A dynamic observation method, device and storage medium for sperm three-dimensional morphology

Abstract

The present invention discloses a method, device, and storage medium for dynamic observation of the three-dimensional morphology of sperm. The method comprises: obtaining a holographic image of the sperm, performing three-dimensional light intensity reconstruction on the holographic image, and obtaining a three-dimensional spatial light intensity distribution; processing the three-dimensional spatial light intensity distribution using a difference method to eliminate background noise interference and enhance the scattered light signal of the sperm flagellum; obtaining the node distribution of the sperm flagellum on the two-dimensional projection plane xy based on the three-dimensional spatial light intensity distribution processed by the difference method, and obtaining the position of the sperm flagellum on the z-axis one by one to achieve three-dimensional reconstruction of the sperm flagellum. The present invention reconstructs the morphology and position of the focused head, and simultaneously uses the difference method to enhance the flagellum signal and then searches for the three-dimensional coordinates of the modified sperm flagellum nodes, thereby achieving three-dimensional reconstruction of the sperm head and flagellum. The method is suitable for observing sperm from various animals and humans, and can obtain the dynamic three-dimensional position of multiple sperm cells in situ and without damage. The present invention can be widely used in the field of microscopic computational imaging.

Description

Technical Field

[0001] The present invention relates to the field of microscopic computational imaging, and in particular to a method, device and storage medium for dynamic observation of the three-dimensional morphology of sperm. Background Art

[0002] The motility and morphology of sperm samples are key parameters for clinical sperm screening and assessment of fertilization success rates. Currently, the traditional method for observing sperm morphology involves observing the projection of sperm onto a two-dimensional plane using an optical microscope. However, since sperm consist of two components, the head and flagellum, they often do not lie in the same plane during movement. Only by fully evaluating these two components can we gain a comprehensive assessment and understanding of sperm motility. Conventional microscopic imaging, however, typically focuses on the sperm head, making it difficult to observe the flagellum, where the scattering signal is weaker. Fluorescent staining can enhance the signal-to-noise ratio of the flagellum, but sperm cannot be stained to avoid irreversible damage to fertilization. Furthermore, scanning electron microscopy and transmission electron microscopy methods can provide detailed observation of sperm micromorphology and internal structure, but they cannot maintain cell viability during sample preparation, making them ineffective for real-time dynamic observation of sperm. Newly developed digital holographic microscopy methods can achieve three-dimensional localization of sperm particles while maintaining cell viability, but cannot capture flagellar information.

[0003] As can be seen from the above, there is an urgent need for a non-destructive, non-labeling, real-time technical solution to obtain the complete three-dimensional morphology of sperm including the head and flagella when swimming. This is of great significance to clinical reproductive medicine, animal husbandry and other fields. Summary of the Invention

[0004] In order to solve at least one of the technical problems existing in the prior art to a certain extent, the present invention aims to provide a method, device and storage medium for dynamic observation of the three-dimensional morphology of sperm.

[0005] The technical solution adopted in the present invention is:

[0006] A method for dynamic observation of sperm three-dimensional morphology comprises the following steps:

[0007] Acquire a holographic image of sperm, perform three-dimensional light intensity reconstruction on the holographic image, and obtain three-dimensional spatial light intensity distribution;

[0008] The difference method is used to process the three-dimensional spatial light intensity distribution, eliminate background noise interference, and enhance the scattered light signal of sperm flagella;

[0009] According to the three-dimensional spatial light intensity distribution processed by the difference method, the node distribution of the sperm flagellum on the two-dimensional projection plane xy is obtained, and the position of the sperm flagellum on the z axis is obtained one by one to achieve three-dimensional reconstruction of the sperm flagellum.

[0010] Furthermore, the holographic image is a defocused image, which is reconstructed using a light field propagation formula; the reconstruction process of the holographic image includes a process of defocusing, focusing, and defocusing the sperm cell image;

[0011] In three-dimensional light intensity reconstruction, the differences in corresponding propagation distances between adjacent reconstructed images are equal.

[0012] Furthermore, the processing of the three-dimensional spatial light intensity distribution using the difference method includes:

[0013] Select the center point of the sperm cell head as the feature point and obtain the projection coordinates of the feature point;

[0014] Analyze the distribution law of the light intensity reconstructed by the characteristic points in the propagation direction, and obtain the distance d between the maximum and minimum values of the light intensity in the propagation direction;

[0015] Each reconstructed image in the three-dimensional light intensity distribution is subtracted from the reconstructed image with a propagation distance difference d from the image, so that the scattered light signal of the sperm flagellum in the light intensity distribution image is enhanced and the background signal approaches zero.

[0016] Furthermore, the step of obtaining the node distribution of the sperm flagellum on the two-dimensional projection plane xy according to the three-dimensional spatial light intensity distribution processed by the difference method includes:

[0017] According to the three-dimensional spatial light intensity distribution processed by the difference method, the center point A0 of the sperm head is found, and the reconstructed focused image layer corresponding to this point is extracted to obtain the outline of the sperm head;

[0018] The orientation of the sperm head is obtained by superimposing and matching the images in the propagation direction, and the orientation of the sperm flagellum is predicted;

[0019] Taking the center point A0 as the starting point, find the point with the maximum light intensity within the range where the angle with the direction of the sperm head is less than the preset angle θ0. The two-dimensional coordinate of the point with the maximum light intensity is the first flagellum two-dimensional projection node A1;

[0020] With node A1 as the vertex and the flagellar direction A0A1 as the symmetry axis, the point with the maximum light intensity is found within the range where the angle with the flagellar direction is less than the preset angle θ1 as the next flagellar two-dimensional projection node A2;

[0021] Update the vertex and flagella orientation according to the previous flagella 2D projection node, obtain the search range according to the flagella orientation and obtain the next flagella 2D projection node until the preset search conditions are met, and output the obtained flagella 2D projection nodes A1, A2, ..., A n .

[0022] Furthermore, in the step of finding the center point A0 of the sperm head and extracting the reconstructed focused image layer corresponding to this point to obtain the outline of the sperm head, the coordinates of the point with the maximum light intensity are used as the basis for determining whether the sperm head is focused;

[0023] The step of finding the point with the maximum light intensity with the center point A0 as the starting point and the angle with the direction of the sperm head being less than the preset angle θ0 also includes the following steps:

[0024] When searching for the two-dimensional projection node of the sperm flagellum, the points within a preset distance from the starting point are discarded to avoid interference of the strong signal near the node on the acquisition of the next two-dimensional projection node of the flagellum.

[0025] Furthermore, the z-axis positions of the flagella are obtained one by one to achieve three-dimensional reconstruction of the sperm flagella, including:

[0026] The three-dimensional spatial light intensity distribution is processed according to the difference method to obtain the light intensity distribution curve corresponding to each two-dimensional projection node of the sperm flagellum in the light intensity propagation direction;

[0027] The peak value of each node in the propagation direction is obtained by fitting the light intensity distribution curve, and the three-dimensional coordinates corresponding to the peak value are regarded as the three-dimensional position of the sperm flagellum corresponding to the projection node;

[0028] The three-dimensional coordinates of each flagellar node are connected, and the spatial distribution of the flagella is calculated using the connecting line segments as the skeleton to achieve three-dimensional reconstruction of the sperm flagellum.

[0029] Furthermore, the calculation of obtaining the spatial distribution of flagella using the connecting line segments as skeletons includes:

[0030] The reconstructed sperm flagellum is skeletonized, expanded, and reconstructed by using the centroid method or clustering algorithm to obtain a new reconstruction result. The process is iterated until convergence to obtain the optimal solution.

[0031] Furthermore, the method for dynamic observation of sperm three-dimensional morphology further comprises the following steps:

[0032] Holographic images are continuously acquired, and each frame of the image is reconstructed to obtain the spatial motion parameters of sperm and the changes in morphology in the time dimension.

[0033] Another technical solution adopted in the present invention is:

[0034] A dynamic observation device for three-dimensional sperm morphology, comprising:

[0035] at least one processor;

[0036] at least one memory for storing at least one program;

[0037] When the at least one program is executed by the at least one processor, the at least one processor implements the above method.

[0038] Another technical solution adopted in the present invention is:

[0039] A computer-readable storage medium stores a program executable by a processor, wherein the program executable by the processor is used to perform the method described above when executed by the processor.

[0040] The beneficial effects of the present invention are as follows: the present invention reconstructs the focused head morphology and position, and uses the difference method to enhance the flagella signal and then searches for the three-dimensional coordinates of the sperm flagella node, thereby realizing three-dimensional reconstruction of the sperm head and flagella. The present invention is suitable for observing sperm from various animals and humans, and can obtain the dynamic three-dimensional positions of multiple sperm cells in situ and without damage. It can be widely used in clinical reproductive testing, animal husbandry and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following introduction is made to the drawings of the embodiments of the present invention or the related technical solutions in the prior art. It should be understood that the drawings introduced below are only for the convenience of clearly describing some embodiments of the technical solutions of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative work.

[0042] Figure 1 This is a flow chart of a method for dynamic observation of sperm three-dimensional morphology according to an embodiment of the present invention;

[0043] Figure 2 is the original sperm cell holographic defocused image in the embodiment of the present invention;

[0044] Figure 3 is a focused image of sperm after light intensity reconstruction and difference processing in an embodiment of the present invention;

[0045] Figure 4 : is a schematic diagram of the sperm light intensity superposition diagram and its fitting results in an embodiment of the present invention; wherein the dotted line represents the direction of the sperm head;

[0046] Figure 5 is a schematic diagram of the two-dimensional projection nodes of the sperm head and flagellum in optional embodiment 1;

[0047] Figure 6 is a schematic diagram of the three-dimensional reconstructed skeleton of sperm in optional embodiment 1;

[0048] Figure 7 is a partial schematic diagram of a normal sperm cell holographic defocused image in an embodiment of the present invention;

[0049] Figure 8 is a schematic diagram of the two-dimensional projection nodes of the sperm head and flagellum in optional embodiment 2;

[0050] Figure 9 is a schematic diagram of the three-dimensional reconstructed skeleton of sperm in optional embodiment 2;

[0051] Figure 10 1 is a partial schematic diagram of a holographic defocused image of a sperm cell with too short flagella in an embodiment of the present invention;

[0052] Figure 11 is a schematic diagram of the two-dimensional projection nodes of the sperm head and flagellum in optional embodiment 3;

[0053] Figure 12 This is a schematic diagram of the three-dimensional reconstructed skeleton of sperm in optional embodiment 3. DETAILED DESCRIPTION

[0054] The embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and are not to be construed as limiting the present invention. The step numbers in the following embodiments are provided for ease of explanation only and do not limit the order of the steps. The order of execution of the steps in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0055] In the description of the present invention, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on the present invention.

[0056] In the description of the present invention, "several" means one or more, "many" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0057] In the description of the present invention, unless otherwise clearly defined, terms such as setting, installing, and connecting should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention based on the specific content of the technical solution.

[0058] like Figure 1 As shown in the figure, this embodiment provides a method for dynamic observation of the three-dimensional morphology of sperm. This method uses a digital holographic microscope for imaging, calculates the spatial distribution of light intensity through diffraction theory, reconstructs the morphology and position of the focused head, and uses the difference method to enhance the flagellar signal and then searches for the three-dimensional coordinates of the sperm flagella node, thereby achieving three-dimensional reconstruction of the sperm head and flagella. The method specifically includes the following steps:

[0059] S1. Obtain a holographic image of sperm, perform three-dimensional light intensity reconstruction on the holographic image, and obtain a three-dimensional spatial light intensity distribution.

[0060] Reconstruct the three-dimensional spatial light intensity distribution of multiple sperm. Observe using a digital holographic microscope, then reconstruct the holographic image in three dimensions based on diffraction theory, calculating the three-dimensional spatial light intensity distribution at different distances from the imaging surface. The holographic image is a defocused image, and reconstruction calculations are performed using the light field propagation formula. The reconstruction result should include the defocus-focus-defocus process of the sperm cell image. In the three-dimensional light intensity reconstruction, the difference in corresponding propagation distances between adjacent reconstructed images is equal.

[0061] S2. Use the difference method to process the three-dimensional spatial light intensity distribution, eliminate background noise interference, and enhance the scattered light signal of sperm flagella.

[0062] Wherein, step S2 specifically includes steps S21-S23:

[0063] S21. Select the center point of the sperm cell head as a feature point, and obtain the projection coordinates of the feature point.

[0064] Find the projection coordinates of the feature point. The feature point is generally the center of the sperm cell head, where the light intensity is relatively high and is less affected by background interference. This can more objectively show the variation of the reconstructed light intensity in space.

[0065] S22. Analyze the distribution pattern of the light intensity reconstructed from the characteristic points in the propagation direction, and obtain the distance d between the maximum and minimum values of the light intensity in the propagation direction.

[0066] S23. Subtract each reconstructed image in the three-dimensional spatial light intensity distribution from the reconstructed image with a propagation distance difference d from the image, so that the scattered light signal of the sperm flagellum in the light intensity distribution image is enhanced and the background signal approaches zero.

[0067] Perform difference subtraction. Since the light intensity variation of the flagellum in the propagation direction coincides with the characteristic points, each reconstructed image in the light intensity distribution can be subtracted from the reconstructed image with a propagation distance difference d from the image. The flagellar information of the obtained light intensity distribution image is enhanced, and the background signal approaches zero.

[0068] S3. According to the three-dimensional spatial light intensity distribution processed by the difference method, the node distribution of the sperm flagellum on the two-dimensional projection surface xy is obtained, and the position of the sperm flagellum on the z axis is obtained one by one to achieve three-dimensional reconstruction of the sperm flagellum.

[0069] Find the focal plane and obtain the sperm head outline. Step S3 specifically includes steps S31-S38:

[0070] S31. Find the center point A0 of the sperm head according to the three-dimensional spatial light intensity distribution processed by the difference method, extract the reconstructed focused image layer corresponding to this point, and obtain the outline of the sperm head.

[0071] According to the acquired spatial light intensity distribution, the center point A0 of the sperm head is found, the reconstructed focused image layer corresponding to this point is extracted, and the sperm head contour is obtained using the threshold binarization method.

[0072] When there is no major interference, the coordinates of the point with maximum light intensity can be used as the basis for determining the focus of the sperm head.

[0073] S32. Obtain the orientation of the sperm head by superimposing and matching the images in the propagation direction, and predict the orientation of the sperm flagellum.

[0074] Obtain the orientation of the sperm head; since the sperm cell head is ellipsoidal and has a strong halo in the left and right directions, the orientation of the sperm head can be matched and obtained by superimposing images in the propagation direction, and then the orientation of the sperm flagellum can be predicted.

[0075] S33, starting from the center point A0, searching for the point with the maximum light intensity within a range where the angle with the direction of the sperm head is less than a preset angle θ0, and the two-dimensional coordinates of the point with the maximum light intensity are the first flagella two-dimensional projection node A1.

[0076] Obtain the first two-dimensional projection node A1 of the sperm flagellum; take the sperm head coordinate A0 as the starting point, and find the point with the maximum light intensity within the range where the angle with the direction of the sperm head is less than the preset angle θ0. The two-dimensional coordinate of this point is the first flagellum two-dimensional projection node A1.

[0077] As an optional implementation, when acquiring the two-dimensional projection node of the sperm flagellum, it is necessary to discard points within a certain distance from the starting point to avoid interference of a strong signal near the acquisition node with the acquisition of the next two-dimensional projection node of the flagellum.

[0078] S34, taking node A1 as the vertex and the flagellar direction A0A1 as the symmetry axis, searching for the point with the maximum light intensity within the range where the angle with the flagellar direction is less than the preset angle θ1 as the next flagellar two-dimensional projection node A2.

[0079] S35, update the vertex and flagella direction according to the previous flagella two-dimensional projection node, obtain the search range according to the flagella direction and obtain the next flagella two-dimensional projection node, until the preset search conditions are met, and output the obtained flagella two-dimensional projection nodes A1, A2, ..., A n .

[0080] With A1 as the vertex and the flagellar direction A0A1 as the symmetry axis, find the point with the maximum light intensity within the range of the angle with the flagellar direction less than the preset angle θ1 as the next flagellar two-dimensional projection node A2, repeat this step until the obtained flagellar node light intensity value is less than the preset limit or the node spacing is greater than the preset limit length, and output the obtained flagellar nodes A1, A2, ..., A n .

[0081] S36. Process the three-dimensional spatial light intensity distribution according to the difference method to obtain the light intensity distribution curve corresponding to each two-dimensional projection node of the sperm flagellum in the light intensity propagation direction.

[0082] S37. Obtain the peak value of each node in the propagation direction by fitting the light intensity distribution curve, and the three-dimensional coordinates corresponding to the peak value are regarded as the three-dimensional position of the part of the sperm flagellum corresponding to the projection node.

[0083] S38. Connect the three-dimensional coordinates of each flagella node, use the connecting line segments as the skeleton to calculate and obtain the spatial distribution of the flagella, and realize the three-dimensional reconstruction of the sperm flagellum.

[0084] When performing three-dimensional reconstruction of sperm flagella based on flagellar nodes, the reconstructed sperm flagella can be skeletonized, expanded, and reconstructed using methods such as the centroid method and clustering to obtain new reconstruction results. The process is iterated until convergence to obtain the optimal solution.

[0085] As a further optional implementation, in the above method, each three-dimensional reconstruction of the sperm flagellum relies only on a single holographic image. For a system capable of continuous imaging, the sperm flagellum can be reconstructed for each frame of the image, thereby obtaining its spatial motion parameters and morphological changes in the time dimension.

[0086] Optional embodiment 1

[0087] This example presents the experimental results of a three-dimensional reconstruction of human sperm flagella using a holographic imaging system. The light source used in the experiment was a light source with a wavelength of λ = 505nm, and the magnification of the holographic imaging system was 10×. The observation sample was sperm cells that had been removed from the seminal plasma and resuspended in culture medium. These cells were placed in a 150μm thick sample cell. A camera with a pixel count of 1024×1024 and a pixel side length of 6.5μm was used to perform defocused holographic imaging of the human sperm sample, obtaining an image such as Figure 2 shown.

[0088] Taking into account the actual defocus distance, the embodiment of the present invention reconstructs and calculates the light intensity distribution within the range of 0.5 to 60.5 μm from the imaging surface. The difference method is used for processing, and the number of reconstructed images with subtraction image difference is 15; the center of the sperm cell head is found based on the point of maximum light intensity, and a focused sperm image centered on this point is obtained, such as Figure 3 shown.

[0089] The search for sperm flagella nodes is based on the light intensity spatial distribution processed by the difference method. The light intensity distribution in the propagation direction of the area near the sperm head is superimposed and fitted to obtain the direction of the sperm head, such as Figure 4 As shown. Starting from the center of the sperm head, search for the first flagella node. After excluding the 5×5 range centered on the starting point, find the point with the maximum light intensity in the image within the range of 90° with the sperm head direction as the symmetry axis as the first flagella node, and then search for the next flagella node from this node as the starting point until the light intensity value is less than the preset threshold or the distance to the next node exceeds the preset range. The obtained flagella two-dimensional projection node is as follows Figure 5 shown.

[0090] The light intensity distribution of each flagellar node corresponding to the position in the propagation direction of the light field is peaked, and the three-dimensional height corresponding to the peak point is obtained, which is used as the position of the flagellar node in the propagation direction, thereby realizing the three-dimensional reconstruction of the sperm flagellum. Its spatial distribution is shown in the figure below. Figure 6 shown.

[0091] Optional embodiment 2

[0092] This example presents the results of an experiment using a holographic imaging system to perform three-dimensional reconstruction of human sperm flagella. The light source used in the experiment was a light source with a wavelength of λ = 505 nm, and the magnification of the holographic imaging system was 10×. The observation sample was sperm cells that had been removed from the seminal plasma and resuspended in culture medium. These cells were placed in a 150 μm thick sample cell. A camera with a pixel side length of 6.5 μm was used to perform defocused holographic imaging of the human sperm sample. The image of the sperm position is shown in Figure 1. Figure 7 shown.

[0093] The light intensity distribution within the range of 0.5 to 60.5 μm from the imaging surface was reconstructed and calculated. The difference method was used for processing, and the number of reconstructed images with subtraction difference was 15. The center of the sperm cell head was found based on the point of maximum light intensity to obtain a focused sperm image.

[0094] The light intensity distribution in the propagation direction of the area near the sperm head is superimposed and fitted to obtain the orientation of the sperm head. The first flagellar node is searched with the center of the sperm head as the starting point. After excluding the 5×5 range centered on the starting point, the point with the maximum light intensity is found in the image within the range of 90° with the sperm head orientation as the symmetry axis as the first flagellar node, and then the next flagellar node is searched with this node as the starting point until the light intensity value is less than the preset threshold or the distance to the next node exceeds the preset range. The obtained flagellar two-dimensional projection node is as follows Figure 8 The light intensity distribution corresponding to each flagellar node in the propagation direction of the light field is peaked, and the three-dimensional height corresponding to the peak point is obtained, which is used as the position of the flagellar node in the propagation direction, thereby realizing the three-dimensional reconstruction of the sperm flagellum. Its spatial distribution is shown in Figure 9 shown.

[0095] Optional embodiment three

[0096] This example presents the results of an experiment using a holographic imaging system to perform three-dimensional reconstruction of human sperm flagella. The light source used in the experiment was a light source with a wavelength of λ = 505 nm, and the magnification of the holographic imaging system was 10×. The observation sample was sperm cells that had been removed from the seminal plasma and resuspended in culture medium. These cells were placed in a 150 μm thick sample cell. A camera with a pixel side length of 6.5 μm was used to perform defocused holographic imaging of the human sperm sample. The image of the sperm position is shown in Figure 1. Figure 10 In this example, sperm were observed to have an abnormal flagella that were too short.

[0097] The light intensity distribution within the range of 0.5 to 60.5 μm from the imaging surface was reconstructed and calculated. The difference method was used for processing, and the number of reconstructed images with subtraction difference was 15. The center of the sperm cell head was found based on the point of maximum light intensity to obtain a focused sperm image.

[0098] The light intensity distribution in the propagation direction of the area near the sperm head is superimposed and fitted to obtain the orientation of the sperm head. The first flagellar node is searched with the center of the sperm head as the starting point. After excluding the 5×5 range centered on the starting point, the point with the maximum light intensity is found in the image within the range of 90° with the sperm head orientation as the symmetry axis as the first flagellar node, and then the next flagellar node is searched with this node as the starting point until the light intensity value is less than the preset threshold or the distance to the next node exceeds the preset range. The obtained flagellar two-dimensional projection node is as follows Figure 11 The light intensity distribution corresponding to each flagellar node in the propagation direction of the light field is peaked, and the three-dimensional height corresponding to the peak point is obtained, which is used as the position of the flagellar node in the propagation direction, thereby realizing the three-dimensional reconstruction of the sperm flagellum. Its spatial distribution is shown in Figure 12 shown.

[0099] In summary, the method of this embodiment has the following advantages and beneficial effects compared to the prior art:

[0100] (1) The method of this embodiment achieves reconstruction of the three-dimensional morphology of sperm through optical observation and calculation, enabling in situ dynamic observation of the sperm movement process. The method of this embodiment can provide the real-time status of the sperm flagellum, corresponding to the real-time swinging process and fluid dynamics, and intuitively reflect its movement pattern, sperm motility, sperm deformity rate and other information, providing an important reference for analysis and research on various aspects such as reproductive potential and health assessment of organisms.

[0101] (2) The method of this embodiment uses a digital holographic microscope for observation, which can calculate and reconstruct the three-dimensional morphology of sperm on different focal planes based on the principle of optical diffraction. In practical applications, it can quickly and accurately achieve three-dimensional tracking of multiple sperm.

[0102] (3) The method of this embodiment uses the difference method to process the spatial light intensity distribution, which greatly enhances the flagellar signal that is close to the background noise signal, providing conditions for the extraction of flagellar information and subsequent three-dimensional reconstruction.

[0103] (4) The method of this embodiment introduces a flagellar node search method in the flagellar three-dimensional reconstruction process. The positioning of flagellar nodes in the light field propagation direction is relatively independent and has good parallelism. Moreover, mutual comparison can effectively eliminate erroneous nodes with large errors, thereby improving the robustness of the flagellar three-dimensional reconstruction process.

[0104] (5) The method of this embodiment can be extended to the three-dimensional reconstruction of surrounding microstructures under specific shapes, which has a high reference value for solving similar problems.

[0105] This embodiment further provides a device for dynamic observation of sperm three-dimensional morphology, comprising:

[0106] at least one processor;

[0107] at least one memory for storing at least one program;

[0108] When the at least one program is executed by the at least one processor, the at least one processor implements Figure 1 The method shown.

[0109] A dynamic observation device for the three-dimensional morphology of sperm in this embodiment can execute a dynamic observation method for the three-dimensional morphology of sperm 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.

[0110] The present application also discloses a computer program product or computer program, which includes computer instructions stored in a computer-readable storage medium. A processor of a 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 performs Figure 1 The method shown.

[0111] This embodiment also provides a storage medium storing instructions or programs that can execute a method for dynamic observation of the three-dimensional morphology of sperm provided by an embodiment of the method of the present invention. When the instructions or program are run, any combination of implementation steps of the method embodiment can be executed, and the corresponding functions and beneficial effects of the method can be obtained.

[0112] In some optional embodiments, the function / operation mentioned in the block diagram may not occur in the order mentioned in the operation diagram. For example, depending on the function / operation involved, the two boxes shown in succession can actually be executed substantially simultaneously or the boxes can sometimes be executed in reverse order. In addition, the embodiment presented and described in the flow chart of the present invention is provided in an exemplary manner for the purpose of providing a more comprehensive understanding of the technology. The disclosed method is not limited to the operation and logic flow presented herein. Optional embodiments are contemplated in which the order of the various operations is changed and the sub-operations described as a part of a larger operation are performed independently.

[0113] Furthermore, although the present invention is described in the context of functional modules, it should be understood that, unless otherwise indicated, one or more of the functions and / or features described may be integrated into 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 will also be understood that a detailed discussion of the actual implementation of each module is not necessary for understanding the present invention. More specifically, given the properties, functions, and internal relationships of the various functional modules in the devices disclosed herein, the actual implementation of the module will be understood within the ordinary skill of an engineer. Therefore, a person skilled in the art using ordinary skill will be able to implement the present invention set forth in the claims without undue experimentation. It will 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.

[0114] If the functions are implemented in the form of software functional 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, or the part that contributes to the prior art, or the part of the technical solution, can be embodied in the form of a software product. The computer software product is stored in a storage medium and includes several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes various media that can store program codes, such as a USB flash drive, a mobile hard disk, a read-only memory (ROM), a random access memory (RAM), a magnetic disk, or an optical disk.

[0115] The logic and / or steps represented in the flowcharts or otherwise described herein, for example, can be considered as an ordered list of executable instructions for implementing the logical functions, and can be embodied in any computer-readable medium for use by, or in conjunction with, an instruction execution system, apparatus, or device (e.g., a computer-based system, a system including a processor, or other system that can fetch and execute instructions from an instruction execution system, apparatus, or device). For 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 conjunction with, an instruction execution system, apparatus, or device.

[0116] More specific examples (a non-exhaustive list) of computer-readable media include the following: an electrical connection with one or more wires (electronic devices), a portable computer disk cartridge (magnetic devices), a random access memory (RAM), a read-only memory (ROM), an erasable and programmable read-only memory (EPROM or flash memory), a fiber optic device, and a portable compact disc read-only memory (CDROM). In addition, the computer-readable medium may even be paper or other suitable medium on which the program is printed, since the program may be obtained electronically, for example, by optically scanning the paper or other medium, followed by editing, deciphering, or processing in another suitable manner as necessary, and then stored in a computer memory.

[0117] It should be understood that various parts of the present invention can be implemented using hardware, software, firmware, or a combination thereof. In the above-described embodiments, multiple steps or methods can be implemented using software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented using hardware, as in another embodiment, any one of the following technologies known in the art or a combination thereof can be used: a discrete logic circuit having a logic gate circuit for implementing a logic function on a data signal, an application-specific integrated circuit having a suitable combination of logic gate circuits, a programmable gate array (PGA), a field programmable gate array (FPGA), etc.

[0118] In the above description of this specification, reference to the terms "one embodiment / example," "another embodiment / example," or "certain embodiments / examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0119] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

[0120] The above is a specific description of the preferred implementation of the present invention, but the present invention is not limited to the above embodiments. Those skilled in the art can make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for dynamic observation of sperm three-dimensional morphology, characterized in that: The following steps are involved: Acquire a holographic image of sperm, perform three-dimensional light intensity reconstruction on the holographic image, and obtain three-dimensional spatial light intensity distribution; The difference method is used to process the three-dimensional spatial light intensity distribution, eliminate background noise interference, and enhance the scattered light signal of sperm flagella; According to the three-dimensional spatial light intensity distribution processed by the difference method, the node distribution of the sperm flagellum on the two-dimensional projection plane xy is obtained, and the position of the sperm flagellum on the z axis is obtained one by one to achieve three-dimensional reconstruction of the sperm flagellum; The step of obtaining the node distribution of the sperm flagellum on the two-dimensional projection plane xy according to the three-dimensional spatial light intensity distribution processed by the difference method includes: According to the three-dimensional spatial light intensity distribution processed by the difference method, the center point A0 of the sperm head is found, and the reconstructed focused image layer corresponding to this point is extracted to obtain the outline of the sperm head; The orientation of the sperm head is obtained by superimposing and matching the images in the propagation direction, and the orientation of the sperm flagellum is predicted; Taking the center point A0 as the starting point, when the angle with the sperm head is less than the preset angle θ 0, and the two-dimensional coordinate of the maximum light intensity point is the first flagellum two-dimensional projection node A1; With node A1 as the vertex and flagella direction A0A1 as the symmetry axis, when the angle with the flagella direction is less than the preset angle θ 1 to find the point with the maximum light intensity as the next flagellum two-dimensional projection node A2; Update the vertex and flagella orientation according to the previous flagella 2D projection node, obtain the search range according to the flagella orientation and obtain the next flagella 2D projection node until the preset search conditions are met, and output the obtained flagella 2D projection nodes A1, A2, ..., A n ; The step of obtaining the z-axis positions of the flagella one by one to achieve three-dimensional reconstruction of the sperm flagella includes: The three-dimensional spatial light intensity distribution is processed according to the difference method to obtain the light intensity distribution curve corresponding to each two-dimensional projection node of the sperm flagellum in the light intensity propagation direction; The peak value of each node in the propagation direction is obtained by fitting the light intensity distribution curve, and the three-dimensional coordinates corresponding to the peak value are regarded as the three-dimensional position of the sperm flagellum corresponding to the projection node; The three-dimensional coordinates of each flagellar node are connected, and the spatial distribution of the flagella is calculated using the connecting line segments as the skeleton to achieve three-dimensional reconstruction of the sperm flagellum.

2. The method for dynamic observation of sperm three-dimensional morphology according to claim 1, characterized in that: The holographic image is a defocused image, which is reconstructed using a light field propagation formula; the reconstruction process of the holographic image includes a process of defocusing, focusing, and defocusing the sperm cell image; In three-dimensional light intensity reconstruction, the differences in corresponding propagation distances between adjacent reconstructed images are equal.

3. The method for dynamic observation of sperm three-dimensional morphology according to claim 1, characterized in that: The method of processing the three-dimensional spatial light intensity distribution using the difference method includes: Select the center point of the sperm cell head as the feature point and obtain the projection coordinates of the feature point; Analyze the distribution law of the light intensity reconstructed by the characteristic points in the propagation direction, and obtain the distance d between the maximum and minimum values of the light intensity in the propagation direction; Each reconstructed image in the three-dimensional light intensity distribution is subtracted from the reconstructed image with a propagation distance difference d from the image, so that the scattered light signal of the sperm flagellum in the light intensity distribution image is enhanced and the background signal approaches zero.

4. The method for dynamic observation of sperm three-dimensional morphology according to claim 1, characterized in that: In the step of finding the center point A0 of the sperm head and extracting the reconstructed focused image layer corresponding to this point to obtain the outline of the sperm head, the coordinates of the point with maximum light intensity are used as the basis for determining the focus of the sperm head; Starting from the center point A0, when the angle with the direction of the sperm head is less than the preset angle θ The step of finding the maximum light intensity point within the range of 0 also includes the following steps: When searching for the two-dimensional projection node of the sperm flagellum, the points within a preset distance from the starting point are discarded to avoid interference of the strong signal near the node on the acquisition of the next two-dimensional projection node of the flagellum.

5. The method for dynamic observation of sperm three-dimensional morphology according to claim 1, characterized in that: The method of calculating and obtaining the spatial distribution of flagella using the connecting line segments as skeletons includes: The reconstructed sperm flagellum is skeletonized, expanded, and reconstructed by using the centroid method or clustering algorithm to obtain a new reconstruction result. The process is iterated until convergence to obtain the optimal solution.

6. The method for dynamic observation of sperm three-dimensional morphology according to claim 1, characterized in that: The method for dynamic observation of sperm three-dimensional morphology further comprises the following steps: Holographic images are continuously acquired, and each frame of the image is reconstructed to obtain the spatial motion parameters of sperm and the changes in morphology in the time dimension.

7. A dynamic observation device for three-dimensional sperm morphology, characterized in that: include: 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 to 6.

8. A computer-readable storage medium storing a program executable by a processor, characterized in that: The processor-executable program is used to perform the method according to any one of claims 1 to 6 when executed by the processor.

Images