Cell communication signal acquisition method and system

Abstract

The application discloses a cell communication signal acquisition method and system, and belongs to the technical field of three-dimensional cell optical microscopic imaging. The application aims at the problem that the axial depth of field of a fluorescence microscope is small, and a clear cell communication signal image cannot be obtained through single shooting. The method comprises the following steps: emitting an excitation light beam to a dyed fluorescence cell, and determining three focusing positions of the dyed fluorescence cell along a depth direction; after the fluorescence emitted by the dyed fluorescence cell is split, four to-be-acquired light beams are obtained: the first light beam is collected to obtain light field fluorescence data after passing through a microlens array; the second to fourth light beams are collected to obtain fluorescence images at the first position to the third position respectively; and cell communication signal reconstruction is carried out based on the light field fluorescence data, the fluorescence image at the first position, the fluorescence image at the second position and the fluorescence image at the third position, so that a visualized three-dimensional cell communication signal is obtained. The application is used for acquiring cell communication signals.

Description

TECHNICAL FIELD

[0001] The application relates to a cell communication signal acquisition method and system, and belongs to the technical field of three-dimensional cell optical microscopic imaging. BACKGROUND

[0002] The axial depth of field of a traditional fluorescence microscope is small, and only a small depth range can be clearly imaged at a time, or multiple axial depth scans need to be performed on cells to splice complete depth information, which requires a precise axial motion system, resulting in a complex system, high cost and long time consumption. SUMMARY

[0003] In view of the problem that the axial depth of field of a fluorescence microscope is small and a clear image of cell communication signals cannot be obtained through single shooting, the application provides a cell communication signal acquisition method and system.

[0004] The cell communication signal acquisition method provided by the application comprises the following steps:

[0005] The excitation light beam emitted by the dyed fluorescent cell is used to make the dyed fluorescent cell emit fluorescence, and three focusing positions of the dyed fluorescent cell along the depth direction are determined; the fluorescence emitted by the dyed fluorescent cell is sequentially split to obtain four to-be-acquired light beams:

[0006] The first to-be-acquired light beam is imaged through a microlens array to acquire light field fluorescence data; the second to-be-acquired light beam corresponds to the first focusing position to acquire a first position fluorescence image; the third to-be-acquired light beam corresponds to the second focusing position to acquire a second position fluorescence image; and the fourth to-be-acquired light beam corresponds to the third focusing position to acquire a third position fluorescence image.

[0007] Based on the light field fluorescence data, the first position fluorescence image, the second position fluorescence image and the third position fluorescence image, cell communication signal reconstruction is performed to obtain visualized three-dimensional cell communication signals.

[0008] According to the cell communication signal acquisition method, the method for making the dyed fluorescent cell emit fluorescence is as follows:

[0009] The excitation light beam emitted by the fluorescent light source is sequentially shaped, filtered and focused, and then converted into parallel light to irradiate the dyed fluorescent cell, so that the dyed fluorescent cell emits fluorescence.

[0010] According to the cell communication signal acquisition method, the method for obtaining four to-be-acquired light beams is as follows:

[0011] The fluorescence emitted by the fluorescently dyed cells is split into beams to obtain the original light beams of the first beam of to-be-collected light beams and the second beam of to-be-collected light beams; the original light beams of the second beam of to-be-collected light beams are split into beams to obtain the original light beams of the second beam of to-be-collected light beams and the third beam of to-be-collected light beams; the original light beams of the third beam of to-be-collected light beams are split into beams to obtain the third beam of to-be-collected light beams and the fourth beam of to-be-collected light beams.

[0012] According to the cell communication signal acquisition method, the method for obtaining the visualized three-dimensional cell communication signal is:

[0013] A predicted three-dimensional fluorescence point cloud is generated from the first position fluorescence image, the second position fluorescence image, and the third position fluorescence image;

[0014] A first mathematical model taking the predicted three-dimensional fluorescence point cloud as input and the first position fluorescence image, the second position fluorescence image, and the third position fluorescence image as output is established, and a second mathematical model taking the predicted three-dimensional fluorescence point cloud as input and the light field fluorescence data as output is established;

[0015] The predicted three-dimensional fluorescence point cloud is subjected to nonlinear optimization based on the first mathematical model and the second mathematical model in combination with a regularization term and a constraint term conforming to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and the target three-dimensional fluorescence point cloud is taken as the visualized three-dimensional cell communication signal.

[0016] According to the cell communication signal acquisition method, the three focusing positions of the fluorescently dyed cells along the depth direction are spaced apart by 0-80 um.

[0017] The cell communication signal acquisition system comprises:

[0018] A fluorescence excitation unit is configured to emit an excitation light beam to the fluorescently dyed cells to make them emit fluorescence; the fluorescently dyed cells have three focusing positions along a depth direction;

[0019] A first beam of to-be-collected light beams acquisition unit is configured to split the fluorescence emitted by the fluorescently dyed cells into the original light beams of the first beam of to-be-collected light beams and the second beam of to-be-collected light beams; and to use a microlens array to image the first beam of to-be-collected light beams to acquire light field fluorescence data;

[0020] A second beam of to-be-collected light beams acquisition unit is configured to split the original light beams of the second beam of to-be-collected light beams to obtain the original light beams of the second beam of to-be-collected light beams and the third beam of to-be-collected light beams; and to image the second beam of to-be-collected light beams corresponding to a first focusing position to acquire a first position fluorescence image;

[0021] The third beam of to-be-acquired light beam acquisition unit is used for splitting the original light beam of the third beam of to-be-acquired light beam, obtaining the third beam of to-be-acquired light beam and the fourth beam of to-be-acquired light beam; and imaging the third beam of to-be-acquired light beam corresponding to the second focusing position, acquiring the second position fluorescence image;

[0022] The fourth beam of to-be-acquired light beam acquisition unit is used for imaging the fourth beam of to-be-acquired light beam corresponding to the third focusing position, acquiring the third position fluorescence image;

[0023] The cell communication signal reconstruction unit is used for performing cell communication signal reconstruction on the light field fluorescence data, the first position fluorescence image, the second position fluorescence image and the third position fluorescence image, and obtaining visualized three-dimensional cell communication signals.

[0024] According to the cell communication signal acquisition system, the fluorescence excitation unit comprises a fluorescence light source, a shaping lens group, a fluorescence filter group and a microscope objective; the fluorescence filter group comprises an excitation filter, a dichroic mirror and an emission filter;

[0025] The excitation light beam emitted by the fluorescence light source is shaped by the shaping lens group, is sequentially focused to the back focal plane of the microscope objective through the excitation filter and the dichroic mirror of the fluorescence filter group, and parallel light is obtained through the microscope objective to irradiate the stained fluorescent cells, and fluorescence is obtained.

[0026] The fluorescence is collected by the microscope objective, and then passes through the dichroic mirror and the emission filter of the fluorescence filter group, and the to-be-acquired fluorescence beam is obtained.

[0027] According to the cell communication signal acquisition system, the first beam of to-be-acquired light beam acquisition unit further comprises a first beam splitting prism, a first tube lens group and an optical sensor;

[0028] The to-be-acquired fluorescence beam is divided into two beams by the first beam splitting prism; the first light beam after the splitting of the first beam splitting prism is used as the first beam of to-be-acquired light beam, is converged to the microlens array through the first tube lens group, and is collected by the optical sensor to obtain the light field fluorescence data;

[0029] The second light beam after the splitting of the first beam splitting prism is used as the original light beam of the second beam of to-be-acquired light beam.

[0030] According to the cell communication signal acquisition system, the microlens array is located at the focal plane of the first tube lens group; and the effective photosensitive surface source of the optical sensor is located at the focal plane of the microlens array.

[0031] According to the cell communication signal acquisition system, the second beam of to-be-acquired light beam acquisition unit comprises a second beam splitting prism, a second tube lens group and a first camera,

[0032] The original light beam of the second bundle of to-be-acquired light beams is split into two light beams by the second beam-splitting prism; the first light beam exiting the second beam-splitting prism is converged onto the first camera by the second tube lens group, to obtain the first-position fluorescence image; and the second light beam exiting the second beam-splitting prism serves as the original light beam of the third bundle of to-be-acquired light beams.

[0033] According to the cell communication signal acquisition system, the third bundle of to-be-acquired light beams includes a third beam-splitting prism, a first imaging lens group, and a second camera,

[0034] The original light beam of the third bundle of to-be-acquired light beams is split into two light beams by the third beam-splitting prism; the first light beam exiting the third beam-splitting prism is imaged on the second camera by the first imaging lens group, to obtain a second-position fluorescence image; and the second light beam exiting the third beam-splitting prism serves as the fourth bundle of to-be-acquired light beams.

[0035] According to the cell communication signal acquisition system, the fourth bundle of to-be-acquired light beams includes a second imaging lens group and a third camera,

[0036] The fourth bundle of to-be-acquired light beams is imaged on the third camera by the second imaging lens group, to obtain a third-position fluorescence image.

[0037] According to the cell communication signal acquisition system, the imaging of the first camera, the second camera, and the third camera respectively corresponds to the first focusing position, the second focusing position, and the third focusing position of the fluorescently dyed cells.

[0038] According to the cell communication signal acquisition system, the cell communication signal reconstruction unit includes a predicted three-dimensional fluorescence point cloud generation module, a first mathematical model, and a second mathematical model,

[0039] The predicted three-dimensional fluorescence point cloud generation module generates a predicted three-dimensional fluorescence point cloud based on the first-position fluorescence image, the second-position fluorescence image, and the third-position fluorescence image;

[0040] The first mathematical model is used to output the first-position fluorescence image, the second-position fluorescence image, and the third-position fluorescence image based on the predicted three-dimensional fluorescence point cloud;

[0041] The second mathematical model is used to output the light field fluorescence data based on the predicted three-dimensional fluorescence point cloud;

[0042] The first mathematical model and the second mathematical model perform nonlinear optimization on the predicted three-dimensional fluorescence point cloud in combination with a regularization term and a constraint term conforming to prior knowledge, to obtain a target three-dimensional fluorescence point cloud, and the target three-dimensional fluorescence point cloud is taken as a visualized three-dimensional cell communication signal.

[0043] The cell communication signal acquisition system according to the present application further comprises a culture dish, and the fluorescently dyed cells are placed in the culture dish.

[0044] The cell communication signal acquisition system according to the present application, wherein the optical sensor comprises a CCD camera and / or a CMOS camera.

[0045] The cell communication signal acquisition system according to the present application, wherein the three focusing positions of the fluorescently dyed cells along the depth direction are spaced apart by 0-80 um.

[0046] The present application has the following advantages: the present application is based on microlens array light field imaging, and does not need a precise axial motion system; clear imaging of cells in a large depth range can be obtained through single photographing, corresponding super-resolution reconstruction algorithm and 4-light-path fusion algorithm.

[0047] The present application adopts a light field method for digital refocusing, and can realize single three-dimensional imaging with a large depth of field; single photographing imaging is not needed, and the axial layer scanning is not needed, so that the efficiency is high; a complex and precise axial motion mechanism is not needed, so that the cost is effectively reduced; meanwhile, the present application has an algorithm advantage, and in the image reconstruction process, a variety of sensor data can be fused, and the optimal three-dimensional fluorescent data can be obtained according to the prior constraint. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is a light path principle diagram of the cell communication signal acquisition method according to the present application;

[0049] Figure 2 is a three focusing position diagram of the fluorescently dyed cells;

[0050] Figure 3 is a traditional microscopic imaging principle diagram; in the diagram, v is the horizontal coordinate of the lens exit pupil coordinate system, mu is the vertical coordinate of the lens exit pupil coordinate system, x is the horizontal coordinate of the camera coordinate system, and y is the vertical coordinate of the camera coordinate system;

[0051] Figure 4 is an imaging diagram of the present application using a microlens array; in the diagram, v is the horizontal coordinate of the microlens array coordinate system, mu is the vertical coordinate of the microlens array coordinate system, s is the horizontal coordinate of the camera coordinate system, and t is the vertical coordinate of the camera coordinate system. DETAILED DESCRIPTION

[0052] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be clearly and completely described below, 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 skilled in the art without creative work belong to the protection scope of the present application.

[0053] 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.

[0054] The present application will be further described below in combination with the accompanying drawings and specific embodiments, but not as a limitation of the present application.

[0055] Specific implementation Figures 1 to 4 As shown in the specific implementation, the present application provides a cell communication signal acquisition method, comprising:

[0056] The excitation light beam emitted by the stained fluorescent cell is used to make the stained fluorescent cell emit fluorescence, and three focusing positions of the stained fluorescent cell along the depth direction are determined; after the fluorescence emitted by the stained fluorescent cell is sequentially split, four to-be-acquired light beams are obtained:

[0057] Among them, the first to-be-acquired light beam is imaged through a microlens array to acquire light field fluorescence data; the second to-be-acquired light beam corresponds to the first focusing position to acquire a first position fluorescence image; the third to-be-acquired light beam corresponds to the second focusing position to acquire a second position fluorescence image; and the fourth to-be-acquired light beam corresponds to the third focusing position to acquire a third position fluorescence image.

[0058] Based on the light field fluorescence data, the first position fluorescence image, the second position fluorescence image and the third position fluorescence image, cell communication signal reconstruction is performed to obtain visualized three-dimensional cell communication signals.

[0059] In combination with Figure 1 As shown in the specific implementation, the method for making the stained fluorescent cell emit fluorescence is:

[0060] The excitation light beam emitted by the fluorescent light source is sequentially shaped, filtered and focused by the host computer control, and then converted into parallel light to irradiate the stained fluorescent cell, so that the stained fluorescent cell emits fluorescence.

[0061] The first focusing position of the stained fluorescent cell is a position of the stained fluorescent cell away from the fluorescent light source along the depth direction.

[0062] In the embodiment, the fluorescence emitted by the fluorescently dyed cells is split into beams to obtain the original light beams of the first and second beams of to-be-collected light beams; the original light beams of the second beam of to-be-collected light beams are split into beams to obtain the original light beams of the second and third beams of to-be-collected light beams; and the original light beams of the third beam of to-be-collected light beams are split into beams to obtain the third and fourth beams of to-be-collected light beams.

[0063] The method for obtaining the visualized three-dimensional cell communication signal in the embodiment is as follows:

[0064] A predicted three-dimensional fluorescence point cloud is generated from the first, second, and third position fluorescence images;

[0065] A first mathematical model taking the predicted three-dimensional fluorescence point cloud as input and the first, second, and third position fluorescence images as output and a second mathematical model taking the predicted three-dimensional fluorescence point cloud as input and the light field fluorescence data as output are established;

[0066] The predicted three-dimensional fluorescence point cloud is subjected to nonlinear optimization based on the first and second mathematical models in combination with a regularization term and a constraint term conforming to prior knowledge to obtain a target three-dimensional fluorescence point cloud, which is taken as the visualized three-dimensional cell communication signal.

[0067] As an example, the three focusing positions of the fluorescently dyed cells along the depth direction are spaced apart by 0-80 um.

[0068] Specific implementation two, in combination Figures 1 to 4 The application also provides a cell communication signal collection system, which comprises:

[0069] A fluorescence excitation unit is configured to emit an excitation light beam to fluorescently dyed cells to make the fluorescently dyed cells emit fluorescence; and the fluorescently dyed cells have three focusing positions along a depth direction.

[0070] A first beam of to-be-collected light beam collection unit is configured to split the fluorescence emitted by the fluorescently dyed cells into the original light beams of the first and second beams of to-be-collected light beams; and the first beam of to-be-collected light beams is imaged by a microlens array 1 to obtain light field fluorescence data.

[0071] A second beam of to-be-collected light beam collection unit is configured to split the original light beams of the second beam of to-be-collected light beams into the original light beams of the second and third beams of to-be-collected light beams; and the second beam of to-be-collected light beams is imaged at a first focusing position to obtain a first position fluorescence image.

[0072] The third beam of to-be-acquired light beam acquisition unit is used for splitting the original light beam of the third beam of to-be-acquired light beam to obtain the third beam of to-be-acquired light beam and the fourth beam of to-be-acquired light beam; and imaging the third beam of to-be-acquired light beam corresponding to the second focusing position to acquire the second position fluorescence image;

[0073] The fourth beam of to-be-acquired light beam acquisition unit is used for imaging the fourth beam of to-be-acquired light beam corresponding to the third focusing position to acquire the third position fluorescence image;

[0074] The cell communication signal reconstruction unit is used for performing cell communication signal reconstruction on the light field fluorescence data, the first position fluorescence image, the second position fluorescence image and the third position fluorescence image to obtain visualized three-dimensional cell communication signals.

[0075] Further, in combination with Figure 1 As shown in the figure, the fluorescence excitation unit includes a fluorescence light source 4, a shaping mirror group 5, a fluorescence filter group 6 and a microscope objective 7; the fluorescence filter group 6 includes an excitation filter 6-1, a dichroic mirror 6-2 and an emission filter 6-3;

[0076] The excitation light beam emitted by the fluorescence light source 4 is shaped by the shaping mirror group 5, then sequentially focused to the back focal plane of the microscope objective 7 through the excitation filter 6-1 and the dichroic mirror 6-2 of the fluorescence filter group 6, and parallel light is obtained by the microscope objective 7 to irradiate the stained fluorescent cells to obtain fluorescence; the fluorescence excitation of the stained fluorescent cells is completed.

[0077] The fluorescence is collected by the microscope objective 7, then passes through the dichroic mirror 6-2 and the emission filter 6-3 of the fluorescence filter group 6 to obtain the to-be-acquired fluorescence beam.

[0078] The shaping mirror group 5 is used for shaping the light beam emitted by the fluorescence light source. The fluorescence light source 4 is an LED light source or an LD light source. The fluorescence filter group 6 is used for filtering out unnecessary wavebands and only retaining the concerned fluorescence waveband to improve the signal-to-noise ratio of the system.

[0079] The first beam of to-be-acquired light beam acquisition unit further includes a first beam splitting prism 8-1, a first tube lens mirror group 9-1 and an optical sensor 2;

[0080] The to-be-acquired fluorescence beam is split into two light beams by the first beam splitting prism 8-1; the first light beam after splitting of the first beam splitting prism 8-1 is used as the first beam of to-be-acquired light beam, converges to the microlens array 1 through the first tube lens mirror group 9-1, and is acquired by the optical sensor 2 to obtain the light field fluorescence data;

[0081] The second light beam after splitting of the first beam splitting prism 8-1 is used as the original light beam of the second beam of to-be-acquired light beam.

[0082] The first focused position of the fluorescently dyed cell is a position of the fluorescently dyed cell away from the fluorescent light source 4 in the depth direction.

[0083] The microlens array 1 is located at the focal plane of the first tube lens group 9-1; the effective photosensitive surface source of the optical sensor 2 is located at the focal plane of the microlens array 1. The relative position relationship of the first tube lens group 9-1, the microlens array 1 and the optical sensor 2 needs to be strictly guaranteed.

[0084] The second beam to be collected light beam collection unit includes a second beam splitting prism 8-2, a second tube lens group 9-2 and a first camera 10-1,

[0085] The original light beam of the second beam to be collected light beam is split into two light beams by the second beam splitting prism 8-2; the first light beam emitted by the second beam splitting prism 8-2 is converged onto the first camera 10-1 by the second tube lens group 9-2, thereby obtaining the first position fluorescent image; the second light beam emitted by the second beam splitting prism 8-2 serves as the original light beam of the third beam to be collected light beam.

[0086] All parameters of the first tube lens group 9-1 and the second tube lens group 9-2 are the same.

[0087] The third beam to be collected light beam collection unit includes a third beam splitting prism 8-3, a first imaging lens group 11-1 and a second camera 10-2,

[0088] The original light beam of the third beam to be collected light beam is split into two light beams by the third beam splitting prism 8-3; the first light beam emitted by the third beam splitting prism 8-3 is imaged onto the second camera 10-2 by the first imaging lens group 11-1, thereby obtaining the second position fluorescent image; the second light beam emitted by the third beam splitting prism 8-3 serves as the fourth beam to be collected light beam.

[0089] The fourth beam to be collected light beam collection unit includes a second imaging lens group 11-2 and a third camera 10-3,

[0090] The fourth beam to be collected light beam is imaged onto the third camera 10-3 by the second imaging lens group 11-2, thereby obtaining the third position fluorescent image.

[0091] Further, the imaging of the first camera 10-1, the second camera 10-2 and the third camera 10-3 respectively corresponds to the first focused position, the second focused position and the third focused position of the fluorescently dyed cell.

[0092] In combination Figure 2As shown, by reasonable design and matching of each optical component, each light path imaging unit is focused on three positions of the cell, and clear imaging of the entire cell depth range is completed through the selected three positions, and the amplification of the three optical units is close to each other.

[0093] Further, the cell communication signal reconstruction unit comprises a predicted three-dimensional fluorescent point cloud generation module, a first mathematical model and a second mathematical model.

[0094] The predicted three-dimensional fluorescent point cloud generation module generates a predicted three-dimensional fluorescent point cloud based on the first position fluorescent image, the second position fluorescent image and the third position fluorescent image.

[0095] The first mathematical model is used to output the first position fluorescent image, the second position fluorescent image and the third position fluorescent image based on the predicted three-dimensional fluorescent point cloud.

[0096] The second mathematical model is used to output the light field fluorescent data based on the predicted three-dimensional fluorescent point cloud.

[0097] The first mathematical model and the second mathematical model combine a regularization term and a constraint term conforming to prior knowledge to perform nonlinear optimization on the predicted three-dimensional fluorescent point cloud, obtain a target three-dimensional fluorescent point cloud, and take the target three-dimensional fluorescent point cloud as a visualized three-dimensional cell communication signal.

[0098] In this embodiment, after single image acquisition is completed, the acquired data is reconstructed to obtain a visualized three-dimensional cell communication signal with higher precision. Different position fluorescent images are respectively projections of the observed fluorescent cell object on different focal planes, and the generated fluorescent point cloud is actually three planes, not a real three-dimensional fluorescent structure. The target three-dimensional fluorescent point cloud obtained after final optimization conforms to visual characteristics such as smoothness, continuity and spatial correlation. Since the imaging model is highly nonlinear and not convex, it cannot be quickly globally optimized by using an optimization algorithm, and therefore the predicted three-dimensional fluorescent point cloud obtained by image processing is used as an initial value for optimization to avoid that the nonlinear optimization falls into a local extreme value with a large gap from the real three-dimensional point cloud.

[0099] The system of the present application also comprises a culture dish 3, and the dyed fluorescent cells are placed in the culture dish 3.

[0100] As an example, the optical sensor 2 comprises a CCD camera and / or a CMOS camera.

[0101] As an example, the three focusing positions of the dyed fluorescent cells along the depth direction are spaced apart by 0-80 um.

[0102] In this embodiment, three focusing positions can be selected with a sequential interval of 50µm.

[0103] The specific imaging process of this invention: [Combined with...] Figure 1 and Figure 2 As shown, after the stained cells are excited, they emit fluorescence at a specific wavelength, and the fluorescence signals at three locations are collected by the microscope objective. After the light at the first focusing position is collected, it passes through the fluorescence filter group 6 and is split into two beams by the first beam splitter prism 8-1. The beam splitting ratio of the first beam splitter prism 8-1 can be 1:1 or 1:3. One of the beams is converged by the first tube endoscope group 9-1 onto the microlens array and received by the optical sensor 2, completing the acquisition of light field fluorescence data. The optical sensor 2 then images the entire stained cell. The other beam is split into two beams by the second beam splitter prism 8-2. One beam is converged onto the first camera 10-1 by the second tube mirror group 9-2, and the first camera 10-1 only clearly images the first focusing position. The other beam is split into two beams by the third beam splitter prism 8-3. One beam is imaged onto the second camera 10-2 by the first imaging mirror group 11-1, and the second camera 10-2 only clearly images the second focusing position. The other beam is imaged onto the third camera 10-3 by the second imaging mirror group 11-2, and the third camera 10-3 only clearly images the third focusing position.

[0104] like Figure 3 As shown, in traditional microscopic imaging systems, the light rays exiting the system's exit pupil eventually converge at the same position on the camera. This means only the sample's position information can be acquired, not the angle of the light rays. Consequently, clear imaging is only possible within a very small area of ​​the focal point, which is unsuitable for applications requiring 3D depth information testing or devices demanding precise and complex axial scanning. When using the method described in this invention… Figure 4 When performing light field imaging using a microlens array, the plane containing the microlens array and the plane containing the camera form a 4-dimensional space. In this space, each microlens array corresponds to n*n camera pixels, which form a macro-pixel. This macro-pixel corresponds to the spatial position information of the cell. At the same time, each sub-pixel in the n*n array corresponds to an angular direction information, thereby enabling the acquisition of angular information of different cell positions. Combined with a super-resolution refocusing algorithm, cells at different depths can be refocused to achieve clear imaging over a large depth range and 3D state reconstruction.

[0105] While the application has been described with reference to particular embodiments thereof, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present application. It will be apparent to those skilled in the art that numerous modifications can be made within the scope of the present application as defined by the appended claims. It is intended that all such modification fall within the spirit and scope of the present application. It will be understood that the features described in connection with one embodiment can be used in connection with another embodiment.

Claims

1. A method for collecting cell communication signals, the method comprising: receiving a plurality of signals from a plurality of cells; and determining a plurality of cell communication signals from the plurality of signals. comprising: emitting an excitation light beam to the stained fluorescent cell to make it emit fluorescence, and determining three focusing positions of the stained fluorescent cell along the depth direction; the fluorescence emitted by the stained fluorescent cell is split into four beams of to-be-acquired light beams in sequence: wherein the first beam of to-be-acquired light beams is imaged via a microlens array to acquire light field fluorescence data; the second beam of to-be-acquired light beams corresponds to the first focusing position to acquire a first position fluorescence image; the third beam of to-be-acquired light beams corresponds to the second focusing position to acquire a second position fluorescence image; the fourth beam of to-be-acquired light beams corresponds to the third focusing position to acquire a third position fluorescence image; reconstructing a cell communication signal based on the light field fluorescence data, the first position fluorescence image, the second position fluorescence image, and the third position fluorescence image to obtain a visualized three-dimensional cell communication signal; the method for obtaining the visualized three-dimensional cell communication signal comprises: generating a predicted three-dimensional fluorescence point cloud from the first position fluorescence image, the second position fluorescence image, and the third position fluorescence image; establishing a first mathematical model taking the predicted three-dimensional fluorescence point cloud as input and the first position fluorescence image, the second position fluorescence image, and the third position fluorescence image as output, and a second mathematical model taking the predicted three-dimensional fluorescence point cloud as input and the light field fluorescence data as output; performing nonlinear optimization on the predicted three-dimensional fluorescence point cloud based on the first mathematical model and the second mathematical model, combining a regularization term and a constraint term conforming to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and taking the target three-dimensional fluorescence point cloud as the visualized three-dimensional cell communication signal.

2. The cell communication signal acquisition method according to claim 1, characterized in that, The method for making the stained fluorescent cell emit fluorescence comprises: shaping, filtering, and focusing the excitation light beam emitted by a fluorescent light source in sequence, and then converting it into parallel light to irradiate the stained fluorescent cell to make the stained fluorescent cell emit fluorescence.

3. The cell communication signal acquisition method according to claim 1, characterized in that, The method for obtaining the four beams of to-be-acquired light beams comprises: splitting the fluorescence emitted by the stained fluorescent cell to obtain the original light beams of the first beam of to-be-acquired light beams and the second beam of to-be-acquired light beams; splitting the original light beams of the second beam of to-be-acquired light beams to obtain the original light beams of the second beam of to-be-acquired light beams and the third beam of to-be-acquired light beams; and splitting the original light beams of the third beam of to-be-acquired light beams to obtain the third beam of to-be-acquired light beams and the fourth beam of to-be-acquired light beams.

4. The cell communication signal acquisition method according to claim 1, wherein the three focusing positions of the stained fluorescent cell along the depth direction are spaced apart by 0-80 um.

5. A cellular communication signal acquisition system, comprising: comprising: a fluorescence excitation unit for emitting an excitation light beam to the stained fluorescent cell to make it emit fluorescence; the stained fluorescent cell has three focusing positions along the depth direction; a first beam of to-be-acquired light beams acquisition unit for splitting the fluorescence emitted by the stained fluorescent cell to obtain the original light beams of the first beam of to-be-acquired light beams and the second beam of to-be-acquired light beams; and imaging the first beam of to-be-acquired light beams using a microlens array (1) to acquire light field fluorescence data; The second-beam-to-be-collected light beam collecting unit is configured to split the original light beam of the second-beam-to-be-collected light beam to obtain original light beams of the second-beam-to-be-collected light beam and the third-beam-to-be-collected light beam, and to image the second-beam-to-be-collected light beam at a first focus position to obtain a first-position fluorescence image; The third-beam-to-be-collected light beam collecting unit is configured to split the original light beam of the third-beam-to-be-collected light beam to obtain original light beams of the third-beam-to-be-collected light beam and the fourth-beam-to-be-collected light beam, and to image the third-beam-to-be-collected light beam at a second focus position to obtain a second-position fluorescence image; The fourth-beam-to-be-collected light beam collecting unit is configured to image the fourth-beam-to-be-collected light beam at a third focus position to obtain a third-position fluorescence image; The cell communication signal reconstruction unit is configured to reconstruct cell communication signals based on the light field fluorescence data, the first-position fluorescence image, the second-position fluorescence image, and the third-position fluorescence image to obtain visualized three-dimensional cell communication signals; The cell communication signal reconstruction unit includes a predicted three-dimensional fluorescence point cloud generation module, a first mathematical model, and a second mathematical model, The predicted three-dimensional fluorescence point cloud generation module generates a predicted three-dimensional fluorescence point cloud based on the first-position fluorescence image, the second-position fluorescence image, and the third-position fluorescence image; The first mathematical model is configured to output the first-position fluorescence image, the second-position fluorescence image, and the third-position fluorescence image based on the predicted three-dimensional fluorescence point cloud; The second mathematical model is configured to output the light field fluorescence data based on the predicted three-dimensional fluorescence point cloud; The first mathematical model and the second mathematical model perform nonlinear optimization on the predicted three-dimensional fluorescence point cloud in combination with a regularization term and a constraint term conforming to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and the target three-dimensional fluorescence point cloud is taken as the visualized three-dimensional cell communication signal.

6. The cell communication signal acquisition system according to claim 5, wherein The fluorescence excitation unit includes a fluorescence light source (4), a shaping lens group (5), a fluorescence filter group (6), and a microscope objective (7); the fluorescence filter group (6) includes an excitation filter (6-1), a dichroic mirror (6-2), and an emission filter (6-3); The excitation light beam emitted by the fluorescence light source (4) is shaped by the shaping lens group (5), and then sequentially focused to the back focal plane of the microscope objective (7) through the excitation filter (6-1) and the dichroic mirror (6-2) of the fluorescence filter group (6), and parallel light is obtained through the microscope objective (7) to irradiate the stained fluorescent cells to obtain fluorescence; The fluorescence is collected by the microscope objective (7), and then passes through the dichroic mirror (6-2) and the emission filter (6-3) of the fluorescence filter group (6) to obtain a to-be-collected fluorescence beam.

7. The cell communication signal acquisition system according to claim 6, wherein The first-beam-to-be-collected light beam collecting unit further includes a first beam-splitting prism (8-1), a first tube lens group (9-1), and an optical sensor (2). The to-be-collected fluorescence beam is split into two beams by a first beam-splitting prism (8-1); the first beam after splitting by the first beam-splitting prism (8-1) is used as the first to-be-collected light beam and is converged by a first tube lens group (9-1) to the microlens array (1), and then is collected by the optical sensor (2) to obtain the light field fluorescence data. The second beam after splitting by the first beam-splitting prism (8-1) is used as the original light beam of the second to-be-collected light beam.

8. The cell communication signal collection system according to claim 7, wherein The microlens array (1) is located at the focal plane of the first tube lens group (9-1); and the effective photosensitive surface of the optical sensor (2) is located at the focal plane of the microlens array (1).

9. The cell communication signal collection system according to claim 8, wherein The second to-be-collected light beam collection unit comprises a second beam-splitting prism (8-2), a second tube lens group (9-2) and a first camera (10-1); The original light beam of the second to-be-collected light beam is split into two beams by the second beam-splitting prism (8-2); the first beam emitted by the second beam-splitting prism (8-2) is converged by the second tube lens group (9-2) to the first camera (10-1) to obtain the first position fluorescence image; and the second beam emitted by the second beam-splitting prism (8-2) is used as the original light beam of the third to-be-collected light beam.

10. The cellular communication signal collection system of claim 9, wherein, The third to-be-collected light beam collection unit comprises a third beam-splitting prism (8-3), a first imaging lens group (11-1) and a second camera (10-2); The original light beam of the third to-be-collected light beam is split into two beams by the third beam-splitting prism (8-3); the first beam emitted by the third beam-splitting prism (8-3) is imaged on the second camera (10-2) by the first imaging lens group (11-1) to obtain the second position fluorescence image; and the second beam emitted by the third beam-splitting prism (8-3) is used as the fourth to-be-collected light beam.

11. The cellular communication signal collection system of claim 10, wherein, The fourth to-be-collected light beam collection unit comprises a second imaging lens group (11-2) and a third camera (10-3); The fourth to-be-collected light beam is imaged on the third camera (10-3) by the second imaging lens group (11-2) to obtain the third position fluorescence image.

12. The cellular communication signal collection system of claim 5, wherein, A culture dish (3) is further included, and the fluorescently dyed cells are placed in the culture dish (3).

13. The cellular communication signal collection system of claim 7, wherein, The optical sensor (2) comprises a CCD camera and / or a CMOS camera.

14. The cell communication signal collection system according to claim 5, wherein The three focusing positions of the fluorescently dyed cells along the depth direction are spaced apart by 0-80 um.

Images