the other light signal enters the two-dimensional image acquisition module. The optical sensor acquires low-resolution three-dimensional image information based on one optical signal, and the two-dimensional image acquisition module acquires two-dimensional image information based on the other optical signal.

7. The imaging apparatus according to claim 6, characterized in that, The optical sensor includes a first tube mirror group, a microlens array, and a first camera; The optical signal passes sequentially through the No. 1 tube lens group and the microlens array before being acquired by the No. 1 camera. The image acquired by the No. 1 camera is a low-resolution three-dimensional image information.

8. The imaging apparatus according to claim 6, characterized in that, The two-dimensional image acquisition module includes a second tube mirror group and a second camera; The other optical signal is acquired by camera No. 2 through lens group No. 2, and the image acquired by camera No. 2 is two-dimensional image information.

Images