A method, device and storage medium for positioning and tracking the three-dimensional movement of cells

Abstract

The present invention discloses a method, device and storage medium for positioning and tracking the three-dimensional movement of cells, belonging to the field of imaging measurement. The method includes: acquiring the real-time three-dimensional scattered light field distribution of multiple cells in the field of view in space; performing two-dimensional positioning of single-frame cell images; connecting the two-dimensional trajectories of the previous and subsequent frames for the cell individuals that have achieved two-dimensional positioning in the field of view according to the spatio-temporal consistency of their positions; performing longitudinal positioning on each position point on the two-dimensional trajectory of a single cell; and obtaining the three-dimensional continuous space movement trajectory of the same cell according to the relative longitudinal displacement and the spatio-temporal consistency of the two-dimensional trajectory. The present invention uses the interference optical imaging method to acquire the scattered light field distribution of cells in three-dimensional space, and then realizes the three-dimensional real-time positioning and tracking of multiple cells by first performing two-dimensional cell positioning and then performing longitudinal positioning based on the relative displacement of the three-dimensional cell light field at this position; it has the advantages of high positioning accuracy and good robustness.

Description

Technical Field

[0001] The present invention relates to the field of imaging measurement, and in particular, to a method, device, and storage medium for positioning and tracking the three-dimensional movement of cells. Background Art

[0002] As the smallest unit of life, the study of the three-dimensional movement and migration behavior of cells is of great significance for fields such as life science, medicine, and agricultural production. However, the optical contrast between cells themselves and the surrounding medium is weak, and there are complex organelle structures inside; at the same time, complex morphological changes can occur during the movement and migration process of cells. Therefore, it is extremely challenging to perform in-situ and real-time three-dimensional positioning and tracking of cells, especially non-adherent cells.

[0003] Currently, cell dynamic observation methods usually rely on bright-field illumination or fluorescent real-time imaging of labeled cells. After obtaining the images, the two-dimensional trajectories of the cells are obtained and analyzed. These methods are suitable for adherent cells migrating on the surface. After imaging the cells and combining image segmentation and other processing methods, the center of the cells or specifically labeled organelles can be identified, and then the positioning and tracking of cells in the two-dimensional direction can be achieved. However, such methods cannot reflect the movement and migration patterns of cells in the real three-dimensional space, especially cells in a non-adherent state, especially cells migrating in a complex three-dimensional environment, which have great limitations. Currently, to achieve three-dimensional tracking of cells, it often depends on a high-speed confocal fluorescence microscope to stain and scan the cells layer by layer at different focal planes. The relevant hardware costs are high, and in-situ monitoring cannot be achieved. And special staining treatment of cells may damage some cells, such as the health status of immune cells.

[0004] It can be seen that there is an urgent need for a high-precision, label-free, and robust method for three-dimensional cell positioning and tracking to achieve real-time research on the movement laws of cells and provide inspiration for related fields. Summary of the Invention

[0005] To solve at least one of the technical problems existing in the prior art to a certain extent, the purpose of the present invention is to provide a method, device, and storage medium for positioning and tracking the three-dimensional movement of cells.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A method for positioning and tracking the three-dimensional movement of cells includes the following steps:

[0008] S1. Use the interference optical imaging method to photograph a sample containing multiple cells, numerically reconstruct the photographed cell interference image, and calculate the scattered light intensity distribution of the cell sample in three-dimensional space;

[0009] S2. Obtain the projection position of the cell center on each frame of the interference image by superimposing the longitudinal light intensity values and searching for local maxima, so as to realize the two-dimensional positioning of a single-frame cell image;

[0010] S3. For the cell individuals that have achieved two-dimensional positioning, connect the two-dimensional trajectories of the front and back frames according to the spatio-temporal consistency of their positions;

[0011] S4. Obtain the longitudinal light intensity change curve of the cell center at different times according to the two-dimensional positioning coordinates and the scattered light intensity distribution, and calculate the relative longitudinal displacement between adjacent frames of the same cell according to the longitudinal light intensity change curve;

[0012] S5. Obtain the three-dimensional continuous spatial motion trajectory of the same cell according to the relative longitudinal displacement and the spatio-temporal consistency of the two-dimensional trajectory.

[0013] Furthermore, in step S1, the interference optical imaging method is performed by illuminating with a plane wave or a spherical wave, and the obtained image is a defocused optical interference pattern;

[0014] The scattered light intensity distribution in the three-dimensional space is obtained from the defocused optical interference image according to the propagation simulation formula of the optical diffraction principle. To ensure that the three-dimensional position of the cell can be obtained, the reconstruction distance should cover the entire range of defocus - focus - defocus.

[0015] Furthermore, in the two-dimensional positioning step of step S2, the method of superimposing longitudinal light intensity values is applied, including:

[0016] Calculate the discrete values of the scattered light intensity distribution obtained from a single-frame image along the longitudinal direction and superimpose them, and then obtain a longitudinal light intensity superimposed image with the same pixel size as the original image. Based on this longitudinal light intensity superimposed image, subsequent two-dimensional positioning calculations are performed.

[0017] Furthermore, step S2 includes:

[0018] Set the determination range of local maxima according to the cell size and magnification, and traverse the entire superimposed image for searching;

[0019] When the value at a certain position on the image is greater than the other points within the preset determination range defined with this point as the center, it is determined that this point is the two-dimensional projection position corresponding to a single cell.

[0020] Furthermore, step S3 includes:

[0021] S31. Set the maximum distance for trajectory connection. When the distance between the two-dimensional positioning points of adjacent images is greater than the preset value, it is determined that the cells in the two frames of images are not the same cell;

[0022] S32. Arrange all two-dimensional positioning results in chronological order. Taking the two-dimensional positioning points of the first frame as the center, search for corresponding points in the next frame. When there are no positioning points within the preset distance range, terminate the search for this trajectory. When there is only one positioning point within the preset distance, consider this point to be on the same trajectory as the starting point. When there are two or more positioning points within the preset distance, first assume that all positioning points are corresponding points, and determine the connection method of the two-dimensional trajectories of the front and rear frames according to the parameters calculated subsequently.

[0023] S33. Repeat step S32, and judge the connection relationship between the two-dimensional positioning points of all adjacent frames. According to the connection relationship, organize all the obtained positioning points into the trajectory of the two-dimensional projection of the cell center moving over time, discard the unconnected positioning points and overly short trajectories among them, and obtain the two-dimensional projection trajectory of the cell.

[0024] Further, the step S4 includes:

[0025] S41. Determine two intensity curves I1 and I2 that need to be matched according to the two-dimensional trajectory.

[0026] S42. Obtain the cell intensity characteristic curve from the intensity curve I1.

[0027] S43. Match the intensity characteristic curve with the intensity curve I2: Starting from the endpoints of the intensity curve I2, intercept a line segment of the same length as the intensity characteristic curve, and calculate the evaluation parameter r1 of the similarity between this line segment and the intensity characteristic curve. Translate one discrete data point and intercept another line segment of the same length as the characteristic curve, and calculate the evaluation parameter r2 of the new line segment. Repeat this process step by step until the end point of the intercepted line segment exceeds the range of the intensity curve I2, obtaining a series of parameters r3, r4, ……, rn. Obtain the displacement relationship of the curves according to the parameter variation law, and then calculate the relative movement distance of the cell between two frames of images.

[0028] S44. Use the position of the maximum spatial intensity to determine the three-dimensional position of the trajectory starting point, and superimpose the pairwise relative displacements to obtain the movement trajectory of the cell in space, thereby realizing the longitudinal positioning and three-dimensional trajectory tracking of the cell.

[0029] Further, the intensity characteristic curve includes the cell center position, and this intensity characteristic curve is obtained by discarding the data at both ends far from the focal plane of the intensity curve I1; according to the estimated maximum longitudinal displacement of the cell and the reconstruction range, intercept a part of the intensity curve including the focused range of the intensity curve I1 as the cell positioning feature.

[0030] Further, the evaluation parameter in step S43 is not only used to obtain the longitudinal displacement of the cell, but also used for the secondary verification and evaluation of whether the two-dimensional trajectory positioning points are the same cell; this evaluation parameter is obtained by calculating the correlation coefficient or calculating the sum of the squared differences of the corresponding position values.

[0031] Another technical solution adopted by the present invention is as follows:

[0032] A device for positioning and tracking the three-dimensional movement of cells, comprising:

[0033] At least one processor;

[0034] At least one memory for storing at least one program;

[0035] When the at least one program is executed by the at least one processor, the at least one processor implements the method as described above.

[0036] Another technical solution adopted by the present invention is as follows:

[0037] A computer-readable storage medium storing a program executable by a processor, and the program executable by the processor is used to execute the method as described above when executed by the processor.

[0038] The beneficial effects of the present invention are as follows: The present invention uses the interference optical imaging method to obtain the scattered light field distribution of cells in three-dimensional space, and then realizes the three-dimensional real-time positioning and tracking of multiple cells by the method of first performing two-dimensional cell positioning and then performing longitudinal positioning based on the relative displacement of the three-dimensional cell light field at this position. The present invention can realize the three-dimensional real-time positioning and tracking of various types and different morphologies of cells, has high positioning accuracy, has good robustness, and can be applied to the characterization and research of the three-dimensional movement and migration of cells with complex three-dimensional morphological changes, including adherent or non-adherent cells. Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following introduces the accompanying drawings related to the technical solutions in the embodiments of the present invention or the prior art. It should be understood that the accompanying drawings introduced below are only for conveniently and clearly expressing some embodiments of the technical solutions in the present invention, and those skilled in the art can also obtain other accompanying drawings based on these drawings without creative efforts.

[0040] Figure 1 It is a flowchart of a method for positioning and tracking the three-dimensional movement of cells in an embodiment of the present invention;

[0041] Figure 2 It is a defocus interference image of T cells obtained by the interference optical imaging method in an embodiment of the present invention;

[0042] Figure 3 It is a two-dimensional projection trajectory diagram of cells in an embodiment of the present invention;

[0043] Figure 4 It is a curve of the correlation coefficient changing with the step length in an embodiment of the present invention;

[0044] Figure 5 It is a three-dimensional trajectory image of T cells obtained in the embodiments of the present invention. Detailed implementation manners

[0045] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention. For the step numbers in the following embodiments, they are only set for the convenience of explanation and illustration, and no limitation is imposed on the order between the steps. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0046] In the description of the present invention, it should be understood that for the orientation description, such as the orientation or positional relationship indicated by up, down, front, back, left, right, etc., is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0047] In the description of the present invention, the meaning of several is one or more, the meaning of multiple is two or more, and understandings such as greater than, less than, exceeding, etc. do not include the present number, and understandings such as above, below, within, etc. include the present number. If there is a description of first and second, it is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features or implicitly indicating the sequence relationship of the indicated technical features.

[0048] In addition, in the description of the present invention, unless otherwise specified, "multiple" means two or more. "And / or" describes the association relationship of associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0049] In the description of the present invention, unless otherwise clearly defined, words such as setting, installing, connecting, etc. should be understood in a broad sense, and those skilled in the art can reasonably determine the specific meanings of the above words in the present invention in combination with the specific content of the technical solution.

[0050] Such as Figure 1As shown in the figure, this embodiment provides a method for positioning and tracking the three-dimensional movement of cells. This method performs two-dimensional positioning of cells by superimposing longitudinal light intensity values and searching for local maxima, and then obtains the longitudinal displacement through the matching between the longitudinal light intensity change curves corresponding to the two-dimensional positioning points, so as to achieve pixel-level three-dimensional positioning and tracking of cells. The method specifically includes the following steps:

[0051] S1. Obtain the real-time three-dimensional scattered light field distribution of multiple cells within the field of view in space.

[0052] The acquisition method is to use the interference optical imaging method to perform real-time and continuous shooting on a sample containing multiple cells, numerically reconstruct the captured cell interference images, and calculate the scattered light intensity distribution of the cell sample in three-dimensional space; in order to ensure that the three-dimensional positions of the cells can be obtained, the acquired images are defocused optical interference images, and the reconstruction distance should cover the entire range from defocus to focus and then to defocus.

[0053] S2. Obtain the projection position of the cell center on each frame of the interference image by superimposing longitudinal light intensity values and searching for local maxima, so as to achieve two-dimensional positioning of a single-frame cell image.

[0054] Superimpose the discrete numerical values of the scattered light intensity distribution calculated for a single-frame image in step S1 along the longitudinal direction to obtain a longitudinal light intensity superimposed image with the same pixel size as the original image; set the determination range of local maxima according to the cell size and magnification, and traverse the entire superimposed image for searching; when the value at a certain position on the image is greater than the other points within the preset determination range defined with this point as the center, then this point is determined as the two-dimensional projection position corresponding to a single cell. Due to the existence of factors such as noise, this two-dimensional positioning method will inevitably obtain some false determinations on the blank background, which can be excluded by introducing methods such as the threshold method. In addition, this two-dimensional positioning can also be corrected by introducing methods such as the centroid method to further improve the positioning accuracy.

[0055] S3. Connect the two-dimensional trajectories of the cells that have been two-dimensionally positioned within the field of view according to the space-time consistency of their positions, etc., for the front and rear frames.

[0056] The method for connecting two-dimensional trajectories specifically includes the following steps S31 - S33:

[0057] S31. Set the maximum distance for trajectory connection according to parameters such as the cell movement speed and the image shooting speed. When the distance between two-dimensional positioning points in adjacent images is greater than the preset value, it is considered that they cannot be the same cell.

[0058] S32. Arrange all two-dimensional positioning results in chronological order. Taking the two-dimensional positioning points of the first frame as the center, search for corresponding points in the next frame. When there are no positioning points within the preset distance range, terminate the search for this trajectory; when there is only one positioning point within the preset distance, consider this point to be on the same trajectory as the starting point; when there are two or more positioning points within the preset distance, first assume that they are all corresponding points, and determine the connection method of the two-dimensional trajectories of the front and rear frames based on parameters such as the distance between points obtained from subsequent calculations, the spatio-temporal consistency of movement, and the finally obtained trajectory length.

[0059] S33. Repeat the second step to judge the connection relationship between the two-dimensional positioning points of all adjacent frames, organize them into the trajectory of the two-dimensional projection of the cell center moving over time, and discard the unconnected positioning points and overly short trajectories among them to obtain the two-dimensional projection trajectory of the cell.

[0060] S4. Perform longitudinal positioning on each position point on the two-dimensional trajectory of a single cell.

[0061] Obtain the longitudinal light intensity change curve of the cell center at different times based on the two-dimensional positioning coordinates and the scattered light intensity distribution obtained in step S1, and calculate the relative longitudinal displacement between adjacent frames of the same cell according to these curves. This process specifically includes:

[0062] S41. Determine two light intensity curves I1 and I2 that need to be matched according to the two-dimensional trajectory.

[0063] S42. Obtain the cell light intensity characteristic curve from the light intensity curve I1. Since the distance for reconstructing the light field distribution is large, according to the estimated maximum longitudinal displacement of the cell and the reconstruction range, intercept a part of the light intensity curve that includes the focusing range of the light intensity curve I1 as the cell positioning characteristic.

[0064] S43. Match the characteristic curve with the light intensity curve I2. Starting from the endpoint of the light intensity curve I2, intercept a line segment of the same length as the characteristic curve and calculate the evaluation parameter r1 of the similarity between this line segment and the characteristic curve; translate one discrete data point and intercept a line segment of the same length as the characteristic curve again, and calculate the evaluation parameter r2 of the new line segment; repeat this process step by step until the end point of the intercepted line segment exceeds the range of the light intensity curve I2, obtaining a series of parameters r3, r4, ……, r n , and according to the variation law of the parameters, the displacement relationship of the curve can be obtained, and then the relative movement distance of the cell between two frames of images can be calculated.

[0065] The evaluation parameters obtained by the characteristic curve matching are not only used to obtain the longitudinal displacement of the cells, but also used for the secondary verification and evaluation of whether the two-dimensional trajectory positioning points are the same cell. This parameter can be obtained by calculating the correlation coefficient, calculating the sum of squared differences of the corresponding position values, etc., and can be further extended according to the actual situation. The characteristic curve matching can achieve a more accurate matching effect through fitting and other methods, so as to improve the positioning and tracking accuracy.

[0066] S44. Use the position of the maximum spatial light intensity to determine the three-dimensional position of the trajectory starting point, and superimpose the pairwise relative displacements to obtain the movement trajectory of the cells in space, so as to achieve the longitudinal positioning and three-dimensional trajectory tracking of the cells.

[0067] S5. Obtain the three-dimensional continuous space movement trajectory of the same cell according to the relative longitudinal displacement and the spatio-temporal consistency of the two-dimensional trajectory.

[0068] The obtained three-dimensional trajectory of the cells can be used to calculate the movement parameters such as the movement speed and acceleration of the cells, so as to further analyze the movement behavior of the cells.

[0069] As an alternative implementation, in the processes of obtaining the two-dimensional projection position in step S2, calculating the longitudinal displacement in step S4, etc., algorithms including but not limited to the centroid method, clustering, fitting, etc. can be introduced in the calculation process to optimize the results, so as to achieve higher-precision three-dimensional positioning and tracking.

[0070] The above method will be explained in detail below in conjunction with the accompanying drawings and specific embodiments.

[0071] This embodiment of the present invention shows an experimental result of three-dimensional positioning and tracking of the movement of T cells using a coaxial holographic imaging system. The light source used in the experiment is an LED light source with a wavelength of λ = 505 nm, and the magnification of the holographic imaging system is 10x. The camera used for imaging is a black-and-white camera with a pixel side length of 6.5 μm. The camera is used to image the T cells to obtain their defocus interference images as Figure 2 . 100 frames of images are recorded, and the time interval is 5 s.

[0072] Considering the calculation speed and accuracy, this embodiment of the present invention reconstructs the three-dimensional light field in the range from 3.5 μm to 603.5 μm away from the imaging plane. According to the pixel side length and magnification, the distance between adjacent reconstructed light intensity images should be consistent with the imaging range of the pixel unit size, that is, 0.65 μm. That is, the three-dimensional discrete light field is obtained by simulating the optical propagation process using the Rayleigh Sommerfeld diffraction formula at distances of 3.5 μm, 4.15 μm, 4.8 μm, ……

[0073] The three-dimensional light field obtained for a single-frame image is superimposed longitudinally to obtain a longitudinal light intensity superimposed image with the same pixel size as the original image. According to the cell size and imaging hardware parameters, a range of 50*50 pixels is set as the determination range for local maxima, and the entire superimposed image is traversed for searching; in order to exclude false determination points on the blank background, 5% of the average value of all discrete points on the superimposed image is used as the threshold, and two-dimensional positioning points smaller than this value are discarded.

[0074] The obtained two-dimensional positioning points are connected. Points within a distance of 13 μm are assumed to be on the same trajectory. Taking the two-dimensional positioning points of the first frame as the center, search for corresponding points in the next frame. When there are no positioning points within the preset distance range, the search for this trajectory is terminated; when there is only one positioning point within the preset distance, this point is considered to be on the same trajectory as the starting point; when there are two or more positioning points within the preset distance, first assume that they are all corresponding points, and determine the connection method of the two-dimensional trajectories of the front and rear frames according to parameters such as the distance between points, the spatio-temporal consistency of movement, and the finally obtained trajectory length calculated subsequently; repeat this step to judge the connection relationship between the two-dimensional positioning points of all adjacent frames to obtain the two-dimensional projection trajectory of the cell, and the result is as Figure 3 shown.

[0075] Calculate the longitudinal position of the cell. Obtain the light intensity change curve when the two-dimensional projection trajectory of the cell propagates longitudinally. Taking the first and second frame images as an example, obtain the light intensity curves I1 and I2 of the cell center in the longitudinal axis direction, and discard 30 discrete points at both ends of I1 as the cell light intensity characteristic curve. Starting from the end point of I2, intercept a line segment of the same length as the characteristic curve to calculate the correlation coefficient r1; translate one discrete data point and intercept the line segment, and calculate the correlation coefficient r2 again; repeat this process step by step until the end point of the intercepted line segment exceeds the range of I2, and obtain a series of correlation coefficients r3, r4,..., r n , the change of this series of correlation coefficients is as Figure 4 shown, that is, the two are most matched when the displacement is 5, so it is considered that the longitudinal displacement of the cell is 3.25 μm at this time. After the calculation is completed, take the second and third frames again to obtain new I1 and I2, and calculate the correlation coefficient again. Repeat this process to obtain the longitudinal relative movement distance of the cell between all images, and then the three-dimensional movement trajectory of the cell can be obtained, and its schematic diagram is as Figure 5 shown. Further, according to the cell displacement and imaging time, parameters such as the movement speed vector and acceleration of the cell can be calculated frame by frame.

[0076] In summary, the embodiments of the present invention have at least the following advantages and beneficial effects compared with the prior art:

[0077] (1) The method of this embodiment applies the method of matching the light intensity change curve for longitudinal positioning, so as to obtain the relative position change of cells. This method is still effective for cell samples with complex internal structures that are difficult to focus and image.

[0078] (2) The method of this embodiment applies the method of matching the light intensity change curve for longitudinal positioning. The relative displacement is only related to the three-dimensional light fields corresponding to these two images, which can effectively reduce the path dependence in the analysis process when analyzing motion problems, and is more universal in data processing, and can more objectively reflect the cell motion law.

[0079] (3) The method of this embodiment applies the method of matching the light intensity change curve, and can also evaluate whether the positioning points are the same cell while performing cell axial positioning, which can preferably reduce the results of misidentification in practical applications.

[0080] (4) The method of this embodiment realizes the three-dimensional spatial positioning of cells by first performing two-dimensional positioning and then longitudinal positioning. The traditional two-dimensional tracking research can conveniently introduce this method, which has extremely high optimization value.

[0081] (5) The method of this embodiment can be conveniently extended and applied to the three-dimensional tracking of samples with complex structures, which has high reference significance for solving the positioning and tracking problems of a class of special samples.

[0082] This embodiment also provides a device for positioning and tracking the three-dimensional motion of cells, including:

[0083] At least one processor;

[0084] At least one memory for storing at least one program;

[0085] When the at least one program is executed by the at least one processor, the at least one processor is caused to implement as Figure 1 the method shown.

[0086] A device for positioning and tracking the three-dimensional motion of cells in this embodiment can execute a method for positioning and tracking the three-dimensional motion of cells provided by the method embodiment of the present invention, can execute any combination of implementation steps of the method embodiment, and has the corresponding functions and beneficial effects of the method.

[0087] This application embodiment also discloses a computer program product or a computer program. The computer program product or the computer program includes computer instructions, and the computer instructions are stored in a computer-readable storage medium. The processor of the computer device can read the computer instructions from the computer-readable storage medium, and the processor executes the computer instructions, so that the computer device executes Figure 1 the method shown.

[0088] This embodiment also provides a storage medium storing instructions or programs that can execute a method for positioning and tracking the three-dimensional movement of cells provided by the method embodiment of the present invention. When the instructions or programs are run, any combination of implementation steps of the method embodiment can be executed, and the corresponding functions and beneficial effects of the method are achieved.

[0089] In some alternative embodiments, the functions / operations mentioned in the block diagrams may not occur in the order mentioned in the operation diagrams. For example, depending on the functions / operations involved, two consecutive blocks shown may actually be executed substantially simultaneously or the blocks can sometimes be executed in the reverse order. Additionally, the embodiments presented and described in the flowcharts of the present invention are provided by way of example for the purpose of providing a more comprehensive understanding of the technology. The disclosed method is not limited to the operations and logic flows presented herein. Alternative embodiments are contemplated where the order of various operations is changed and where sub-operations described as part of a larger operation are executed independently.

[0090] Furthermore, although the present invention has been described in the context of functional modules, it should be understood that, unless otherwise stated to the contrary, one or more of the functions and / or features may be integrated in a single physical device and / or software module, or one or more functions and / or features may be implemented in separate physical devices or software modules. It can also be understood that a detailed discussion of the actual implementation of each module is not necessary for understanding the present invention. Rather, given the attributes, functions, and internal relationships of the various functional modules in the devices disclosed herein, the actual implementation of the modules will be understood within the ordinary skills of an engineer. Thus, those skilled in the art can implement the present invention as set forth in the claims without undue experimentation. It can also be understood that the specific concepts disclosed are merely illustrative and are not intended to limit the scope of the present invention, which is determined by the full scope of the appended claims and their equivalents.

[0091] If the above-described functions are implemented in the form of software function units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or a part of this technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the methods described in various embodiments of the present invention. The aforementioned storage medium includes: USB flash drives, mobile hard disks, read-only memories (ROMs), random access memories (RAMs), magnetic disks, or optical discs, etc., which can store program codes of various kinds.

[0092] The logic and / or steps represented in the flowchart or described in other ways herein, for example, can be considered as a predefined sequence list of executable instructions for implementing logical functions, and can be specifically implemented in any computer-readable medium for use by an instruction execution system, apparatus, or device (such as a computer-based system, a system including a processor, or other systems that can fetch and execute instructions from the instruction execution system, apparatus, or device), or in combination with these instruction execution systems, apparatus, or devices. For the purposes of this specification, a "computer-readable medium" can be any device that can contain, store, communicate, propagate, or transport a program for use by or in combination with an instruction execution system, apparatus, or device.

[0093] More specific examples (non-exhaustive list) of computer-readable media include the following: electrical connection parts (electronic devices) having one or more wirings, portable computer disk cartridges (magnetic devices), random access memories (RAMs), read-only memories (ROMs), erasable programmable read-only memories (EPROMs or flash memories), optical fiber devices, and portable compact disc read-only memories (CDROMs). Additionally, the computer-readable medium can even be paper or other suitable media on which the program can be printed, because the program can be obtained electronically, for example, by optically scanning the paper or other media, followed by editing, interpretation, or, if necessary, other appropriate processing, and then storing it in a computer memory.

[0094] It should be understood that various parts of the present invention can be implemented by hardware, software, firmware, or a combination thereof. In the above-described embodiments, multiple steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented by hardware, as in another embodiment, any one or a combination of the following techniques well known in the art can be used: discrete logic circuits having logic gate circuits for implementing logical functions on data signals, application specific integrated circuits having appropriate combinational logic gate circuits, programmable gate arrays (PGAs), field programmable gate arrays (FPGAs), and the like.

[0095] In the above description of this specification, the descriptions referring to the terms "one embodiment / example", "another embodiment / example", or "certain embodiments / examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0096] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0097] The above has specifically described the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Those skilled in the art can also make various equivalent deformations or substitutions without departing from the spirit of the present invention, and these equivalent deformations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for positioning and tracking the three-dimensional movement of cells, characterized in that, It includes the following steps: S1. Use the interference optical imaging method to photograph a sample containing multiple cells, numerically reconstruct the photographed cell interference image, and calculate the scattered light intensity distribution of the cell sample in three-dimensional space; S2. Obtain the projection position of the cell center on each frame of the interference image by superimposing the longitudinal light intensity values and searching for local maxima, so as to achieve two-dimensional positioning of a single-frame cell image; S3. For the cell individuals that have achieved two-dimensional positioning, connect the two-dimensional trajectories of the front and back frames according to the spatio-temporal consistency of their positions; S4. Obtain the longitudinal light intensity change curve of the cell center at different times according to the two-dimensional positioning coordinates and the scattered light intensity distribution, and calculate the relative longitudinal displacement of adjacent frames of the same cell according to the longitudinal light intensity change curve; S5. Obtain the three-dimensional continuous space motion trajectory of the same cell according to the relative longitudinal displacement and the spatio-temporal consistency of the two-dimensional trajectory.

2. The method for positioning and tracking the three-dimensional movement of cells according to claim 1, characterized in that In step S1, the interference optical imaging method is used for illumination in the form of plane waves or spherical waves, and the obtained image is a defocused optical interference pattern; The scattered light intensity distribution in the three-dimensional space is obtained from the defocused optical interference image according to the propagation simulation formula of the optical diffraction principle.

3. A method for positioning and tracking the three-dimensional movement of cells according to claim 1, characterized in that, In the step of two-dimensional positioning in step S2, the method of superimposing longitudinal light intensity values is applied, including: Calculate the discrete values of the scattered light intensity distribution obtained from a single-frame image along the longitudinal direction and superimpose them, so as to obtain a longitudinal light intensity superimposed image with the same pixel size as the original image, and perform subsequent two-dimensional positioning calculations on the basis of this longitudinal light intensity superimposed image.

4. A method for positioning and tracking the three-dimensional movement of cells according to claim 1, characterized in that, The said step S2 includes: Set the determination range of local maxima according to the cell size and magnification factor, and traverse the entire superimposed image for searching; When the value at a certain position on the image is greater than the remaining points within the preset determination range defined with this point as the center, it is determined that this point is the two-dimensional projection position corresponding to a single cell.

5. A method for positioning and tracking the three-dimensional movement of cells according to claim 1, characterized in that, The said step S3 includes: S31. Set the maximum distance for trajectory connection. When the distance between two-dimensional positioning points in adjacent images is greater than the preset value, it is determined that the cells in the two frames of images are not the same cell; S32. Arrange all two-dimensional positioning results in chronological order. With the two-dimensional positioning point of the first frame as the center, search for whether there is a corresponding point in the next frame; when there is no positioning point within the preset distance range, terminate the search for this trajectory; when there is only one positioning point within the preset distance, it is considered that this positioning point and the starting point are the same trajectory; when there are two or more positioning points within the preset distance, first assume that the positioning points are all corresponding points, and determine the connection method of the two-dimensional trajectories of the front and back frames according to the parameters calculated subsequently; S33. Repeat step S32, and judge the connection relationship between the two-dimensional positioning points of all adjacent frames. According to the connection relationship, organize all the obtained positioning points into the trajectory of the two-dimensional projection of the cell center moving with time, discard the unconnected positioning points among them, and obtain the two-dimensional projection trajectory of the cell.

6. A method for positioning and tracking the three-dimensional movement of cells according to claim 1, characterized in that, The said step S4 includes: S41. Determine two light intensity curves I1 and I2 that need to be matched according to the two-dimensional trajectory; S42. Obtain the cell light intensity characteristic curve from the light intensity curve I1; S43. Match the light intensity feature curve with the light intensity curve I2: Starting from the endpoint of the light intensity curve I2, intercept a line segment of the same length as the light intensity feature curve, and calculate the evaluation parameter r1 of the similarity between this line segment and the light intensity feature curve; translate one discrete data point and then intercept a line segment of the same length as the feature curve again, and calculate the evaluation parameter r2 of the new line segment; repeat this process step by step until the endpoint of the intercepted line segment exceeds the range of the light intensity curve I2, and obtain a series of parameters r3, r4, ……, r n , obtain the displacement relationship of the curve according to the variation law of the parameters, and then calculate the relative movement distance of the cells between two frames of images; S44. Determine the three-dimensional position of the trajectory starting point using the position of the maximum spatial light intensity, and superimpose the pairwise relative displacements to obtain the movement trajectory of the cell in space, thereby realizing the longitudinal positioning and three-dimensional trajectory tracking of the cell.

7. A method for positioning and tracking the three-dimensional movement of cells according to claim 6, characterized in that, The light intensity characteristic curve includes the cell center position, and this light intensity characteristic curve is obtained by discarding the data at both ends far from the focal plane from the light intensity curve I1; according to the estimated maximum longitudinal displacement of the cell and the reconstruction range, a part of the light intensity curve including the focusing range of the light intensity curve I1 is intercepted as the cell positioning feature.

8. The method for positioning and tracking the three-dimensional movement of cells according to claim 6, wherein, The evaluation parameter in step S43 is not only used to obtain the longitudinal displacement of the cell, but also used for the secondary verification and evaluation of whether the two-dimensional trajectory positioning points are the same cell; this evaluation parameter is obtained by calculating the correlation coefficient or calculating the sum of the squared differences of the corresponding position values.

9. A device for positioning and tracking the three-dimensional movement of cells, characterized in that, Comprising: At least one processor; At least one memory for storing at least one program; When the at least one program is executed by the at least one processor, the at least one processor implements the method according to any one of claims 1-8.

10. A computer-readable storage medium storing a program executable by a processor, characterized in that, The program executable by the processor is used to execute the method according to any one of claims 1-8 when executed by the processor.

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