Methods for assessing cardiac mesothelial cell distribution based on immunofluorescence images

Abstract

This invention relates to the field of image analysis technology, specifically to a method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images. The method includes: acquiring multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the assessment group; determining initial cardiac mesothelial cell regions based on the staining status of the isotype control group and the differences in staining depth among multiple staining regions in the immunofluorescence images; analyzing non-specific staining based on changes in the staining area and irregular cell boundaries of the initial cardiac mesothelial cell regions, and adjusting the antibody concentration accordingly; determining multiple target cardiac mesothelial cell regions, and obtaining the location information of the cardiac mesothelial cells based on the degree of overlap and similarity between these regions; and analyzing the spacing distribution of cardiac mesothelial cells and their distribution in the immunofluorescence images based on the location information to obtain the uniformity of the cardiac mesothelial cell distribution.

Description

Technical Field

[0001] This invention relates to the field of image analysis technology, and specifically to a method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images. Background Technology

[0002] Accurate identification of cardiac mesothelial cell distribution using immunofluorescence imaging enables detailed analysis of the cardiac tissue microenvironment. Assessing cardiac mesothelial cell distribution through immunofluorescence imaging avoids structural interference caused by traditional tissue sections, allowing direct observation of cytoskeleton and molecular colocalization, thus enhancing insights into cell function and interactions. Furthermore, immunofluorescence can track the dynamic distribution of cells after transplantation, providing crucial data support for cardiac regenerative medicine. Its high resolution and multi-channel imaging capabilities help elucidate the mechanisms of abnormal cell distribution in cardiac diseases, driving the development of precision treatment strategies.

[0003] However, judging the non-specific staining in the evaluation group solely based on the staining in the isotype control group, without considering the weakening of mesothelial cell signals caused by excessive dilution, will lead to inaccurate identification of the distribution of mesothelial cells.

[0004] Therefore, improving the accuracy of cardiac mesothelial cell distribution identification is an urgent problem to be solved. Summary of the Invention

[0005] To address the technical problem of improving the accuracy of cardiac mesothelial cell distribution identification, the present invention aims to provide a method for assessing cardiac mesothelial cell distribution based on immunofluorescence images. The specific technical solution adopted is as follows:

[0006] This application provides a method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images, the method comprising:

[0007] Multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated were obtained.

[0008] For each of the immunofluorescence images, based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence images, the initial cardiac mesothelial cell region is determined from the immunofluorescence images;

[0009] Based on the changes in the staining area and irregularities of the cell boundaries in the initial cardiac mesothelial cell region, the non-specific staining was analyzed, and the antibody concentration was adjusted according to the analysis results.

[0010] Multiple target cardiac mesothelial cell regions are identified from multiple immunofluorescence images corresponding to the adjusted antibody concentration, and the location information of the cardiac mesothelial cells is obtained based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions.

[0011] Based on the location information of the cardiac mesothelial cells, the spacing distribution of the cardiac mesothelial cells and their distribution in the immunofluorescence image are analyzed to obtain the uniformity of the distribution of the cardiac mesothelial cells.

[0012] In some embodiments, acquiring multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated includes:

[0013] Obtain cardiac mesothelial cell sections stained with different concentrations of antibodies from the group to be evaluated. The cardiac mesothelial cell sections are fixed with an aldehyde fixative and obtained by cryostat sectioning. The antibodies include specific and non-specific markers.

[0014] Multiple immunofluorescence images of the cardiac mesothelial cells at different antibody concentrations were acquired using a pre-configured two-photon microscope and independent fluorescence channels.

[0015] In some embodiments, determining the initial cardiac mesothelial cell region from the immunofluorescence image based on the staining status of the isotype control group and the difference in staining depth of multiple stained regions in the immunofluorescence image includes:

[0016] Based on the difference in staining depth of multiple stained regions in the immunofluorescence image, suspected cardiac mesothelial cells were identified from the immunofluorescence image;

[0017] Obtain the staining status of the isotype control group corresponding to the immunofluorescence image. The specific antibody of the isotype control group is the same subtype as the specific antibody of the group to be evaluated. The non-specific antibody of the isotype control group is the same as the non-specific antibody of the group to be evaluated.

[0018] The staining of the suspected cardiac mesothelial cells was compared with that of the isotype control group, and the accuracy of the judgment of suspected cardiac mesothelial cells was obtained based on the comparison results.

[0019] Based on the accuracy of the identification of the suspected cardiac mesothelial cells, the initial cardiac mesothelial cell region is determined from the immunofluorescence image.

[0020] In some embodiments, before identifying suspected cardiac mesothelial cells from the immunofluorescence image based on the difference in staining depth of multiple stained regions in the immunofluorescence image, the method further includes:

[0021] The immunofluorescence image is converted to grayscale and segmented cells are filled through wells to obtain a preprocessed immunofluorescence image;

[0022] Based on the preprocessed immunofluorescence image, the average gray level of each cell within a preset cell range is obtained, and the area of ​​each cell within the preset cell range is used to indicate the stained area.

[0023] Based on the average gray level of each cell within a preset cell range, a gray level decrease curve is generated;

[0024] Obtain the absolute value of the slope of the line connecting the point where the previous gray level and the next gray level are located in the gray level decrease curve. The absolute value of the slope is used to indicate the difference in staining depth of the multiple staining regions.

[0025] In some embodiments, the step of analyzing non-specific staining based on changes in the staining area and irregularities of cell boundaries in the initial cardiac mesothelial cell region, and adjusting the antibody concentration based on the analysis results of non-specific staining, includes:

[0026] The staining area changes of the initial cardiac mesothelial cell region under different antibody concentrations were obtained;

[0027] Based on the boundary pixels of the initial cardiac mesothelial cell region at different antibody concentrations, the proportion of irregularly shaped cells is determined.

[0028] The degree of antibody concentration reduction is determined based on the changes in the stained area and the proportion of cells with irregular boundaries.

[0029] The antibody concentration is adjusted according to the degree of decrease in antibody concentration.

[0030] In some embodiments, when the different antibody concentrations are diluted based on a preset dilution concentration, obtaining the change in staining area of ​​the initial cardiac mesothelial cell region at different antibody concentrations includes:

[0031] Sequentially obtain the staining area of ​​the initial cardiac mesothelial cell region in the immunofluorescence images corresponding to two adjacent antibody concentrations that have been diluted and reduced;

[0032] The change in staining area is obtained based on the difference between the staining areas of the initial cardiac mesothelial cell regions.

[0033] In some embodiments, determining the proportion of irregularly shaped cells based on the boundary pixels of the initial cardiac mesothelial cell region at different antibody concentrations includes:

[0034] A rectangular coordinate system is established with the lower left corner of the immunofluorescence image corresponding to the initial cardiac mesothelial cell region as the origin, the vertical upward direction as the positive direction of the ordinate, and the horizontal rightward direction as the positive direction of the abscissa.

[0035] In the Cartesian coordinate system, obtain the absolute value of the difference between the left and right slopes of the boundary pixels of a single cell in the initial cardiac mesothelial cell region;

[0036] The absolute value of the difference is compared with a preset absolute value threshold, and the first proportion of boundary pixels with an absolute value of difference greater than the preset absolute value threshold is determined based on the comparison result. The first proportion is used to indicate the degree of boundary regularity of the single cell.

[0037] Cells with a first proportion greater than a preset threshold for the proportion of boundary pixels are defined as irregular boundary cells. The number of irregular boundary cells is compared with the number of all cells in the immunofluorescence image to obtain a second proportion, which is used to indicate the proportion of irregular boundary cells.

[0038] In some embodiments, obtaining the location information of the cardiac mesothelial cells based on the degree of overlap similarity between the plurality of target cardiac mesothelial cell regions includes:

[0039] The number of times the same cell appeared in the multiple target cardiac mesothelial cell regions was obtained, as well as the minimum antibody concentration corresponding to the appearance of different cells;

[0040] The degree to which each cell is classified as a cardiac mesothelial cell is determined by combining the frequency of occurrence and the minimum antibody concentration.

[0041] Cells with a degree of attribution greater than a preset degree of attribution are identified as cardiac mesothelial cells, and the location information of the cardiac mesothelial cells is determined.

[0042] In some embodiments, the step of analyzing the spacing distribution of the cardiac mesothelial cells and their distribution in the immunofluorescence image based on the location information of the cardiac mesothelial cells to obtain the uniformity of the cardiac mesothelial cell distribution includes:

[0043] The location of the center point of each cardiac mesothelial cell is determined based on the location information of each cardiac mesothelial cell.

[0044] Based on the location of the center point of each cardiac mesothelial cell, determine the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells;

[0045] Based on the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells, the mean of the shortest distance is determined, and the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells is compared with the mean of the shortest distance to obtain the distance difference of all cardiac mesothelial cells.

[0046] Clustering is performed on the center point locations of each cardiac mesothelial cell to obtain multiple clusters, and the area of ​​the cluster containing the most center points is obtained as a percentage of the immunofluorescence image.

[0047] The uniformity of the distribution of the cardiac mesothelial cells is obtained by combining the distance difference of all the cardiac mesothelial cells with the area of ​​the cluster containing the most centroids and the proportion of the immunofluorescence image.

[0048] In some embodiments, after the step of obtaining the uniformity of the distribution of the cardiac mesothelial cells, the method further includes:

[0049] The uniformity of the distribution of the cardiac mesothelial cells is transmitted to a database for storage.

[0050] In response to the display command, the distribution uniformity of the target cardiac mesothelial cells is queried from the database, and the distribution uniformity of the cardiac mesothelial cells is displayed in a table;

[0051] From the plurality of immunofluorescence images, the immunofluorescence image containing cardiac mesothelial cells and containing the fewest non-cardiac mesothelial cells is selected to demonstrate the distribution of the cardiac mesothelial cells.

[0052] The present invention has the following beneficial effects:

[0053] First, multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the evaluation group were acquired. Then, for each immunofluorescence image, initial cardiac mesothelial cell regions were determined from the immunofluorescence images based on the staining status of the isotype control group and the differences in staining depth among multiple stained regions. Next, non-specific staining was analyzed based on the changes in staining area and irregular cell boundaries of the initial cardiac mesothelial cell regions, and the antibody concentration was adjusted based on the analysis results. Then, multiple target cardiac mesothelial cell regions were determined from the multiple immunofluorescence images corresponding to the adjusted antibody concentrations, and the location information of the cardiac mesothelial cells was obtained based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions. Finally, based on the location information of the cardiac mesothelial cells, the spacing distribution of the cardiac mesothelial cells and the distribution of the cardiac mesothelial cells in the immunofluorescence images were analyzed to obtain the uniformity of the cardiac mesothelial cell distribution. In this application, by acquiring images at different antibody concentrations, a comprehensive understanding of the staining performance of cardiac mesothelial cells under different antibody environments can be achieved, making the analysis results more comprehensive and accurate. This is because different antibody concentrations may result in different staining effects for mesothelial cells. Only by combining images from multiple concentrations can the biased judgment caused by a single concentration image be avoided, thereby improving the accuracy of identification. The initial region is determined by combining the staining results of the isotype control group with the difference in staining depth in the immunofluorescence images. The isotype control group can serve as a reference standard to help eliminate interference from non-specific staining; while the difference in staining depth can reflect the staining differences between mesothelial cells and other cells due to different antibody binding strengths, thus more accurately delineating the area where mesothelial cells are located and improving the accuracy of identification. By focusing on changes in staining area and irregular cell boundaries, the influence of non-specific staining can be keenly detected. Non-specific staining causes irregular boundaries and abnormal changes in area of ​​the stained region. Adjusting the antibody concentration based on these conditions can optimize the staining effect and prevent non-specific staining from masking the true staining of mesothelial cells, thereby improving the accuracy of mesothelial cell distribution identification. After adjusting the antibody concentration, the location information is determined based on the degree of overlap and similarity among multiple target regions. After multiple adjustments, the overlapping parts between images often more accurately reflect the true location of mesothelial cells, reducing errors caused by improper antibody concentration or other factors, and improving the accuracy of mesothelial cell location identification. Analyzing the distribution of intercellular distances and the overall distribution of mesothelial cells allows for a comprehensive assessment of their distribution, further exploring distribution characteristics from location information to accurately determine the uniformity of distribution and improve the accuracy of mesothelial cell distribution identification. Attached Figure Description

[0054] To more clearly illustrate the technical solutions and advantages in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is a schematic diagram of the implementation environment for a method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images, provided in one embodiment of the present invention.

[0056] Figure 2 This is a schematic flowchart of a method for assessing cardiac mesothelial cell distribution based on immunofluorescence images, provided in one embodiment of the present invention.

[0057] Figure 3 This is a schematic diagram illustrating the distribution of cardiac mesothelial cells by selecting an immunofluorescence image containing cardiac mesothelial cells and containing the fewest non-cardiac mesothelial cells, as provided in an embodiment of the present invention.

[0058] Figure 4 This is a schematic diagram of a cardiac mesothelial cell distribution assessment device based on immunofluorescence imaging, provided as an embodiment of the present invention. Detailed Implementation

[0059] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the following, in conjunction with the accompanying drawings and preferred embodiments, details the specific implementation, structure, features, and effects of a method for assessing cardiac mesothelial cell distribution based on immunofluorescence images proposed according to the present invention. In the following description, different "one embodiment" or "another embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0060] It should be noted that the terms "first," "second," etc., in the specification and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, apparatus, product, or device that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] The following describes in detail, with reference to the accompanying drawings, a specific scheme for evaluating the distribution of cardiac mesothelial cells based on immunofluorescence images provided by the present invention.

[0063] Please see Figure 1 , Figure 1 This is a schematic diagram illustrating the implementation environment of a method for assessing cardiac mesothelial cell distribution based on immunofluorescence images, as provided in one embodiment of the present invention. Figure 1 As shown, the implementation environment includes a distributed assessment terminal 101 and an image acquisition device 102. The distributed assessment terminal 101 can be a terminal device equipped with a cardiac mesothelial cell distribution assessment platform, including but not limited to mobile devices, laptops, tablets, PDAs, desktop computers, etc., with local computing capabilities. The cardiac mesothelial cell distribution assessment platform can be implemented as a target client, which can be a video client, instant messaging client, browser client, or other client supporting leakage current monitoring. The distributed assessment terminal 101 can communicate with the image acquisition device 102 via a network, including but not limited to wired networks and wireless networks. The wired network includes local area networks (LANs), metropolitan area networks (MANs), and wide area networks (WANs), while the wireless network includes Bluetooth, Wi-Fi, and other networks enabling wireless communication. The distributed assessment terminal 101 may include, but is not limited to, a human-computer interaction screen, a processor, and a memory. The processor can be used, but is not limited to, to respond to human-computer interaction operations, execute corresponding operations, or generate corresponding instructions.

[0064] As an alternative, the distributed evaluation terminal 101 can be a computer, which can acquire in real time multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the evaluation group acquired by the image acquisition device 102.

[0065] As an optional approach, the distributed evaluation terminal 101 can be a server. The server can be a single server, a server cluster consisting of multiple servers, or a cloud server. The above is merely an example, and this embodiment does not impose any limitations on it.

[0066] Alternatively, the following steps of the cardiac mesothelial cell distribution assessment method based on immunofluorescence images can be performed on the distribution assessment terminal 101:

[0067] Multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated were obtained.

[0068] For each of the immunofluorescence images, based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence images, the initial cardiac mesothelial cell region is determined from the immunofluorescence images;

[0069] Based on the changes in the staining area and irregularities of the cell boundaries in the initial cardiac mesothelial cell region, the non-specific staining was analyzed, and the antibody concentration was adjusted according to the analysis results.

[0070] Multiple target cardiac mesothelial cell regions are identified from multiple immunofluorescence images corresponding to the adjusted antibody concentration, and the location information of the cardiac mesothelial cells is obtained based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions.

[0071] Based on the location information of the cardiac mesothelial cells, the spacing distribution of the cardiac mesothelial cells and their distribution in the immunofluorescence image are analyzed to obtain the uniformity of the distribution of the cardiac mesothelial cells.

[0072] The above method, by acquiring images at different antibody concentrations, provides a comprehensive understanding of the staining behavior of cardiac mesothelial cells under various antibody environments. This makes the analysis results more comprehensive and accurate, as different antibody concentrations can lead to different staining effects in mesothelial cells. Only by combining images from multiple concentrations can we avoid biased judgments caused by images from a single concentration, thereby improving identification accuracy. The initial region is determined by combining the staining results of the isotype control group with the difference in staining depth in the immunofluorescence images. The isotype control group serves as a reference standard to help eliminate interference from non-specific staining; while the difference in staining depth reflects the staining differences between mesothelial cells and other cells due to different antibody binding strengths, thus more accurately delineating the area where mesothelial cells are located and improving identification accuracy. By focusing on changes in staining area and irregular cell boundaries, the influence of non-specific staining can be keenly detected. Non-specific staining causes irregular boundaries and abnormal changes in area of ​​the stained region. Adjusting the antibody concentration based on these characteristics can optimize the staining effect and prevent non-specific staining from masking the true staining of mesothelial cells, thereby improving the accuracy of mesothelial cell distribution identification. After adjusting the antibody concentration, the location information is determined based on the degree of overlap and similarity among multiple target regions. After multiple adjustments, the overlapping parts between images often more accurately reflect the true location of mesothelial cells, reducing errors caused by improper antibody concentration or other factors, and improving the accuracy of mesothelial cell location identification. Analyzing the distribution of intercellular distances and the overall distribution of mesothelial cells allows for a comprehensive assessment of their distribution, further exploring distribution characteristics from location information to accurately determine the uniformity of distribution and improve the accuracy of mesothelial cell distribution identification.

[0073] As an optional example, this embodiment does not limit the executing entity of the above-described method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images. The above-described method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images can be executed on the distribution assessment terminal 101. For example, if the distribution assessment terminal 101 is a computer, some or all of the steps of the above-described method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images can be executed on the computer.

[0074] The above section introduced the exemplary implementation environment of the technical solution of this application. Next, we will continue to introduce the method of assessing the distribution of cardiac mesothelial cells based on immunofluorescence images of this application.

[0075] To address the problem of improving the accuracy of cardiac mesothelial cell distribution identification in the prior art, embodiments of this application propose a method for assessing cardiac mesothelial cell distribution based on immunofluorescence images, a device for assessing cardiac mesothelial cell distribution based on immunofluorescence images, an electronic device, a computer-readable storage medium, and a computer program product. These embodiments will be described in detail below.

[0076] Please see Figure 2 , Figure 2 This is a schematic flowchart of a method for assessing cardiac mesothelial cell distribution based on immunofluorescence images, provided in one embodiment of the present invention. This method can be applied to… Figure 1 The implementation environment is shown. It should be understood that this method can also be applied to other exemplary implementation environments and specifically executed by devices in other implementation environments. This embodiment does not limit the implementation environment to which the method is applicable.

[0077] like Figure 2 As shown, in an exemplary embodiment, the method for assessing cardiac mesothelial cell distribution based on immunofluorescence images includes at least steps S210 to S250, which are detailed below:

[0078] In step S210, multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated are acquired.

[0079] The evaluation group refers to the sample group whose cardiac mesothelial cell distribution needs to be evaluated. It usually includes multiple cardiac samples and is the main target of the entire evaluation method.

[0080] Different antibody concentrations refer to the use of different amounts of antibodies to stain cardiac mesothelial cells during the experiment. Changes in antibody concentration affect the staining effect, and the staining appearance of mesothelial cells varies under different concentrations. This helps to analyze the cell condition more comprehensively. Immunofluorescence images with different staining effects can be obtained by changing the antibody concentration, thereby enabling in-depth research on mesothelial cells.

[0081] Among them, the immunofluorescence image is a cell image obtained by staining cardiac mesothelial cells with immunofluorescence technology and then using a specific microscope or other equipment. The image contains cell staining information, and the embodiments of this application rely on these images to analyze various characteristics of cardiac mesothelial cells.

[0082] In step S220, for each of the immunofluorescence images, based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence image, the initial cardiac mesothelial cell region is determined from the immunofluorescence image.

[0083] Among them, the isotype control group is the control group set up in the experiment. Its specific antibody is of the same subtype as the specific antibody of the group to be evaluated, and its non-specific antibody is also the same as that of the group to be evaluated. It is used for comparison and reference to help determine whether the staining in the group to be evaluated is specific staining.

[0084] The staining condition refers to the state of cells during immunofluorescence staining, including the depth of staining, uniformity, and presence of fluorescence. By observing the staining condition, the cell type and staining effect can be preliminarily determined.

[0085] The stained area refers to a specific region in an immunofluorescence image that appears due to the fluorescent reaction caused by the binding of antibodies to cells. Different cells or cell types may correspond to different stained areas.

[0086] Among them, the difference in staining depth refers to the difference in the degree of staining in different staining areas. Mesothelial cells may exhibit differences in staining depth because their binding strength with specific antibodies may differ from that of other cells, which can be used as one of the criteria for identifying mesothelial cells.

[0087] In step S230, the non-specific staining is analyzed based on the changes in the staining area and irregularities of the cell boundaries in the initial cardiac mesothelial cell region, and the antibody concentration is adjusted based on the analysis results of the non-specific staining.

[0088] Among them, the change in staining area refers to the change in the area of ​​the staining region corresponding to the initial cardiac mesothelial cell region under different antibody concentrations. This change can reflect the influence of antibody concentration on the staining effect and help to determine whether non-specific staining exists.

[0089] Among them, irregular cell boundary refers to the degree of irregularity of cell boundaries in immunofluorescence images. Non-specific staining may cause irregular cell boundaries, which can be used as an indicator for judging non-specific staining.

[0090] Non-specific staining refers to the staining reaction between the antibody and non-target cells or tissue components. This staining can interfere with the accurate identification of target cells (cardiac mesothelial cells) and needs to be identified and eliminated through analysis. Based on the analysis of non-specific staining, such as changes in staining area and irregular cell boundaries, the antibody concentration can be appropriately adjusted to optimize the staining effect, reduce non-specific staining interference, and improve the accuracy of mesothelial cell identification.

[0091] In step S240, multiple target cardiac mesothelial cell regions are determined from multiple immunofluorescence images corresponding to the adjusted antibody concentration, and the location information of the cardiac mesothelial cells is obtained based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions.

[0092] The target cardiac mesothelial cell region was a more accurate region that likely contains cardiac mesothelial cells, determined from new immunofluorescence images after adjusting the antibody concentration; this region was more precise than the initial region.

[0093] Among them, the degree of overlap similarity refers to the degree of similarity in location, morphology and other aspects between multiple target cardiac mesothelial cell regions identified at different antibody concentrations. By analyzing the degree of overlap similarity, the true location of cardiac mesothelial cells can be determined.

[0094] Among them, the location information of cardiac mesothelial cells refers to the specific location of cardiac mesothelial cells in immunofluorescence images. Clarifying this information helps in analyzing the distribution of mesothelial cells.

[0095] In step S250, based on the location information of the cardiac mesothelial cells, the spacing distribution of the cardiac mesothelial cells and the distribution of the cardiac mesothelial cells in the immunofluorescence image are analyzed to obtain the uniformity of the distribution of the cardiac mesothelial cells.

[0096] Among them, the spacing distribution refers to the distance distribution between cardiac mesothelial cells. By analyzing the spacing distribution, we can understand the spatial distribution characteristics of mesothelial cells.

[0097] The distribution of cardiac mesothelial cells in the immunofluorescence image refers to the distribution status of cardiac mesothelial cells throughout the entire immunofluorescence image range, including whether they are concentrated or dispersed, and is used together with the spacing distribution to analyze the uniformity of mesothelial cell distribution.

[0098] Among them, the uniformity of cardiac mesothelial cell distribution is a quantitative description of whether cardiac mesothelial cells are evenly distributed in immunofluorescence images, reflecting the distribution characteristics of mesothelial cells in cardiac tissue.

[0099] For example, a group of heart samples was used as the evaluation group. An antibody was initially applied at a concentration of 1 mg / mL, and then diluted at 100 μg / mL intervals to obtain different antibody concentrations. These different antibody concentrations were used to stain cardiac mesothelial cell sections, and multiple immunofluorescence images were acquired using two-photon microscopy and independent fluorescence channels. An isotype control group was set up, with antibodies consistent with the evaluation group. The acquired immunofluorescence images were analyzed, and some regions that might be mesothelial cells were initially identified as the initial cardiac mesothelial cell regions based on differences in staining depth. Changes in the staining area of ​​these regions were observed; for example, the staining area gradually decreased as the antibody concentration decreased. Irregularity of cell boundaries was also observed, with some regions showing more irregular cell boundaries. Based on these non-specific staining analysis results, the antibody concentration dilution was appropriately reduced. Immunofluorescence images were acquired again after adjusting the antibody concentration to identify the target cardiac mesothelial cell regions. It was found that certain regions frequently appeared in images with different antibody concentrations and similar locations, i.e., a high degree of overlap and similarity, thus confirming these regions as the location information of cardiac mesothelial cells. Further analysis of the spacing distribution of cardiac mesothelial cells, such as calculating the distance between adjacent cells and observing the overall distribution of cells in immunofluorescence images, can determine the uniformity of the distribution of cardiac mesothelial cells.

[0100] As can be seen from steps S210 to S250 above, the proposed scheme in this embodiment, by acquiring images at different antibody concentrations, can comprehensively understand the staining performance of cardiac mesothelial cells under different antibody environments, making the analysis results more comprehensive and accurate. This is because different antibody concentrations may cause different staining effects in mesothelial cells. Only by combining images from multiple concentrations can we avoid biased judgments caused by images of a single concentration, thereby improving the accuracy of identification. The initial region is determined by combining the staining results of the isotype control group with the difference in staining depth in the immunofluorescence images. The isotype control group can serve as a reference standard to help eliminate interference from non-specific staining; while the difference in staining depth can reflect the staining differences between mesothelial cells and other cells due to different antibody binding strengths, thus more accurately delineating the area where mesothelial cells are located and improving the accuracy of identification. By paying attention to changes in staining area and irregular cell boundaries, the influence of non-specific staining can be keenly detected. Non-specific staining can cause irregular boundaries and abnormal changes in area of ​​the stained region. Adjusting the antibody concentration based on these conditions can optimize the staining effect and avoid non-specific staining from masking the true staining of mesothelial cells, thereby improving the accuracy of mesothelial cell distribution identification. After adjusting the antibody concentration, the location information is determined based on the degree of overlap and similarity among multiple target regions. After multiple adjustments, the overlapping parts between images often more accurately reflect the true location of mesothelial cells, reducing errors caused by improper antibody concentration or other factors, and improving the accuracy of mesothelial cell location identification. Analyzing the distribution of intercellular distances and the overall distribution of mesothelial cells allows for a comprehensive assessment of their distribution, further exploring distribution characteristics from location information to accurately determine the uniformity of distribution and improve the accuracy of mesothelial cell distribution identification.

[0101] In one embodiment of this application, acquiring multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated includes:

[0102] Obtain cardiac mesothelial cell sections stained with different concentrations of antibodies from the group to be evaluated. The cardiac mesothelial cell sections are fixed with an aldehyde fixative and obtained by cryostat sectioning. The antibodies include specific and non-specific markers.

[0103] Multiple immunofluorescence images of the cardiac mesothelial cells at different antibody concentrations were acquired using a pre-configured two-photon microscope and independent fluorescence channels.

[0104] Aldehyde fixatives are commonly used reagents in biological sample processing. Their function is to maintain the morphology and antigenicity of cells and tissues by cross-linking proteins and other biomolecules, preventing structural changes and antigen loss during subsequent processing. In this embodiment, aldehyde fixatives (such as formaldehyde) can be used to fix cardiac tissue, with the fixation time controlled within 10 minutes. This aims to preserve the protein structure of cardiac mesothelial cell membranes, ensure uniform fixation, reduce edge effects, and provide a stable sample basis for accurate observation and analysis of cardiac mesothelial cells.

[0105] A cryostat is a device used to cut biological tissues into thin slices at low temperatures. It allows the tissue to retain its original shape while frozen, producing slices of uniform thickness, which helps maintain the integrity of cell morphology and facilitates microscopic observation and immunofluorescence staining.

[0106] Specific markers are substances that can specifically recognize and bind to specific antigens within target cells (here, cardiac mesothelial cells). Due to their high affinity binding to target cells, these cells exhibit a unique fluorescent signal after immunofluorescence staining, thus distinguishing them from other cells. In this embodiment, mesothelial cell-specific markers (such as Calretinin and WT-1) can be used as primary antibodies to specifically label cardiac mesothelial cells, aiding in accurate identification of these cells.

[0107] Non-specific markers are the opposite of specific markers. They participate in the staining process in immunofluorescence experiments, but are not specifically targeted at the target cells. In this embodiment, non-specific markers can be used in conjunction with specific markers. By comparing with the isotype control group, it helps to determine which staining in the evaluation group is specifically targeting cardiac mesothelial cells and which may be non-specific staining, thereby improving the accuracy of mesothelial cell identification.

[0108] Two-photon microscopy is a high-resolution microscopy technique that utilizes the principle of two-photon excitation fluorescence to achieve imaging of deep structures in biological tissues. It offers advantages such as minimal sample damage, low photobleaching, and low phototoxicity. In this embodiment, two-photon microscopy can be used to penetrate deep into cardiac tissue to obtain high-resolution immunofluorescence images of cardiac mesothelial cells, facilitating clear observation of the distribution and morphology of mesothelial cells within the tissue.

[0109] In this context, an independent fluorescence channel refers to a separate signal acquisition channel set up for different fluorescent dyes in a microscope imaging system. Different fluorescent dyes have different emission spectra. An independent fluorescence channel allows for the separate acquisition of the fluorescence signal emitted by each fluorescent dye, avoiding interference between signals and ensuring that each fluorescence signal is clearly and accurately recorded. In this embodiment, an independent fluorescence channel can be set up, and the corresponding filters can be adjusted to match the emission spectra of each fluorescent dye, thereby obtaining clear immunofluorescence images of cardiac mesothelial cells, which is beneficial for accurately analyzing the fluorescent labeling of cells.

[0110] For example, aldehyde fixatives (such as formaldehyde) are used to preserve membrane protein structures, with fixation time controlled within 10 minutes to avoid antigen loss. For cardiac tissue, uniform fixation is ensured to reduce edge effects. High-quality sections are obtained using a cryostat (such as a Leica Sp5 / Sp8 confocal microscope), with thickness controlled at 10-20 μm to preserve cell morphology. Staining is performed using mesothelial cell-specific markers (such as Calretinin, WT-1) (primary antibody) combined with markers for other cell types (such as CD31-labeled endothelial cells) (secondary antibody). Two-photon microscopy (TPLSM) is used to penetrate deep into the tissue and acquire high-resolution images. Independent fluorescence channels are set up, and corresponding filters are adjusted to match the emission spectra of each fluorescent dye to ensure clear signals. Image processing is performed using software such as ImageJ, and cells are segmented using well-filling techniques to eliminate noise in small areas. Cardiomyocytes are differentiated based on morphological characteristics (such as irregular shapes).

[0111] In this embodiment, the use of aldehyde fixatives and strict control of fixation time effectively preserved the membrane protein structure and antigenicity of cardiac mesothelial cells, reducing changes in cell structure and antigen loss during sample processing. This provided a stable and reliable sample for accurate observation and analysis of cardiac mesothelial cells, thereby improving the accuracy of cardiac mesothelial cell distribution identification. Using a cryostat to cut heart tissue into high-quality sections of appropriate thickness maintained the integrity of cell morphology, facilitating subsequent immunofluorescence staining and microscopic observation. This helped to more clearly present the characteristics of cardiac mesothelial cells, further improving the accuracy of cardiac mesothelial cell distribution identification. The specific binding of specific markers to specific antigens within cardiac mesothelial cells enabled them to exhibit unique fluorescent signals after immunofluorescence staining, accurately distinguishing them from other cells. This provided crucial evidence for accurate identification of mesothelial cell distribution, improving identification accuracy. The combined use of non-specific markers and specific markers, along with an isotype control group, effectively assessed the non-specific staining status in the evaluation group, avoiding interference from non-specific staining on mesothelial cell identification and further improving the accuracy of cardiac mesothelial cell distribution identification. Two-photon microscopy can penetrate deep into cardiac tissue and acquire high-resolution images, allowing researchers to clearly observe the distribution and morphology of cardiac mesothelial cells within the tissue, providing strong support for a comprehensive and accurate assessment of mesothelial cell distribution. The independent fluorescence channels and filter adjustments ensure that signals from different fluorescent dyes are clearly and accurately acquired, avoiding signal interference and resulting in clear immunofluorescence images, which helps in the accurate analysis of the fluorescent labeling of cardiac mesothelial cells.

[0112] In one embodiment of this application, determining the initial cardiac mesothelial cell region from the immunofluorescence image based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence image includes:

[0113] Based on the difference in staining depth of multiple stained regions in the immunofluorescence image, suspected cardiac mesothelial cells were identified from the immunofluorescence image;

[0114] Obtain the staining status of the isotype control group corresponding to the immunofluorescence image. The specific antibody of the isotype control group is the same subtype as the specific antibody of the group to be evaluated. The non-specific antibody of the isotype control group is the same as the non-specific antibody of the group to be evaluated.

[0115] The staining of the suspected cardiac mesothelial cells was compared with that of the isotype control group, and the accuracy of the judgment of suspected cardiac mesothelial cells was obtained based on the comparison results.

[0116] Based on the accuracy of the identification of the suspected cardiac mesothelial cells, the initial cardiac mesothelial cell region is determined from the immunofluorescence image.

[0117] In acquiring immunofluorescence images, low antibody concentrations can result in insufficient staining of mesothelial cells, while excessive concentrations may lead to nonspecific staining. Therefore, isotype controls are used to identify suspected mesothelial cell distribution areas.

[0118] Suspected cardiac mesothelial cells refer to individual cells or cell populations that are initially screened based on differences in staining depth across multiple stained regions in an immunofluorescence image analysis. Since judging solely by differences in staining depth is insufficient to definitively identify them as cardiac mesothelial cells, they are termed suspected cardiac mesothelial cells.

[0119] The accuracy of identifying suspected cardiac mesothelial cells is a quantitative indicator derived by comparing the staining characteristics of suspected cardiac mesothelial cells with those of the isotype control group. This indicator measures the likelihood that the suspected cells are indeed cardiac mesothelial cells. It comprehensively reflects the degree of difference in staining characteristics between the suspected cells and the isotype control; the greater the difference and the consistency with mesothelial cell staining characteristics, the higher the accuracy of the identification. In this embodiment, based on this accuracy, a more precise initial cardiac mesothelial cell region can be further determined from the immunofluorescence image.

[0120] For example, during the analysis of the acquired immunofluorescence images, it was found that some cells in the images were stained in relatively dark areas. Based on the difference in staining depth, these cells were initially identified as suspected cardiac mesothelial cells. Simultaneously, immunofluorescence images of the corresponding isotype control group were acquired, using the same subtype-specific antibody and the same non-specific antibody as the group being evaluated. Next, the staining of the suspected cardiac mesothelial cells was compared with that of the isotype control group. For example, the difference between the mean grayscale of the suspected cardiac mesothelial cells and the mean grayscale of all cell regions in the isotype control group was calculated, and the absolute value of the slope of the grayscale change in the stained area of ​​the suspected cardiac mesothelial cells was analyzed. If the difference is large and the absolute value of the slope is also large, it indicates that the grayscale difference of the suspected cardiac mesothelial cells is significant compared to the cells in the isotype control, which is more consistent with the characteristic that mesothelial cells have a stronger binding strength to their specific antibodies than other cells, thus indicating a higher accuracy in identifying suspected cardiac mesothelial cells. Finally, based on this accuracy, the areas containing the suspected cardiac mesothelial cells with the highest accuracy were determined as the initial cardiac mesothelial cell regions.

[0121] In this embodiment, suspected cardiac mesothelial cells were identified based on differences in staining depth, providing a preliminary scope for subsequent precise identification of cardiac mesothelial cells, narrowing the analysis target, making subsequent analysis more targeted, and improving the efficiency of identifying cardiac mesothelial cell distribution. Introducing an isotype control group and comparing staining results yielded the accuracy of the suspected cardiac mesothelial cell identification. Using the isotype control as a reference standard effectively eliminated interference from factors such as non-specific staining, making the identification of suspected cardiac mesothelial cells more scientific and accurate. This improved the accuracy of determining the initial cardiac mesothelial cell region from immunofluorescence images, laying a solid foundation for the final accurate assessment of cardiac mesothelial cell distribution. Determining the initial cardiac mesothelial cell region based on the accuracy of the suspected cardiac mesothelial cell identification makes the determined initial region closer to the actual cardiac mesothelial cell distribution region, reducing misjudgments, further improving the accuracy of cardiac mesothelial cell distribution identification, and providing a reliable starting point for subsequent analysis.

[0122] In one embodiment of this application, before determining suspected cardiac mesothelial cells from the immunofluorescence image based on the difference in staining depth of multiple stained regions in the immunofluorescence image, the method further includes:

[0123] The immunofluorescence image is converted to grayscale and segmented cells are filled through wells to obtain a preprocessed immunofluorescence image;

[0124] Based on the preprocessed immunofluorescence image, the average gray level of each cell within a preset cell range is obtained, and the area of ​​each cell within the preset cell range is used to indicate the stained area.

[0125] Based on the average gray level of each cell within a preset cell range, a gray level decrease curve is generated;

[0126] Obtain the absolute value of the slope of the line connecting the point where the previous gray level and the next gray level are located in the gray level decrease curve. The absolute value of the slope is used to indicate the difference in staining depth of the multiple staining regions.

[0127] Grayscale conversion is a common operation in image processing, converting color immunofluorescence images into grayscale images. In immunofluorescence images, color information is complex; grayscale conversion simplifies the image data, focusing on brightness information, making subsequent analysis of features such as staining depth more convenient. Because differences in staining depth are visually reflected in grayscale images through changes in grayscale values, it helps highlight features related to cardiac mesothelial cells, facilitating further analysis.

[0128] One such image segmentation technique is well-filled cell segmentation. In immunofluorescence images, there may be gaps or discontinuous areas between cells. Well-filled cell segmentation fills these gaps, separating the cells from the background and making each cell an independent analyzable unit. This accurately defines the extent of each cell, providing a precise data foundation for obtaining relevant parameters (such as average grayscale) for each cell.

[0129] The preset cell range is a pre-defined specific region surrounding each cell, used to statistically analyze cell-related parameters, primarily calculating the average grayscale. By setting the preset cell range, it ensures that the average grayscale calculation focuses on the grayscale information of the cell itself, without excessive interference from surrounding cells or the background. This ensures that the obtained average grayscale accurately reflects the staining degree of the cell, providing reliable data for analyzing differences in staining depth.

[0130] For example, specific antibodies targeting antigens in mesothelial cells bind more readily to them than to other cells, resulting in a greater color depth at the location of mesothelial cells in the resulting immunofluorescence image. The immunofluorescence image j is converted to grayscale, and cells are segmented using a well-filling technique to obtain a preprocessed immunofluorescence image. The average grayscale h of different cells k within a preset cell range is calculated in the preprocessed immunofluorescence image. k The average gray values ​​within different cell ranges are arranged in descending order. The arrangement index is used as the horizontal axis, with ascending gray values ​​as the positive direction. The corresponding gray values ​​are used as the vertical axis, with ascending gray values ​​as the positive direction. Adjacent data points are connected to obtain the corresponding gray-level decreasing curve. The absolute value of the slope |k| of the line connecting a single gray value m and the next gray value m+1 is calculated. m |

[0131] Use a non-specific antibody of the same dose and subtype as the primary antibody used for staining the group to be evaluated, and follow the same secondary antibody labeling steps as an isotype control. The isotype control setup must be completely identical to the group to be evaluated, including antibody concentration, incubation time, and washing conditions.

[0132] Obtain the staining details of the isotype control group corresponding to the immunofluorescence image. This involves calculating the average grayscale value of all cell regions in the immunofluorescence image of the isotype control group with the same antibody concentration p as the group being evaluated.

[0133] The absolute value of the slope |k m | A gray level m greater than 1 is used as the gray level for classifying suspected cardiac mesothelial cells, and then cells with a gray level greater than or equal to gray level m are identified as suspected cardiac mesothelial cells.

[0134] Next, the staining patterns of suspected cardiac mesothelial cells were compared with those of the isotype control group, which is the basis for calculation. and Size difference in, The average grayscale size of suspected cardiac mesothelial cells. When... The larger |k m The larger the value, the greater the gray level of the suspected cardiac mesothelial cells compared to the cells in the isotype control group, and the greater the gray level compared to other cells in the image obtained in the group to be evaluated. This is more consistent with the characteristic that mesothelial cells bind to their specific antibodies more strongly than other cells, thus resulting in greater fluorescence brightness.

[0135] This allows us to determine the accuracy of identifying suspected cardiac mesothelial cells at antibody concentration p using grayscale m. The suspected cardiac mesothelial cells identified at antibody concentration p with the highest accuracy are used as the reference for this concentration, and the initial cardiac mesothelial cell region is then determined from the immunofluorescence image.

[0136] For example, the accuracy of the judgment of suspected cardiac mesothelial cells can be expressed as follows:

[0137]

[0138] Among them, Q p,m The accuracy of identifying suspected cardiac mesothelial cells obtained by grayscale segmentation at antibody concentration p; The staining results of suspected cardiac mesothelial cells are compared with those of the isotype control group; |k m | represents the absolute value of the slope.

[0139] In this embodiment, grayscale processing simplifies the immunofluorescence image data, highlights brightness information, and allows differences in staining depth to be more intuitively reflected through changes in grayscale values. This facilitates subsequent accurate analysis of staining depth differences, helps to more accurately identify suspected cardiac mesothelial cells, and improves the accuracy of cardiac mesothelial cell distribution identification. Cell segmentation by well filling accurately separates cells from the background and fills in gaps, making each cell an independent analyzable unit. This ensures the accuracy of subsequent calculations of parameters such as the average grayscale of each cell, providing a reliable data foundation for cardiac mesothelial cell identification based on cell characteristics, thereby improving the reliability of cardiac mesothelial cell distribution identification. The preset cell range ensures that the calculation of the average cell grayscale accurately reflects the cell's own staining degree, avoiding interference from other surrounding factors. This provides effective data for generating an accurate grayscale decrease curve reflecting staining depth differences, helping to more accurately identify suspected cardiac mesothelial cells from immunofluorescence images and improving the accuracy of cardiac mesothelial cell distribution identification.

[0140] In one embodiment of this application, the step of analyzing non-specific staining based on changes in the staining area and irregularities of cell boundaries in the initial cardiac mesothelial cell region, and adjusting the antibody concentration based on the analysis results of non-specific staining, includes:

[0141] The staining area changes of the initial cardiac mesothelial cell region under different antibody concentrations were obtained;

[0142] Based on the boundary pixels of the initial cardiac mesothelial cell region at different antibody concentrations, the proportion of irregularly shaped cells is determined.

[0143] The degree of antibody concentration reduction is determined based on the changes in the stained area and the proportion of cells with irregular boundaries.

[0144] The antibody concentration is adjusted according to the degree of decrease in antibody concentration.

[0145] Specific staining of mesothelial cells exhibits a structural distribution with clearly defined staining regions. Non-specific staining, on the other hand, lacks this structural characteristic; its boundaries are more susceptible to influences from experimental conditions (such as fixation, washing, and antibody concentration) and tissue characteristics (such as necrotic areas and edge folds), resulting in irregular morphologies. When the antibody concentration in the group being evaluated is too low, it may lead to weakened staining signals and inaccurate acquisition of specific staining for mesothelial cells. The step size for decreasing the antibody concentration should be adjusted based on the appearance of non-specific staining in the current image.

[0146] In this context, boundary pixels refer to the pixels that form the boundary of each cell within the initial cardiac mesothelial cell region in an immunofluorescence image. The characteristics of these pixels, such as their location and grayscale, reflect the morphology of the cell boundaries.

[0147] Among them, irregularly shaped cells refer to cells within the initial cardiac mesothelial cell region whose boundaries exhibit an irregular morphology. Non-specific staining often leads to irregular cell boundaries, so determining whether a cell boundary is irregular can help determine whether the cell has been affected by non-specific staining.

[0148] The degree of antibody concentration attenuation is a quantitative indicator that comprehensively considers the changes in the stained area of ​​the initial cardiac mesothelial cell region under different antibody concentrations and the proportion of cells with irregular boundaries. It represents the extent to which the current antibody concentration needs to be attenuated to reduce the influence of non-specific staining. Based on the determined degree of antibody concentration attenuation, the currently used antibody concentration is reduced accordingly. Through this adjustment, the antibody concentration is optimized to reduce interference from non-specific staining on the recognition of cardiac mesothelial cells, making the staining effect more conducive to accurately identifying the distribution of cardiac mesothelial cells.

[0149] In this embodiment, irregularly shaped cells are identified by analyzing boundary pixels. Utilizing cell boundary morphology features as one of the criteria for judging non-specific staining increases the dimension of non-specific staining assessment, making the analysis of non-specific staining more comprehensive and facilitating more accurate identification of cardiac mesothelial cells, thus improving identification accuracy. Identifying irregularly shaped cells and calculating their proportion, combined with changes in staining area, determines the degree of antibody concentration reduction. By comprehensively considering multiple factors to quantify the direction and extent of antibody concentration adjustment, a scientific basis is provided for rationally adjusting antibody concentration. This more effectively reduces interference from non-specific staining, optimizes staining effects, and improves the reliability of cardiac mesothelial cell distribution identification. Adjusting antibody concentration based on the degree of reduction makes it more suitable for the staining needs of cardiac mesothelial cells, reducing interference caused by non-specific staining, helping to more clearly present the distribution of cardiac mesothelial cells, improving the accuracy of cardiac mesothelial cell distribution identification, and providing better conditions for subsequent accurate assessment of cardiac mesothelial cell distribution.

[0150] In one embodiment of this application, when the different antibody concentrations are diluted based on a preset dilution concentration, obtaining the change in staining area of ​​the initial cardiac mesothelial cell region at different antibody concentrations includes:

[0151] Sequentially obtain the staining area of ​​the initial cardiac mesothelial cell region in the immunofluorescence images corresponding to two adjacent antibody concentrations that have been diluted and reduced;

[0152] The change in staining area is obtained based on the difference between the staining areas of the initial cardiac mesothelial cell regions.

[0153] The method of reducing antibody concentration based on a preset dilution concentration refers to the gradual reduction of antibody concentration during each dilution to investigate the effect of different antibody concentrations on the staining effect of cardiac mesothelial cells. This method ensures that the change in antibody concentration is regular and controllable, facilitating the observation and analysis of changes in cardiac mesothelial cell staining and related characteristics (such as staining area and cell boundaries) as antibody concentration changes. This provides standardized and comparable experimental conditions for the research.

[0154] In one embodiment of this application, determining the proportion of irregularly shaped cells based on the boundary pixels of the initial cardiac mesothelial cell region at different antibody concentrations includes:

[0155] A rectangular coordinate system is established with the lower left corner of the immunofluorescence image corresponding to the initial cardiac mesothelial cell region as the origin, the vertical upward direction as the positive direction of the ordinate, and the horizontal rightward direction as the positive direction of the abscissa.

[0156] In the Cartesian coordinate system, obtain the absolute value of the difference between the left and right slopes of the boundary pixels of a single cell in the initial cardiac mesothelial cell region;

[0157] The absolute value of the difference is compared with a preset absolute value threshold, and the first proportion of boundary pixels with an absolute value of difference greater than the preset absolute value threshold is determined based on the comparison result. The first proportion is used to indicate the degree of boundary regularity of the single cell.

[0158] Cells with a first proportion greater than a preset threshold for the proportion of boundary pixels are defined as irregular boundary cells. The number of irregular boundary cells is compared with the number of all cells in the immunofluorescence image to obtain a second proportion, which is used to indicate the proportion of irregular boundary cells.

[0159] The preset absolute value threshold for the difference is a pre-defined standard value used to determine the magnitude of the absolute value of the difference in slope between the left and right sides of a single cell boundary pixel within the initial cardiac mesothelial cell region. The degree of irregularity of the cell boundary pixel is determined by comparing the actual acquired absolute value of the difference with this threshold.

[0160] The first proportion refers to the percentage of boundary pixels in a single cell whose absolute difference in slope between the left and right sides exceeds a preset threshold. This proportion reflects the irregularity of the cell boundary; a higher first proportion indicates a higher degree of irregularity, and vice versa.

[0161] The second proportion is the ratio of the number of irregularly shaped cells to the total number of cells in the immunofluorescence image after classifying cells with a first proportion greater than a preset threshold for the proportion of boundary pixels as irregularly shaped cells. The second proportion represents the percentage of irregularly shaped cells among all cells within the entire initial cardiac mesothelial cell region, reflecting the overall irregularity of cell boundaries within this region and providing a quantitative indicator for analyzing the impact of non-specific staining on cell boundaries.

[0162] For example, the initial concentration of purified antibody is set to 1 mg / mL, and then the antibody concentration is reduced by a preset dilution concentration B (set to 100 μg / mL). The size s of the colored area relative to the total image area of ​​the group to be evaluated in the images at the c-th and c-1-th dilutions is obtained. c s c-1 .

[0163] The degree of irregularity of cell boundaries in the images of the evaluation group after each dilution can be determined as follows: A Cartesian coordinate system is established with the bottom left corner of the image as the origin, the vertical upward direction as the positive direction of the ordinate, and the horizontal rightward direction as the positive direction of the abscissa. The absolute value K of the difference between the left and right slopes of the k boundary pixels of a single cell in the image is calculated. k,x '. Calculate K k,x The percentage of boundary pixels with an absolute difference value greater than 1.5 (i.e., the preset threshold for absolute difference) out of all boundary pixels in a single cell k. k (That is, the first proportion). When p k The larger the value, the more irregular the boundary of cell k becomes.

[0164] Then, calculate p k The percentage of cells with >0.6% in the image of the group to be evaluated at the cth dilution (p) c (That is, the second proportion). The difference in the size of the colored area relative to the total image area in the group to be evaluated during the c-th dilution compared to the (c-1)-th dilution (s) c-1 -s c The larger the value of ), and the higher the percentage of irregular cells present in the (c-1)th and (c)th dilutions, the greater the percentage of irregular cells present. c-1 With p c The difference p c When the value of ′ is larger, the staining of the test group after the cth dilution is significantly weakened. To prevent over-dilution and weakening of the staining signal, the dilution intensity should be appropriately reduced.

[0165] Therefore, the degree of decrease in antibody concentration at the (c+1)th dilution can be obtained as follows:

[0166] W c+1 =(s c-1 -s c )×p c ′

[0167] Among them, W c+1 The degree of decrease in antibody concentration at the (c+1)th dilution; s c-1 -s c For changes in staining area; p c ′ represents the proportion of cells with irregular boundaries at the (c-1)th and cth dilutions, p c-1 With p c The difference.

[0168] Then, W is used with maximum-minimum normalization. c+1 After normalization, we get w c+1 w c+1 The range is [0, 0.7]. When w c+1 The larger the concentration, the greater the reduction in dilution strength; therefore, the antibody concentration to be reduced in the (c+1)th dilution should be smaller.

[0169] Therefore, the antibody concentration to be reduced in the (c+1)th dilution is:

[0170] B c+1 =B×(1-w c+1 )

[0171] Among them, B c+1 B represents the antibody concentration to be reduced in the (c+1)th dilution; B represents the preset dilution concentration; w c+1 This represents the degree of decrease in antibody concentration at the (c+1)th dilution after normalization.

[0172] The antibody concentration is adjusted based on the antibody concentration to be reduced during the (c+1)th dilution obtained above.

[0173] In this embodiment, the preset threshold for the absolute value of the difference provides a clear standard for judging the irregularity of individual cell boundary pixels, making the judgment of cell boundary irregularity objective and operable. This helps to accurately screen cells with irregular boundaries and improve the accuracy of analyzing cell boundary irregularities. The first proportion, as a quantitative indicator to measure the regularity of individual cell boundaries, can intuitively reflect the irregularity of individual cell boundaries, providing a clear basis for further judging whether a cell has an irregular boundary. This helps to accurately identify abnormalities in cell boundaries at the microscopic level, thereby improving the accuracy of judging the impact of non-specific staining on cell boundaries. The second proportion reflects the proportion of irregularly shaped cells within the initial cardiac mesothelial cell region. This quantitative indicator allows for a more comprehensive understanding of the degree to which cell boundaries in this region are affected by non-specific staining, providing strong data support for analyzing non-specific staining based on cell boundary irregularities. This improves the comprehensiveness and accuracy of non-specific staining analysis and ultimately helps to more accurately assess the distribution of cardiac mesothelial cells.

[0174] In one embodiment of this application, obtaining the location information of the cardiac mesothelial cells based on the degree of overlap similarity between the plurality of target cardiac mesothelial cell regions includes:

[0175] The number of times the same cell appeared in the multiple target cardiac mesothelial cell regions was obtained, as well as the minimum antibody concentration corresponding to the appearance of different cells;

[0176] The degree to which each cell is classified as a cardiac mesothelial cell is determined by combining the frequency of occurrence and the minimum antibody concentration.

[0177] Cells with a degree of attribution greater than a preset degree of attribution are identified as cardiac mesothelial cells, and the location information of the cardiac mesothelial cells is determined.

[0178] In this study, as the antibody concentration decreases, the number of cells exhibiting fluorescence in the evaluation group gradually decreases, eventually leaving only mesothelial cells. Therefore, cells appearing more frequently during multiple antibody dilutions are more likely to be mesothelial cells. However, considering that the frequency of non-specific staining cells may also increase with higher antibody concentrations, the antibody concentration at which different cells finally appear was used as a weight to determine the degree to which different cells belong to mesothelial cells.

[0179] The minimum antibody concentration refers to the antibody concentration at which a particular cell first appears (i.e., exhibits a fluorescent reaction and can be identified) during experiments with different antibody concentrations. This concentration reflects the ease with which the cell binds to the antibody. Generally, cardiac mesothelial cells have a strong ability to bind to specific antibodies and may appear even at lower antibody concentrations. Recording the minimum antibody concentration helps to distinguish the characteristics of different cells binding to antibodies, thereby assisting in determining whether the cell is a cardiac mesothelial cell.

[0180] The degree to which each cell is classified as a cardiac mesothelial cell is a quantitative indicator of whether the cell is a cardiac mesothelial cell. The more frequently a cell appears, the more stably it is represented at different antibody concentrations. A lower minimum antibody concentration indicates a stronger binding to the specific antibody, making the cell easier to label. Considering both factors, a higher degree of classification suggests that the cell is more likely to be a cardiac mesothelial cell.

[0181] In this context, determining the location information of cardiac mesothelial cells refers to identifying cells with a degree greater than a preset threshold for classification as cardiac mesothelial cells by judging the degree of classification of each cell as cardiac mesothelial cells, and then determining the specific location of these cells in the immunofluorescence image.

[0182] For example, when there is no or very little fluorescence in the images of the isotype control group, there is no nonspecific staining in the group to be evaluated at the current concentration. Therefore, dilution is paused when fewer than 10 fluorescent cells are obtained in the isotype control group. A matching algorithm based on local features is used to identify the same cell at the same location in multiple images obtained at different antibody concentrations. The number of times cell k appears in different images, n... k The minimum antibody concentration f corresponding to the appearance of different cells was obtained. k To obtain the data, the number of times a single cell k appears is n. k The larger the value, the lower the minimum antibody concentration f at which it appears. k The smaller the cell, the tighter the binding between cell K and the specific antibody, and the more likely it is to be a mesothelial cell.

[0183] Therefore, the degree to which cell k belongs to cardiac mesothelial cells can be obtained as follows:

[0184]

[0185] Among them, E k For cells, k represents the degree of attribution to cardiac mesothelial cells; n k f is the number of occurrences. k This represents the minimum antibody concentration.

[0186] Then, using the maximum-minimum normalization pair E k After normalization, we get e k e k The range of e is [0, 1]. k When the value is >0.7, the cell can be identified as a cardiac mesothelial cell.

[0187] In this embodiment, obtaining the minimum antibody concentration provides a basis for determining the binding characteristics of cells to specific antibodies. Combining the frequency of cell occurrence with the likelihood of cells being cardiac mesothelial cells, and considering both binding ability and occurrence stability, makes the judgment more comprehensive and helps improve the accuracy of identifying cardiac mesothelial cells. Cells with a degree of attribution greater than a preset threshold are identified as cardiac mesothelial cells, and their location information is clearly defined. This allows for accurate localization of cardiac mesothelial cells in immunofluorescence images, improving the accuracy and reliability of cardiac mesothelial cell distribution assessment.

[0188] In one embodiment of this application, the step of analyzing the spacing distribution of the cardiac mesothelial cells and their distribution in the immunofluorescence image based on the location information of the cardiac mesothelial cells to obtain the uniformity of the cardiac mesothelial cell distribution includes:

[0189] The location of the center point of each cardiac mesothelial cell is determined based on the location information of each cardiac mesothelial cell.

[0190] Based on the location of the center point of each cardiac mesothelial cell, determine the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells;

[0191] Based on the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells, the mean of the shortest distance is determined, and the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells is compared with the mean of the shortest distance to obtain the distance difference of all cardiac mesothelial cells.

[0192] Clustering is performed on the center point locations of each cardiac mesothelial cell to obtain multiple clusters, and the area of ​​the cluster containing the most center points is obtained as a percentage of the immunofluorescence image.

[0193] The uniformity of the distribution of the cardiac mesothelial cells is obtained by combining the distance difference of all the cardiac mesothelial cells with the area of ​​the cluster containing the most centroids and the proportion of the immunofluorescence image.

[0194] Among them, the more inconsistent the distance distribution of the center points of adjacent mesothelial cells and the more concentrated their presence in the overall image, the more uneven the distribution of mesothelial cells in the heart sample.

[0195] In this context, the center point refers to the point on the two-dimensional immunofluorescence image plane that represents the geometric center of each cardiac mesothelial cell. Using the center point simplifies the description of cell location when analyzing cell distribution, allowing a single point to characterize the approximate location of the cell, facilitating subsequent calculations of distances between cells and analysis of distribution. To determine the center point location of each cardiac mesothelial cell based on its positional information, the specific location information of the cardiac mesothelial cells in the immunofluorescence image can be used, and a specific algorithm (such as the centroid formula) can be employed to calculate the center point location corresponding to each cell.

[0196] The shortest distance from the center point of each cardiac mesothelial cell to the center points of other cardiac mesothelial cells is calculated by finding the closest center point among all the other cardiac mesothelial cells for each cell and determining the value of this shortest distance. This distance reflects the closeness of a single cell to its surrounding cells, and by analyzing these shortest distances of all cells, the spatial density of cell distribution can be understood.

[0197] Clustering is a data analysis method that aims to divide objects in a dataset into different groups or classes based on their similarity. In this embodiment, clustering can be performed on the center point location data of cardiac mesothelial cells, which means grouping center points that are spatially close together to analyze the aggregation of cells in the image and determine whether there are regions where cells are concentrated.

[0198] Clusters are different groups or classes obtained after cluster analysis. Each cluster contains a group of spatially close cardiac mesothelial cell centroids, reflecting the aggregation area of ​​cardiac mesothelial cells in immunofluorescence images. By analyzing the characteristics of the clusters, such as the area proportion of the cluster containing the most centroids, we can understand the overall distribution of cardiac mesothelial cells in the image, whether it is a concentrated or dispersed distribution.

[0199] For example, the centroid formula is used to calculate the location of the center point of different mesothelial cells k in sample g. The Euclidean distance formula is then used to calculate the nearest distance l between the center point of different mesothelial cells k and the center points of other mesothelial cells. k The calculated nearest distance l to all mesothelial cells k is... kThe mean value is calculated, and the absolute value of the difference between k and the mean value for different mesothelial cells is calculated. The absolute values ​​are then summed to obtain L. g The DBSCAN density clustering algorithm was used to cluster the mesothelial cell center points in the image, resulting in multiple clusters. The area p of the cluster containing the most center points was then calculated as a percentage of the total image area. g When p g The smaller L g The larger the value, the more uneven the distribution of mesothelial cells, and the more excessive the aggregation of multiple mesothelial cells, resulting in a more uneven distribution.

[0200] Therefore, the uniformity of the distribution of mesothelial cells in sample g can be obtained as follows:

[0201]

[0202] Among them, R g The uniformity of the distribution of mesothelial cells in sample g; L g p represents the distance difference among all cardiac mesothelial cells. g The area of ​​the cluster containing the most centroids is proportional to the area of ​​the immunofluorescence image.

[0203] Then, using the maximum-minimum normalization method on R g After normalization, we get r g r g The range of its value is [0, 1].

[0204] In this embodiment, determining the center point location of cardiac mesothelial cells simplifies the description of cell location and provides a foundation for accurately calculating intercellular distances and analyzing distribution. This makes the quantitative analysis of cell distribution more feasible and helps improve the accuracy of analyzing the uniformity of cardiac mesothelial cell distribution. By calculating the shortest distance from the center point of each cardiac mesothelial cell to other center points, the density of cell distribution can be intuitively reflected from the spatial relationship between individual cells and surrounding cells. This provides important data support for comprehensively assessing the uniformity of cardiac mesothelial cell distribution and enhances the reliability of the analysis results. The application of cluster analysis can effectively identify clustered areas of cardiac mesothelial cells in the image. By analyzing the characteristics of clusters, such as the area ratio of the cluster containing the most center points, the distribution of cardiac mesothelial cells can be further understood from the perspective of the overall distribution status, enriching the analysis dimensions and improving the comprehensiveness of the assessment of the uniformity of cardiac mesothelial cell distribution.

[0205] In one embodiment of this application, after the step of obtaining the uniformity of the distribution of the cardiac mesothelial cells, the method further includes:

[0206] The uniformity of the distribution of the cardiac mesothelial cells is transmitted to a database for storage.

[0207] In response to the display command, the distribution uniformity of the target cardiac mesothelial cells is queried from the database, and the distribution uniformity of the cardiac mesothelial cells is displayed in a table;

[0208] From the plurality of immunofluorescence images, the immunofluorescence image containing cardiac mesothelial cells and containing the fewest non-cardiac mesothelial cells is selected to demonstrate the distribution of the cardiac mesothelial cells.

[0209] Among them, the database is a repository for organizing, storing and managing data according to data structure. It can be used to store data related to the uniformity of cardiac mesothelial cell distribution, which facilitates long-term data preservation, retrieval and subsequent analysis and processing.

[0210] The display command is a command issued by the user or system to request the display of data on the uniformity of cardiac mesothelial cell distribution. This command triggers the querying and display of relevant data from the database, enabling researchers or relevant personnel to obtain and view the analysis results according to their actual needs, thus achieving data visualization.

[0211] The selection process involved choosing an immunofluorescence image that contained both cardiac mesothelial cells and the fewest non-cardiac mesothelial cells. This was done by selecting one image from multiple acquired immunofluorescence images. This image had to contain both cardiac mesothelial cells and the fewest non-cardiac mesothelial cells. Such an image minimizes interference factors and more clearly and accurately displays the distribution of cardiac mesothelial cells, facilitating intuitive observation and analysis of their true distribution.

[0212] For example, after obtaining the uniformity of the distribution of cardiac mesothelial cells, the uniformity of the distribution of cardiac mesothelial cells is transmitted to the database for corresponding storage. When a user's query command is received, the non-uniformity of the distribution of mesothelial cells in different heart samples is obtained through SQL query statements and visualized in the form of Table 1.

[0213] Table 1. Uniformity of Cardiac Mesothelial Cell Distribution

[0214] Sample number Distribution uniformity 001 0.78 002 0.12 … …

[0215] Then, see Figure 3 , Figure 3 This is a schematic diagram illustrating the distribution of cardiac mesothelial cells by selecting immunofluorescence images containing the fewest non-cardiac mesothelial cells, as provided in one embodiment of the present invention. Figure 3 As shown, the distribution of mesothelial cells is displayed by selecting the immunofluorescence image that best represents the expression of mesothelial cells (the image containing the most mesothelial cells and the image containing the fewest non-mesothelial cells). Mesothelial cells can be marked with arrows during the display.

[0216] In this embodiment, a database is used to store data on the uniformity of cardiac mesothelial cell distribution, ensuring data security and orderliness, facilitating subsequent retrieval and access, providing strong support for long-term research and multi-batch data comparison, and improving the data management efficiency of the entire evaluation method. Immunofluorescence images containing cardiac mesothelial cells with the fewest non-cardiac mesothelial cells are selected for display, minimizing interference from other cells in observing the distribution of cardiac mesothelial cells. This allows for a more intuitive and accurate presentation of the distribution of cardiac mesothelial cells, improving the clarity and accuracy of observation and analysis, and providing a more intuitive and reliable basis for cardiac-related research.

[0217] Figure 4 This is a schematic diagram of a cardiac mesothelial cell distribution assessment device based on immunofluorescence imaging, provided as an embodiment of the present invention. This device can be applied to... Figure 1 The implementation environment shown is not limited to this embodiment. This device can also be applied to other exemplary implementation environments and specifically configured in other devices. This embodiment does not limit the implementation environment to which the device is applicable.

[0218] like Figure 4 As shown, this exemplary device for assessing cardiac mesothelial cell distribution based on immunofluorescence images includes:

[0219] The image acquisition module 401 is used to acquire multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated.

[0220] The region determination module 402 is used to determine the initial cardiac mesothelial cell region from the immunofluorescence image based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence image.

[0221] The antibody concentration adjustment module 403 is used to analyze the non-specific staining situation based on the changes in the staining area and the irregularity of the cell boundaries in the initial cardiac mesothelial cell region, and to adjust the antibody concentration based on the analysis results of the non-specific staining situation.

[0222] The location determination module 404 is used to determine multiple target cardiac mesothelial cell regions from multiple immunofluorescence images corresponding to the adjusted antibody concentration, and to obtain the location information of the cardiac mesothelial cells based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions.

[0223] The distribution analysis module 405 is used to analyze the spacing distribution of the cardiac mesothelial cells and the distribution of the cardiac mesothelial cells in the immunofluorescence image based on the location information of the cardiac mesothelial cells, so as to obtain the uniformity of the distribution of the cardiac mesothelial cells.

[0224] In this exemplary immunofluorescence image-based cardiac mesothelial cell distribution assessment device, by acquiring images at different antibody concentrations, a comprehensive understanding of the staining performance of cardiac mesothelial cells under different antibody environments can be achieved. This makes the analysis results more comprehensive and accurate, as different antibody concentrations may result in different staining effects for mesothelial cells. Only by integrating images from multiple concentrations can the biased judgment caused by a single concentration image be avoided, thereby improving the accuracy of identification. The initial region is determined by combining the staining results of the isotype control group with the staining depth difference in the immunofluorescence image. The isotype control group can serve as a reference standard to help eliminate interference from non-specific staining; while the staining depth difference reflects the staining difference between mesothelial cells and other cells due to different antibody binding strengths, thus more accurately delineating the area where mesothelial cells are located and improving the accuracy of identification. By focusing on changes in staining area and irregular cell boundaries, the influence of non-specific staining can be keenly detected. Non-specific staining causes irregular boundaries and abnormal changes in area of ​​the stained region. Adjusting the antibody concentration based on these conditions can optimize the staining effect and prevent non-specific staining from masking the true staining of mesothelial cells, thereby improving the accuracy of mesothelial cell distribution identification. After adjusting the antibody concentration, the location information is determined based on the degree of overlap and similarity among multiple target regions. After multiple adjustments, the overlapping parts between images often more accurately reflect the true location of mesothelial cells, reducing errors caused by improper antibody concentration or other factors, and improving the accuracy of mesothelial cell location identification. Analyzing the distribution of intercellular distances and the overall distribution of mesothelial cells allows for a comprehensive assessment of their distribution, further exploring distribution characteristics from location information to accurately determine the uniformity of distribution and improve the accuracy of mesothelial cell distribution identification.

[0225] It should be noted that the cardiac mesothelial cell distribution assessment device based on immunofluorescence images provided in the above embodiments and the cardiac mesothelial cell distribution assessment method based on immunofluorescence images provided in the above embodiments belong to the same concept. The specific operation methods of each module and unit have been described in detail in the method embodiments and will not be repeated here. In practical applications, the cardiac mesothelial cell distribution assessment device based on immunofluorescence images provided in the above embodiments can be assigned to different functional modules as needed, that is, the internal structure of the device can be divided into different functional modules to complete all or part of the functions described above. This is not a limitation here.

[0226] Embodiments of this application also provide an electronic device, including: one or more processors; and a storage device for storing one or more programs, which, when executed by the one or more processors, cause the electronic device to implement the method for assessing cardiac mesothelial cell distribution based on immunofluorescence images provided in the above embodiments.

[0227] Another aspect of this application provides a computer-readable storage medium storing a plurality of instructions adapted for loading by a processor to perform steps in any of the methods for assessing cardiac mesothelial cell distribution based on immunofluorescence images provided in the embodiments of this application. This computer-readable storage medium may be included in the electronic device described in the above embodiments, or it may exist independently and not assembled into the electronic device.

[0228] It should be noted that the order of the above embodiments of the present invention is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. The processes depicted in the accompanying drawings do not necessarily require a specific or sequential order to achieve the desired result. In some embodiments, multitasking and parallel processing are possible or may be advantageous.

[0229] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

Claims

1. A method for assessing the distribution of cardiac mesothelial cells based on immunofluorescence images, characterized in that, The method includes: Multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated were obtained. For each of the immunofluorescence images, based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence images, the initial cardiac mesothelial cell region is determined from the immunofluorescence images; Based on the changes in the staining area and irregularities of the cell boundaries in the initial cardiac mesothelial cell region, the non-specific staining was analyzed, and the antibody concentration was adjusted according to the analysis results. Multiple target cardiac mesothelial cell regions are identified from multiple immunofluorescence images corresponding to the adjusted antibody concentration, and the location information of the cardiac mesothelial cells is obtained based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions. Based on the location information of the cardiac mesothelial cells, the spacing distribution of the cardiac mesothelial cells and the distribution of the cardiac mesothelial cells in the immunofluorescence image are analyzed to obtain the uniformity of the distribution of the cardiac mesothelial cells. The process of analyzing non-specific staining based on changes in the staining area and irregularities of cell boundaries in the initial cardiac mesothelial cell region, and adjusting the antibody concentration based on the analysis results of non-specific staining, includes: The staining area changes of the initial cardiac mesothelial cell region under different antibody concentrations were obtained; Based on the boundary pixels of the initial cardiac mesothelial cell region at different antibody concentrations, the proportion of irregularly shaped cells is determined. The degree of antibody concentration reduction is determined based on the changes in the stained area and the proportion of cells with irregular boundaries. The antibody concentration is adjusted according to the degree of decrease in antibody concentration; The step of determining the proportion of irregularly shaped cells based on the boundary pixels of the initial cardiac mesothelial cell region at different antibody concentrations includes: A rectangular coordinate system is established with the lower left corner of the immunofluorescence image corresponding to the initial cardiac mesothelial cell region as the origin, the vertical upward direction as the positive direction of the ordinate, and the horizontal rightward direction as the positive direction of the abscissa. In the Cartesian coordinate system, obtain the absolute value of the difference between the left and right slopes of the boundary pixels of a single cell in the initial cardiac mesothelial cell region; The absolute value of the difference is compared with a preset absolute value threshold, and the first proportion of boundary pixels with an absolute value of difference greater than the preset absolute value threshold is determined based on the comparison result. The first proportion is used to indicate the degree of boundary regularity of the single cell. Cells with a first proportion greater than a preset boundary pixel proportion threshold are defined as boundary irregular cells. The number of boundary irregular cells is compared with the number of all cells in the immunofluorescence image to obtain a second proportion, which is used to indicate the proportion of boundary irregular cells.

2. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 1, characterized in that, The acquisition of multiple immunofluorescence images of cardiac mesothelial cells at different antibody concentrations within the group to be evaluated includes: Obtain cardiac mesothelial cell sections stained with different concentrations of antibodies from the group to be evaluated. The cardiac mesothelial cell sections are fixed with an aldehyde fixative and obtained by cryostat sectioning. The antibodies include specific and non-specific markers. Multiple immunofluorescence images of the cardiac mesothelial cells at different antibody concentrations were acquired using a pre-configured two-photon microscope and independent fluorescence channels.

3. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 2, characterized in that, The step of determining the initial cardiac mesothelial cell region from the immunofluorescence image based on the staining status of the isotype control group and the difference in staining depth of multiple staining regions in the immunofluorescence image includes: Based on the difference in staining depth of multiple stained regions in the immunofluorescence image, suspected cardiac mesothelial cells were identified from the immunofluorescence image; Obtain the staining status of the isotype control group corresponding to the immunofluorescence image. The specific antibody of the isotype control group is the same subtype as the specific antibody of the group to be evaluated. The non-specific antibody of the isotype control group is the same as the non-specific antibody of the group to be evaluated. The staining of the suspected cardiac mesothelial cells was compared with that of the isotype control group, and the accuracy of the judgment of suspected cardiac mesothelial cells was obtained based on the comparison results. Based on the accuracy of the identification of the suspected cardiac mesothelial cells, the initial cardiac mesothelial cell region is determined from the immunofluorescence image.

4. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 3, characterized in that, Before identifying suspected cardiac mesothelial cells from the immunofluorescence image based on the difference in staining depth of multiple stained regions, the method further includes: The immunofluorescence image is converted to grayscale and segmented cells are filled through wells to obtain a preprocessed immunofluorescence image; Based on the preprocessed immunofluorescence image, the average gray level of each cell within a preset cell range is obtained, and the area of ​​each cell within the preset cell range is used to indicate the stained area. Based on the average gray level of each cell within a preset cell range, a gray level decrease curve is generated; Obtain the absolute value of the slope of the line connecting the point where the previous gray level and the next gray level are located in the gray level decrease curve. The absolute value of the slope is used to indicate the difference in staining depth of the multiple staining regions.

5. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 1, characterized in that, When the different antibody concentrations are diluted based on a preset dilution concentration, obtaining the change in staining area of ​​the initial cardiac mesothelial cell region at different antibody concentrations includes: Sequentially obtain the staining area of ​​the initial cardiac mesothelial cell region in the immunofluorescence images corresponding to two adjacent antibody concentrations that have been diluted and reduced; The change in staining area is obtained based on the difference between the staining areas of the initial cardiac mesothelial cell regions.

6. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 1, characterized in that, The step of obtaining the location information of the cardiac mesothelial cells based on the degree of overlap and similarity between the multiple target cardiac mesothelial cell regions includes: The number of times the same cell appeared in the multiple target cardiac mesothelial cell regions was obtained, as well as the minimum antibody concentration corresponding to the appearance of different cells; The degree to which each cell is classified as a cardiac mesothelial cell is determined by combining the frequency of occurrence and the minimum antibody concentration. Cells with a degree of attribution greater than a preset degree of attribution are identified as cardiac mesothelial cells, and the location information of the cardiac mesothelial cells is determined.

7. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 1, characterized in that, The step of analyzing the spacing distribution of the cardiac mesothelial cells and their distribution in the immunofluorescence image based on the location information of the cardiac mesothelial cells to obtain the uniformity of the cardiac mesothelial cell distribution includes: The location of the center point of each cardiac mesothelial cell is determined based on the location information of each cardiac mesothelial cell. Based on the location of the center point of each cardiac mesothelial cell, determine the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells; Based on the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells, the mean of the shortest distance is determined, and the shortest distance from the center point of each cardiac mesothelial cell to the center point of other cardiac mesothelial cells is compared with the mean of the shortest distance to obtain the distance difference of all cardiac mesothelial cells. Clustering is performed on the center point locations of each cardiac mesothelial cell to obtain multiple clusters, and the area of ​​the cluster containing the most center points is obtained as a percentage of the immunofluorescence image. The uniformity of the distribution of the cardiac mesothelial cells is obtained by combining the distance difference of all the cardiac mesothelial cells with the area of ​​the cluster containing the most centroids and the proportion of the immunofluorescence image.

8. The method for assessing cardiac mesothelial cell distribution based on immunofluorescence images as described in claim 1, characterized in that, After the step of obtaining the uniformity of the distribution of the cardiac mesothelial cells, the method further includes: The uniformity of the distribution of the cardiac mesothelial cells is transmitted to a database for storage. In response to the display command, the distribution uniformity of the target cardiac mesothelial cells is queried from the database, and the distribution uniformity of the cardiac mesothelial cells is displayed in a table; From the plurality of immunofluorescence images, the immunofluorescence image containing cardiac mesothelial cells and containing the fewest non-cardiac mesothelial cells is selected to demonstrate the distribution of the cardiac mesothelial cells.

Images