Cell communication signal acquisition method and system

Abstract

The invention discloses a cell communication signal acquisition method and system, and belongs to the technical field of three-dimensional cell optical microscopic imaging. The invention aims to solve the problem that a clear image of a cell communication signal cannot be obtained through single photographing due to small axial depth of field of a fluorescence microscope. The method comprises the following steps: emitting an excitation light beam to a dyed fluorescent cell, and determining three focusing positions of the dyed fluorescent cell along the depth direction; after the fluorescence emitted by the dyed fluorescent cells is split, four light beams to be collected are obtained; the first light beam is collected after passing through the micro-lens array to obtain light field fluorescence data; the second to fourth beams are respectively collected to obtain fluorescence images from the first position to the third position; and performing cell communication signal reconstruction 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 visual three-dimensional cell communication signal. The method is used for acquiring the cell communication signal.

Description

Technical Field

[0001] The invention relates to a cell communication signal collection method and system, and belongs to the technical field of three-dimensional cell optical microscopic imaging. Background Art

[0002] Traditional fluorescence microscopes have a small axial depth of field and can only clearly image a very small depth range at a time, or require multiple axial depth scans of cells to splice complete depth information. This requires a precise axial motion system, which makes the system complex, costly and time-consuming. Summary of the invention

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

[0004] A cell communication signal acquisition method of the present invention comprises:

[0005] An excitation light beam is emitted to the stained fluorescent cell to make it emit fluorescence, and three focusing positions of the stained fluorescent cell along the depth direction are determined; the fluorescence emitted by the stained fluorescent cell is sequentially split to obtain four light beams to be collected:

[0006] The first beam to be collected is imaged through the microlens array to collect light field fluorescence data; the second beam to be collected corresponds to the first focus position, and the fluorescence image of the first position is collected; the third beam to be collected corresponds to the second focus position, and the fluorescence image of the second position is collected; the fourth beam to be collected corresponds to the third focus position, and the fluorescence image of the third position is collected;

[0007] Cell communication signals are reconstructed 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.

[0008] According to the cell communication signal collection method of the present invention, the method of causing the stained fluorescent cells to emit fluorescence is:

[0009] The excitation light beam emitted by the fluorescent light source is shaped, filtered and focused in sequence, and then converted into parallel light to illuminate the stained fluorescent cells, so that the stained fluorescent cells emit fluorescence.

[0010] According to the cell communication signal collection method of the present invention, the method of obtaining four light beams to be collected is:

[0011] The fluorescence emitted by the stained fluorescent cells is split to obtain the original light beams of the first light beam to be collected and the second light beam to be collected; the original light beam of the second light beam to be collected is split to obtain the original light beams of the second light beam to be collected and the third light beam to be collected; the original light beam of the third light beam to be collected is split to obtain the third light beam to be collected and the fourth light beam to be collected.

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

[0013] 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;

[0014] Establishing a first mathematical model with 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 establishing a second mathematical model with the predicted three-dimensional fluorescence point cloud as input and the light field fluorescence data as output;

[0015] Based on the first mathematical model and the second mathematical model, the predicted three-dimensional fluorescence point cloud is nonlinearly optimized in combination with regularization terms and constraint terms that conform to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and the target three-dimensional fluorescence point cloud is used as a visualized three-dimensional cell communication signal.

[0016] According to the cell communication signal acquisition method of the present invention, the three focus positions of the stained fluorescent cells along the depth direction are spaced 0 to 80 um apart.

[0017] A cell communication signal acquisition system of the present invention comprises:

[0018] A fluorescence excitation unit, used for emitting an excitation light beam to the stained fluorescent cells to make them emit fluorescence; the stained fluorescent cells have three focus positions determined along the depth direction;

[0019] A first light beam collection unit is used to split the fluorescence emitted by the stained fluorescent cells to obtain the original light beams of the first light beam to be collected and the second light beam to be collected; and the first light beam to be collected is imaged by a microlens array to collect and obtain light field fluorescence data;

[0020] A second light beam collection unit to be collected is used to split the original light beam of the second light beam to be collected to obtain the original light beams of the second light beam to be collected and the third light beam to be collected; and to image the second light beam to be collected corresponding to the first focus position to collect a fluorescence image of the first position;

[0021] A third light beam collection unit for collecting the third light beam to be collected is used to split the original light beam of the third light beam to be collected to obtain the third light beam to be collected and the fourth light beam to be collected; and to image the third light beam to be collected corresponding to the second focus position to collect a fluorescence image at the second position;

[0022] A fourth light beam to be collected collecting unit, used for imaging the fourth light beam to be collected corresponding to a third focus position, and collecting and obtaining a fluorescence image at the third position;

[0023] The cell communication signal reconstruction unit is used to reconstruct the 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.

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

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

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

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

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

[0029] The second light beam split by the first beam splitting prism serves as the original light beam of the second light beam to be collected.

[0030] According to the cell communication signal acquisition system of the present invention, the microlens array is located at the focal plane of the first tube lens group; 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 of the present invention, the second light beam acquisition unit to be collected includes a second beam splitting prism, a second tube lens group and a first camera.

[0032] The original light beam of the second light beam to be collected is split into two light beams by the second beam splitting prism; the first light beam emitted by the second beam splitting prism is converged onto the first camera by the second tube lens group to obtain the first position fluorescence image; the second light beam emitted by the second beam splitting prism is used as the original light beam of the third light beam to be collected.

[0033] According to the cell communication signal acquisition system of the present invention, the third light beam acquisition unit to be collected includes a third beam splitting prism, a first imaging lens group and a second camera.

[0034] The original light beam of the third light beam to be collected is split into two light beams by the third beam splitting prism. The first light beam emitted by the third beam splitting prism is imaged on the second camera by the first imaging lens group to obtain a second position fluorescence image; the second light beam emitted by the third beam splitting prism is used as the fourth light beam to be collected.

[0035] According to the cell communication signal acquisition system of the present invention, the fourth light beam acquisition unit to be collected includes a second imaging lens group and a third camera.

[0036] The fourth light beam to be collected is imaged on the third camera through the second imaging lens to obtain a third position fluorescence image.

[0037] According to the cell communication signal acquisition system of the present invention, 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 stained fluorescent cells.

[0038] According to the cell communication signal acquisition system of the present invention, 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 regularization terms and constraint terms that conform to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and use the target three-dimensional fluorescence point cloud as a visualized three-dimensional cell communication signal.

[0043] The cell communication signal acquisition system according to the present invention further comprises a culture dish, in which the stained fluorescent cells are placed.

[0044] According to the cell communication signal acquisition system of the present invention, the optical sensor includes a CCD camera and / or a CMOS camera.

[0045] According to the cell communication signal acquisition system of the present invention, the three focusing positions of the stained fluorescent cells along the depth direction are spaced 0 to 80 um apart.

[0046] Beneficial effects of the present invention: The present invention is based on microlens array light field imaging, does not require a precise axial motion system, and can obtain clear imaging of cells in a large depth range through a single shot, a corresponding super-resolution reconstruction algorithm, and a 4-light path fusion algorithm. That is, by adding a microlens array combined with light path beam splitting, four light paths are formed, and a single shot is taken to obtain the four-dimensional parameters of the light field. By fusing the collected four light path data, a high-quality three-dimensional image can be reconstructed.

[0047] The present invention adopts the light field method for digital refocusing, which can realize single-shot three-dimensional imaging with a large depth of field; its single-shot imaging does not require axial layer scanning, and has the advantage of high efficiency; it does not require a complex and precise axial motion mechanism, which effectively reduces the cost; at the same time, the present invention has an algorithm advantage, and in the process of image reconstruction, it can fuse multiple sensor data and obtain the optimized three-dimensional fluorescence data in accordance with the prior constraints. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a principle diagram of the optical path of the cell communication signal collection method of the present invention;

[0049] Figure 2 It is a schematic diagram of the three focal positions of the stained fluorescent cells;

[0050] Figure 3 It is a schematic diagram of the principle of traditional microscopic imaging; in the figure, v is the horizontal coordinate of the lens exit pupil coordinate system, μ 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 Schematic diagram of imaging using a microlens array in the present invention; in the figure, v is the abscissa of the microlens array coordinate system, μ is the ordinate of the microlens array coordinate system, s is the abscissa of the camera coordinate system, and t is the ordinate of the camera coordinate system. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0054] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0055] Specific implementation method 1. Combination Figures 1 to 4 As shown, the present invention provides a method for collecting cell communication signals, comprising:

[0056] An excitation light beam is emitted to the stained fluorescent cell to make it emit fluorescence, and three focusing positions of the stained fluorescent cell along the depth direction are determined; the fluorescence emitted by the stained fluorescent cell is sequentially split to obtain four light beams to be collected:

[0057] The first beam to be collected is imaged through the microlens array to collect light field fluorescence data; the second beam to be collected corresponds to the first focus position, and the fluorescence image of the first position is collected; the third beam to be collected corresponds to the second focus position, and the fluorescence image of the second position is collected; the fourth beam to be collected corresponds to the third focus position, and the fluorescence image of the third position is collected;

[0058] Cell communication signals are reconstructed 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.

[0059] Combination Figure 1 As shown, the method for making the stained fluorescent cells emit fluorescence is:

[0060] The host computer controls the excitation light beam emitted by the fluorescent light source to be shaped, filtered and focused in sequence, and then converted into parallel light to irradiate the stained fluorescent cells, so that the stained fluorescent cells emit fluorescence.

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

[0062] In this embodiment, the fluorescence emitted by the stained fluorescent cells is split to obtain the original beams of the first beam to be collected and the second beam to be collected; the original beam of the second beam to be collected is split to obtain the original beam of the second beam to be collected and the third beam to be collected; the original beam of the third beam to be collected is split to obtain the third beam to be collected and the fourth beam to be collected.

[0063] The method for obtaining a visualized three-dimensional cell communication signal in this embodiment is:

[0064] 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;

[0065] Establishing a first mathematical model with 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 establishing a second mathematical model with the predicted three-dimensional fluorescence point cloud as input and the light field fluorescence data as output;

[0066] Based on the first mathematical model and the second mathematical model, the predicted three-dimensional fluorescence point cloud is nonlinearly optimized in combination with regularization terms and constraint terms that conform to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and the target three-dimensional fluorescence point cloud is used as a visualized three-dimensional cell communication signal.

[0067] As an example, the three focus positions of the stained fluorescent cells along the depth direction are spaced 0 to 80 um apart.

[0068] Specific implementation method 2: Combination Figures 1 to 4 As shown, the present invention also provides a cell communication signal acquisition system, comprising:

[0069] A fluorescence excitation unit, used for emitting an excitation light beam to the stained fluorescent cells to make them emit fluorescence; the stained fluorescent cells have three focus positions determined along the depth direction;

[0070] A first light beam collection unit is used to split the fluorescence emitted by the stained fluorescent cells to obtain the original light beams of the first light beam to be collected and the second light beam to be collected; and the first light beam to be collected is imaged by using a microlens array 1 to collect and obtain light field fluorescence data;

[0071] A second light beam collection unit to be collected is used to split the original light beam of the second light beam to be collected to obtain the original light beams of the second light beam to be collected and the third light beam to be collected; and to image the second light beam to be collected corresponding to the first focus position to collect a fluorescence image of the first position;

[0072] A third light beam collection unit for collecting the third light beam to be collected is used to split the original light beam of the third light beam to be collected to obtain the third light beam to be collected and the fourth light beam to be collected; and to image the third light beam to be collected corresponding to the second focus position to collect a fluorescence image at the second position;

[0073] A fourth light beam to be collected collecting unit, used for imaging the fourth light beam to be collected corresponding to a third focus position, and collecting and obtaining a fluorescence image at the third position;

[0074] The cell communication signal reconstruction unit is used to reconstruct the 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.

[0075] Further, combined with Figure 1 As shown, 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;

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

[0077] 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 fluorescence beam to be collected.

[0078] The shaping lens group 5 is used to shape the light beam emitted by the fluorescent light source. The fluorescent light source 4 is an LED light source or an LD light source. The fluorescent filter group 6 is used to filter out unnecessary bands and only retain the fluorescent band of interest to improve the signal-to-noise ratio of the system.

[0079] The first light beam collection unit to be collected further includes a first beam splitting prism 8-1, a first tube lens assembly 9-1 and an optical sensor 2;

[0080] The fluorescence beam to be collected is divided into two beams by the first beam splitting prism 8-1; the first beam split by the first beam splitting prism 8-1 is used as the first beam to be collected, and then converged to the microlens array 1 by the first tube lens group 9-1, and then collected by the optical sensor 2 to obtain light field fluorescence data;

[0081] The second light beam split by the first beam splitting prism 8 - 1 serves as the original light beam of the second light beam to be collected.

[0082] The first focusing position of the stained fluorescent cell is the position of the stained fluorescent cell far away from the fluorescent light source 4 along 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 between the first tube lens group 9-1, the microlens array 1 and the optical sensor 2 must be strictly guaranteed.

[0084] The second 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 light beam to be collected 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 to obtain the first position fluorescence image; the second light beam emitted by the second beam splitting prism 8-2 is used as the original light beam of the third light beam to be collected.

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

[0087] The third 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 light beam to be collected 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 on the second camera 10-2 by the first imaging lens group 11-1 to obtain a second position fluorescence image; the second light beam emitted by the third beam splitting prism 8-3 is used as the fourth light beam to be collected.

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

[0090] The fourth light beam to be collected is imaged on the third camera 10 - 3 through the second imaging lens group 11 - 2 to obtain a 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 focus position, the second focus position and the third focus position of the stained fluorescent cells.

[0092] Combination Figure 2As shown in the figure, through the reasonable design and matching of each optical component, each optical path imaging unit is focused on three positions of the cell respectively, and the entire cell depth range is clearly imaged through the selected three positions, and the magnification of the three optical units is close. In actual use, the light field optical path can be focused on three target positions according to the application scenario requirements to ensure that the entire depth direction of the cell is covered.

[0093] Furthermore, 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,

[0094] 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;

[0095] 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;

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

[0097] The first mathematical model and the second mathematical model perform nonlinear optimization on the predicted three-dimensional fluorescence point cloud in combination with regularization terms and constraint terms that conform to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and use the target three-dimensional fluorescence point cloud as a visualized three-dimensional cell communication signal.

[0098] In this embodiment, after a single image acquisition is completed, the acquired data is reconstructed to obtain a more accurate visualized three-dimensional cell communication signal. The fluorescence images at different positions are respectively the projections of the observed fluorescent cell objects at different focal planes, and the generated fluorescence point cloud is actually three planes, not a true three-dimensional fluorescence structure; the target three-dimensional fluorescence point cloud finally optimized meets the visual characteristics of smoothness, continuity, and spatial correlation. Since the imaging model is highly nonlinear and not convex, it is impossible to use the optimization algorithm for fast global optimization. Therefore, the predicted three-dimensional fluorescence point cloud obtained by image processing is used as the initial value for optimization to avoid the nonlinear optimization from falling into a local extreme value that is too far away from the real three-dimensional point cloud.

[0099] The system of the present invention further comprises a culture dish 3 , in which the stained fluorescent cells are placed.

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

[0101] As an example, the three focus positions of the stained fluorescent cells along the depth direction are spaced 0 to 80 um apart.

[0102] In this embodiment, three focus positions may be selected with intervals of 50 um in sequence.

[0103] The specific imaging process of the present invention: combining Figure 1 and Figure 2 As shown, after the dyed cells are excited, they emit fluorescence of a specific wavelength, and the fluorescence signals at three positions 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 divided 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 lens group 9-1 onto the microlens array and received by the optical sensor 2 to complete the light field fluorescence data collection. The optical sensor 2 images the entire dyed cell. The other beam is further divided into two beams by the second beam splitting prism 8-2, one of which is converged onto the first camera 10-1 by the second tube lens group 9-2, and the first camera 10-1 only clearly images the first focus position; the other beam is further divided into two beams by the third beam splitting prism 8-3, one of which is imaged onto the second camera 10-2 by the first imaging lens group 11-1, and the second camera 10-2 only clearly images the second focus position; the other beam is imaged onto the third camera 10-3 by the second imaging lens group 11-2, and the third camera 10-3 only clearly images the third focus position.

[0104] like Figure 3 As shown, in the traditional microscopic imaging system, the light emitted from the system exit pupil will eventually converge to the same position of the camera. At this time, only the sample position information can be obtained, and the light angle information cannot be obtained, resulting in clear imaging only within a very small range of the focus position. It cannot meet the application scenarios with 3D depth information testing requirements or devices that require precise and complex axial scanning. When the present invention is used Figure 4 When the microlens array in the image is used for light field imaging, the surface where the microlens array is located and the surface where the camera is located constitute a 4-dimensional space. In this space, each n*n camera pixel corresponding to the microlens array forms a macro pixel, which corresponds to the spatial position information of the cell. At the same time, each sub-pixel in n*n corresponds to an angle direction information, so that the angle information of the cell at different positions can be obtained. Combined with the super-resolution refocusing algorithm, cells at different depths can be refocused to achieve clear imaging in a large depth range and three-dimensional state reconstruction.

[0105] Although the present invention is described herein with reference to specific embodiments, it should be understood that these embodiments are merely examples of the principles and applications of the present invention. It should therefore be understood that many modifications may be made to the exemplary embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims. It should be understood that the various dependent claims and features described herein may be combined in a manner different from that described in the original claims. It should also be understood that the features described in conjunction with a single embodiment may be used in other described embodiments.

Claims

1. A method for collecting cell communication signals, characterized in that: include: An excitation light beam is emitted to the stained fluorescent cell to make it emit fluorescence, and three focusing positions of the stained fluorescent cell along the depth direction are determined; the fluorescence emitted by the stained fluorescent cell is sequentially split to obtain four light beams to be collected: The first beam to be collected is imaged by the microlens array to collect light field fluorescence data; the second beam to be collected corresponds to the first focus position to collect the fluorescence image of the first position; The third light beam to be collected corresponds to the second focusing position, and collects and obtains a fluorescence image of the second position; The fourth light beam to be collected corresponds to the third focusing position, and collects and obtains a fluorescence image at the third position; Cell communication signals are reconstructed 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.

2. The cell communication signal acquisition method according to claim 1, characterized in that: The method for causing the stained fluorescent cells to emit fluorescence is: The excitation light beam emitted by the fluorescent light source is shaped, filtered and focused in sequence, and then converted into parallel light to illuminate the stained fluorescent cells, so that the stained fluorescent cells emit fluorescence.

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

4. The cell communication signal acquisition method according to claim 1, characterized in that: The method for obtaining a visualized three-dimensional cell communication signal is: 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 with 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 establishing a second mathematical model with the predicted three-dimensional fluorescence point cloud as input and the light field fluorescence data as output; Based on the first mathematical model and the second mathematical model, the predicted three-dimensional fluorescence point cloud is nonlinearly optimized in combination with regularization terms and constraint terms that conform to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and the target three-dimensional fluorescence point cloud is used as a visualized three-dimensional cell communication signal.

5. The cell communication signal acquisition method according to claim 1, characterized in that: The three focusing positions of the stained fluorescent cells along the depth direction are spaced 0 to 80 um apart.

6. A cell communication signal acquisition system, characterized in that: include: A fluorescence excitation unit, used for emitting an excitation light beam to the stained fluorescent cells to make them emit fluorescence; the stained fluorescent cells have three focus positions determined along the depth direction; A first light beam to be collected collection unit is used to split the fluorescence emitted by the stained fluorescent cells to obtain the original light beams of the first light beam to be collected and the second light beam to be collected; and the first light beam to be collected is imaged using a microlens array (1) to collect and obtain light field fluorescence data; A second light beam collection unit to be collected is used to split the original light beam of the second light beam to be collected to obtain the original light beams of the second light beam to be collected and the third light beam to be collected; and to image the second light beam to be collected corresponding to the first focus position to collect a fluorescence image of the first position; A third light beam collection unit for collecting the third light beam to be collected is used to split the original light beam of the third light beam to be collected to obtain the third light beam to be collected and the fourth light beam to be collected; and to image the third light beam to be collected corresponding to the second focus position to collect a fluorescence image at the second position; A fourth light beam to be collected collecting unit, used for imaging the fourth light beam to be collected corresponding to a third focus position, and collecting and obtaining a fluorescence image at the third position; The cell communication signal reconstruction unit is used to reconstruct the 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.

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

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

9. The cell communication signal acquisition system according to claim 8, characterized in that: The microlens array (1) is located at the focal plane of the first tube lens group (9-1); and the effective light-sensitive surface source of the optical sensor (2) is located at the focal plane of the microlens array (1).

10. The cell communication signal acquisition system according to claim 9, characterized in that: The second 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 light beam to be collected 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) to obtain the first position fluorescence image; the second light beam emitted by the second beam splitting prism (8-2) is used as the original light beam of the third light beam to be collected.

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

12. The cell communication signal acquisition system according to claim 11, characterized in that: The fourth light beam collection unit comprises a second imaging lens group (11-2) and a third camera (10-3). The fourth light beam to be collected is imaged on a third camera (10-3) via a second imaging lens group (11-2) to obtain a third position fluorescence image.

13. The cell communication signal acquisition system according to claim 12, characterized in that: The imaging of the first camera (10-1), the second camera (10-2) and the third camera (10-3) respectively corresponds to the first focusing position, the second focusing position and the third focusing position of the stained fluorescent cells.

14. The cell communication signal acquisition system according to claim 6, characterized in that: 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 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; The second mathematical model is used 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 regularization terms and constraint terms that conform to prior knowledge to obtain a target three-dimensional fluorescence point cloud, and use the target three-dimensional fluorescence point cloud as a visualized three-dimensional cell communication signal.

15. The cell communication signal acquisition system according to claim 6, characterized in that: It also includes a culture dish (3), in which the stained fluorescent cells are placed.

16. The cell communication signal acquisition system according to claim 8, characterized in that: The optical sensor (2) comprises a CCD camera and / or a CMOS camera.

17. The cell communication signal acquisition system according to claim 6, characterized in that: The three focusing positions of the stained fluorescent cells along the depth direction are spaced 0 to 80 um apart.

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