WO2023071302A1

WO2023071302A1 - Image synthesis method and apparatus, electronic device and storage medium

Info

Publication number: WO2023071302A1
Application number: PCT/CN2022/105773
Authority: WO
Inventors: 李迪迪; 王晓良
Original assignee: 千乘镜像(北京)科技有限公司
Priority date: 2021-10-28
Filing date: 2022-07-14
Publication date: 2023-05-04
Also published as: CN113706433A; CN113706433B

Abstract

The present application provides an image synthesis method and apparatus, an electronic device and a storage medium, comprising: obtaining local three-dimensional images of different areas of a sample, where the local three-dimensional images are taken under different excitation light sources at target beam waist positions of a plurality of light sheets of a light sheet imaging microscope; performing relative reliability screening on the local three-dimensional images to obtain splicing parameters, where the relative reliability is calculated according to the brightness and/or the resolution of the local three-dimensional images; and splicing the three-dimensional images on the basis of different sample areas corresponding to the splicing parameters, so as to obtain a complete three-dimensional image of the sample. The present application solves the problem in the prior art of poor repeatability of a data preprocessing process due to image synthesis parameters being manually obtained, simplifies an operation process, improves the accuracy of the image synthesis process, and saves time and labor.

Description

A picture synthesis method, device, electronic equipment and storage medium

Cross References to Related Applications

This application claims the priority of the Chinese patent application with the application number 202111258879.6 and titled "A method, device, electronic device and storage medium for picture synthesis" filed with the China Patent Office on October 28, 2021, the entire contents of which are incorporated by reference incorporated in this application.

technical field

The present application relates to the technical field of digital image processing, in particular to an image synthesis method, device, electronic equipment and storage medium.

Background technique

Ordinary optical microscopes are only suitable for extremely thin samples, or can only obtain clear images of the sample surface. For larger and thicker samples, it is impossible to analyze the surface and interior of the sample without destroying the overall structure of the sample. Perform sharp optical imaging and obtain the 3D structure of your sample. The light sheet microscope can obtain a micron-scale illumination source with a limited length and a diameter through a laser and lens system. With the help of transparent and fluorescent labeling technology, it can be used to detect local areas of biological tissues or organs while maintaining the integrity of the basic structure. , to achieve micron-scale three-dimensional imaging.

In order to obtain a high-resolution three-dimensional image of a large sample as a whole, the current common practice is to use a light-sheet microscope to collect images multiple times, each time to obtain clear images of different parts of the sample, select clear images and stitch them together, and finally obtain the overall sample. clear image. However, in this process, the parameters required for image stitching rely on manual identification, including the position, range, and quantity of clear images. There are shortcomings such as inconsistent identification standards for different experimenters, large errors, long time-consuming, cumbersome steps, and high labor costs. .

Contents of the invention

In view of this, the purpose of this application is to provide a method for synthesizing pictures of a light sheet imaging microscope, the method comprising:

Acquiring local three-dimensional images of different regions of the sample, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheets of a light sheet imaging microscope;

performing relative reliability screening on the local 3D image to obtain splicing parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the local 3D image;

The three-dimensional images are stitched corresponding to different regions of the sample based on the stitching parameters to obtain a complete three-dimensional image of the sample.

Optionally, the step of acquiring local three-dimensional images of different regions of the sample includes:

Obtaining planar image information of the sample, the planar image information including time stamp label data and planar image;

Calculate the time interval between adjacent planar images based on the time stamp tag data;

Obtaining the physical position of the planar image according to the time interval and the displacement data of the product displacement platform;

According to the time interval and the optical zoom data of the shooting device, the position of the beam waist area of the excitation light sheet is obtained;

The planar image is reconstructed into the local three-dimensional image based on the physical position of the planar image and the position of the target beam waist area of the excitation light sheet.

Optionally, the step of acquiring local three-dimensional images of different regions of the sample further includes:

calculating time intervals between all adjacent planar images based on the timestamp tag data;

Based on the time interval between all adjacent planar images, the support vector machine method is used to cluster the displacement of the optical zoom system of the shooting device and the displacement stage of the sample during the generation interval of adjacent pictures, and obtain all the physical features of the planar images. location and location of the beam waist region of all excitation beams;

The planar image is reconstructed into a local three-dimensional image based on all physical positions of the planar image and target beam waist region positions of all excitation light sheets.

dividing the planar image into at least two small areas according to the direction perpendicular to the light emitted by the optical zoom system of the shooting device;

Evaluating the definition of each small region by using a gradient function to obtain an evaluation result for each small region;

By comparing the evaluation results, the relative definition change trend of the planar image is obtained;

Analyzing the change trend of the relative definition of the planar image to obtain the change direction and change period of the beam waist position of the excitation light target;

Obtain the physical position of the sample corresponding to the planar image according to the displacement direction of the sample displacement stage, the change direction and change period of the beam waist position of the excitation light target;

The planar image is three-dimensionally reconstructed based on the physical position of the sample.

Optionally, the step of performing relative reliability screening on the local 3D images to obtain stitching parameters includes:

calculating relative reliability data of the local 3D image based on the brightness and/or resolution of the 3D image;

Selecting the highest value in the reliability data as the boundary of the preset key area, wherein the preset key area is an overlapping area between the local 3D image and adjacent 3D images;

Selecting a three-dimensional image with a relative reliability difference on both sides of the boundary within a preset range as the overlapping image in the overlapping area;

The boundary and the overlapping image are used as the stitching parameters.

Optionally, the step of calculating the relative reliability data of the local 3D image based on the brightness of the 3D image includes:

Obtaining all effective pixels of the three-dimensional image, and obtaining the brightness ratio of all effective pixels of beam waist regions of different purposes under excitation light irradiation;

The brightness ratio of all effective pixels of the three-dimensional image is normalized, and the normal distribution of the negative exponential power term is used to fit the excitation light energy distribution of different images to obtain a two-dimensional normal distribution;

Then, the variance of the excitation light energy density of each effective pixel point is calculated according to the characteristic that the excitation light energy density obeys the two-dimensional normal distribution;

The variance is used as the relative reliability data.

Optionally, the step of calculating the relative reliability data of the local 3D image based on the resolution of the 3D image includes:

Using a gradient function to compare the image resolutions of different three-dimensional images at the same position in a direction perpendicular to the optical axis of the excitation light;

The resolution is taken as the relative reliability data.

Optionally, the step of stitching the three-dimensional images corresponding to different regions of the sample based on the stitching parameters includes:

Synthesizing overlapping three-dimensional images by interpolation based on the boundary and the overlapping images;

The three-dimensional images are stitched based on the overlapping three-dimensional images.

In the second aspect, the embodiment of the present application provides an image synthesis device of a light-sheet imaging microscope, the device comprising:

The reconstruction module is used to obtain local three-dimensional images of different regions of the sample, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheet objects of a light-sheet imaging microscope;

A screening module, configured to perform relative reliability screening on the partial three-dimensional image to obtain splicing parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the partial three-dimensional image;

A stitching module, configured to stitch the three-dimensional images corresponding to different regions of the sample based on the stitching parameters, to obtain a complete three-dimensional image of the sample.

In a third aspect, an embodiment of the present application provides an electronic device, including: a processor, a storage medium, and a bus, the storage medium stores machine-readable instructions executable by the processor, and when the electronic device is running, the The processor communicates with the storage medium through a bus, and the processor executes the machine-readable instructions, so as to perform the steps of the image synthesis method of the light-sheet imaging microscope as described above.

In a fourth aspect, an embodiment of the present application provides a computer-readable storage medium, on which a computer program is stored, and when the computer program is run by a processor, the image synthesis of the above-mentioned light-sheet imaging microscope is performed. method steps.

The image synthesis method and device of the light-sheet imaging microscope provided in the embodiments of the present application replace manual analysis of original image data and calculation of image synthesis parameters, and realize the automation of the preprocessing process. Compared with the image mosaic method in the prior art, it solves the technical problem that the prior art relying on manual acquisition of image synthesis parameters leads to poor repeatability of the data preprocessing process and is greatly affected by the differences of experimenters, and significantly simplifies the operation process, reducing time and labor costs. The accuracy of the picture stitching is improved, human errors are avoided, and the accuracy of the complete three-dimensional image obtained through the light-sheet imaging microscope is improved.

In order to make the above-mentioned purpose, features and advantages of the present application more comprehensible, preferred embodiments will be described in detail below together with the accompanying drawings.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, so It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

FIG. 1 shows a flow chart of an image synthesis method for a light-sheet imaging microscope provided in an embodiment of the present application;

Fig. 2 shows a schematic structural diagram of an image synthesis device of a light-sheet imaging microscope provided in an embodiment of the present application;

FIG. 3 shows a schematic structural diagram of an electronic device provided by an embodiment of the present application;

FIG. 4 shows a schematic structural diagram of a storage medium provided by an embodiment of the present application.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only It is a part of the embodiments of this application, not all of them. The components of the embodiments of the application generally described and illustrated in the figures herein may be arranged and designed in a variety of different configurations. Accordingly, the following detailed description of the embodiments of the application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. Based on the embodiments of the present application, every other embodiment obtained by those skilled in the art without making creative efforts falls within the protection scope of the present application.

First, the applicable application scenarios of this application are introduced. This application can be applied to a light sheet microscope. Through the laser and lens system, a micron-scale illumination source with a limited length can be obtained. Cooperating with the transparent and fluorescent labeling technology, it can be used on the premise of maintaining the integrity of the basic structure. Biological tissue Or a part of the inside of an organ to achieve micron-scale three-dimensional imaging scenes.

It has been found through research that in order to obtain overall high-resolution three-dimensional images of larger samples, the current common practice is to use a light sheet microscope to collect images multiple times, each time to obtain clear images of different parts of the sample, select clear images and stitch them together, Ultimately, a clear image of the sample as a whole is obtained. However, in this process, the parameters required for image stitching rely on manual identification, including the position, range, and quantity of clear images. High disadvantage.

Based on this, the embodiment of the present application provides an image synthesis method of a light-sheet imaging microscope, which uses a computer to completely replace all the manual links in the image synthesis step, improves the consistency and accuracy of the image processing link, and improves the automation. To a certain extent, the labor cost and time cost are reduced.

Please refer to FIG. 1 . FIG. 1 is a flow chart of an image synthesis method of a light-sheet imaging microscope provided in an embodiment of the present application. As shown in Figure 1, the image synthesis method of the light-sheet imaging microscope provided in the embodiment of the present application includes:

S101. Acquire local three-dimensional images of different regions of the sample, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheets of a light-sheet imaging microscope;

In a possible implementation manner, the step of acquiring local three-dimensional images of different regions of the sample includes:

Exemplarily, if the label information of the planar image information has a time stamp, the time interval between the generation times of adjacent images is obtained, and the time interval between the optical zoom system and the sample displacement stage is determined according to the optical zoom of the microscope and the mechanical characteristics of the sample displacement stage. The physical position of the picture and the position of the target beam waist area of the excitation light sheet can be obtained, based on the physical position of the picture and the position of the target beam waist area of the excitation light sheet, the planar image is reconstructed into the local three-dimensional image.

In a possible implementation manner, the step of acquiring local three-dimensional images of different regions of the sample further includes:

Exemplarily, the SVM (direct vector machine) method is applied to all time intervals, and the actions of the optical zoom system and the sample displacement stage are clustered during the generation intervals of adjacent pictures, and according to the order in which the pictures are taken, the result of the picture can be obtained The physical location and location of the beam waist area of the excitation light sheet. Then, according to the corresponding physical positions of the pictures, the pictures with similar positions of the target beam waist area of the excitation light sheet are reconstructed into three-dimensional images, and several three-dimensional images with different positions of the target beam waist area under the same field of view are obtained.

For example, if there is no time stamp information in the tag information of the image or you choose not to use the time stamp as the basis for 3D image reconstruction, then use the image information of the picture as the basis for 3D reconstruction. In the same field of view, the closer to the target beam waist area Images in the center of the position have higher resolution, sharper image details, and higher peak brightness. Therefore, divide the picture into small areas according to the direction perpendicular to the light, use the Tenengrad gradient function to evaluate the sharpness of each part, and compare the relative sharpness changes of all or part of the pictures in all images to obtain the purpose of excitation light. The change direction and period of the beam waist position, combined with the displacement direction of the sample stage, can be used to know the physical position of the sample corresponding to each picture, so as to realize the three-dimensional reconstruction of the image. Among them, when evaluating the sharpness of each part, there are a variety of non-referenced sharpness evaluation methods to choose from, including but not limited to Tenengrad gradient function, Laplacian gradient function, SMD (gray variance) function, Vollath function, entropy function.

S102. Perform relative reliability screening on the local 3D image to obtain splicing parameters, where the relative reliability is calculated from the brightness and/or resolution of the local 3D image;

Exemplarily, when the excitation light passes through the lens system, there is a relatively concentrated area when the sample is irradiated. The light distribution in this area is relatively concentrated and the resolution is high. The farther away from this area, the more dispersed the light and the lower the resolution. The resolution of each position in a 3D image is different.

In a possible implementation manner, the step of screening the partial three-dimensional image for relative reliability includes:

The boundary and the overlapping image are used as the stitching parameters.

Exemplarily, the excitation light source is a laser, and the energy density of the excitation light obeys a two-dimensional normal distribution. The smaller the distribution variance, the higher the resolution, and the larger the distribution variance, the lower the resolution, because the excitation light is in the eyepiece during imaging. Therefore, on the focal plane of the eyepiece, the energy distribution of the excitation light approximately obeys a one-dimensional normal distribution. Considering that in actual imaging, as the light penetrates the sample, the energy of the excitation light decreases with the increase of the optical path. The relationship between the remaining energy of the light and the optical path is approximately a negative exponential power. Therefore, a normal distribution with a negative exponential power term can be used to fit the excitation light energy distribution in the focal plane of the eyepiece. In order to simplify the calculation, this step can also be directly fitted with a polynomial with a negative degree.

In a possible implementation manner, the step of calculating the relative reliability data of the local 3D image based on the attribute information of the 3D image includes:

performing normalization processing on the brightness ratios of all effective pixels of the three-dimensional image, and fitting the excitation light energy distribution of different images by using a normal distribution with a negative exponential power term to obtain a two-dimensional normal distribution;

The variance is used as the relative reliability data.

Exemplarily, the brightness of a tiny region in the sample in the picture is approximately proportional to the energy density of the excitation light in the tiny region. Exclude the background fluorescence, take all effective pixels, calculate the brightness ratio of all effective pixels under the excitation light in the beam waist area of different purposes, and then normalize the overall brightness, and then use the focal plane of the eyepiece obtained from the above analysis The internal excitation light energy distribution is fitted to the excitation light energy distribution of different images, and then the variance of the excitation light energy density at each position is calculated according to the characteristic that the excitation light energy density obeys the two-dimensional normal distribution. The higher the relative reliability of , the larger the variance, the lower the relative reliability of the image information. In this way, the relative reliability data of all three-dimensional images at any position can be obtained.

Using the gradient function to compare the image clarity of different three-dimensional images at the same position in the direction perpendicular to the optical axis of the excitation light;

The sharpness was taken as the relative reliability data.

Exemplarily, the excitation light is scanned in parallel within the focal plane of the eyepiece, and the optical axis of the eyepiece is perpendicular to the scanning plane of the excitation light. Therefore, from the analysis of the illumination thickness of the sample, the illumination thickness of the target beam waist area of the excitation light is thinner, and the farther away from the target beam waist area The greater the thickness of the illumination, so the difference in image resolution at different positions of the sample is more reflected in the direction parallel to the optical axis of the eyepiece. The closer to the beam waist area of the excitation light, the higher the resolution and the more principle the beam waist of the excitation light. area, the lower the resolution. Use the Tenengrad gradient function to compare the image sharpness of different 3D images at the same position in the direction perpendicular to the optical axis of the excitation light. The difference in sharpness and the difference in resolution have approximately the same trend, so it is calculated with the Tenengrad gradient function The sharpness of different positions can also be used as the relative reliability data of image information. Among them, when evaluating the sharpness of each part, there are a variety of non-referenced sharpness evaluation methods to choose from, including but not limited to Tenengrad gradient function, Laplacian gradient function, SMD (gray variance) function, Vollath function, entropy function.

S103. Stitch the three-dimensional images corresponding to different regions of the sample based on the stitching parameters to obtain a complete three-dimensional image of the sample.

In a possible implementation manner, the step of stitching the three-dimensional images based on different areas of the sample corresponding to the stitching parameters includes:

synthesizing overlapping three-dimensional images by interpolation based on the boundaries and the overlapping images;

Exemplarily, for any position area of the sample, the image at the position with the highest relative reliability on the local three-dimensional image is taken to obtain the boundary between different image data, and according to the relative reliability of the image of the pixel points attached to the boundary data, to the two Take some pixels on each side as the overlapping area of the splicing boundary. In this example, the point where the relative reliability difference of the image reaches 30% to 60% is used as the end point of the overlapping area, which can be adjusted according to the actual imaging situation.

Exemplarily, an interpolation method is used to synthesize images of overlapping regions. The regions on both sides can be interpolated separately. At the boundary point of image synthesis, the data interpolation weights from the two images are equal. At the edge of the overlapping region, the data with higher reliability has a weight of 1, and the data with lower reliability has a weight of 0. The data between the boundary point and the edge are weighted according to the linear interpolation of the weights on both sides, and other interpolation methods can also be used to calculate the weights. Next, for other non-overlapping areas, the data with the highest relative reliability of the image at this position is taken to obtain the synthesized image.

The computer is used for automatic processing, and each step can be carried out continuously. Therefore, the processing of the same image file only needs to be read and written from the memory once, which can significantly save network bandwidth and processing time. Using the computer to calculate the stitching parameters, the stitching parameters obtained from the same image data can be guaranteed to be stable, and will not change due to differences in time, space, and experimenters, significantly reducing error sources, and ensuring high repeatability of experimental data. While calculating the stitching parameters, the relative reliability of the image information of all the images in all parts is compared, so the relative reliability of the stitching parameters can be evaluated, which provides a reference for evaluating the imaging quality of the sample. In the image synthesis step, the overlapping parts of different regions can be adjusted according to the difference in image reliability of the region, so as to preserve as much original image information as possible while ensuring image smoothness, and better restore the three-dimensional structure of the sample.

In a possible implementation, as shown in FIG. 2 , the embodiment of the present application provides an image synthesis device 200 of a light-sheet imaging microscope, and the device includes:

The reconstruction module 201 is used to obtain local three-dimensional images of different regions of the sample, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheets of a light-sheet imaging microscope;

A screening module 202, configured to perform relative reliability screening on the local 3D image to obtain splicing parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the local 3D image;

The stitching module 203 is configured to stitch the target three-dimensional image based on the different regions of the sample corresponding to the stitching parameters to obtain a complete three-dimensional image of the sample.

In a possible implementation, as shown in FIG. 3 , the embodiment of the present application provides an electronic device 300, including: a processor 310, a memory 320, and a bus 330, and the memory 320 stores the information of the processor 310 Executable machine-readable instructions, when the electronic device is running, the processor 310 communicates with the memory 310 through the bus 330, and the processor 310 executes the machine-readable instructions to perform sample acquisition Local three-dimensional images of different regions, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheet objects of a light-sheet imaging microscope; the relative reliability of the local three-dimensional images is screened to obtain Stitching parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the local three-dimensional image; based on the stitching parameters, the three-dimensional images are stitched corresponding to different regions of the sample to obtain a complete image of the sample 3D image.

In a possible implementation, as shown in FIG. 4 , the embodiment of the present application provides a computer-readable storage medium 400, on which a computer program 411 is stored, and the computer program 411 is executed by a processor Perform the acquisition of local three-dimensional images of different regions of the sample during operation, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheets of the light-sheet imaging microscope; compare the local three-dimensional images Reliability screening to obtain splicing parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the local three-dimensional image; splicing the three-dimensional images corresponding to different regions of the sample based on the splicing parameters to obtain A full 3D image of the sample.

Those skilled in the art can clearly understand that for the convenience and brevity of the description, the specific working process of the above-described system, device and unit can refer to the corresponding process in the foregoing method embodiment, which will not be repeated here.

In the several embodiments provided in this application, it should be understood that the disclosed systems, devices and methods may be implemented in other ways. The device embodiments described above are only illustrative. For example, the division of the units is only a logical function division. In actual implementation, there may be other division methods. For example, multiple units or components can be combined or May be integrated into another system, or some features may be ignored, or not implemented. In another point, the mutual coupling or direct coupling or communication connection shown or discussed may be through some communication interfaces, and the indirect coupling or communication connection of devices or units may be in electrical, mechanical or other forms.

The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place, or may be distributed to multiple network units. Part or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, each unit may exist separately physically, or two or more units may be integrated into one unit.

If the functions are realized in the form of software function units and sold or used as independent products, they can be stored in a non-volatile computer-readable storage medium executable by a processor. Based on this understanding, the technical solution of the present application is essentially or the part that contributes to the prior art or the part of the technical solution can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including Several instructions are used to make a computer device (which may be a personal computer, a server, or a network device, etc.) execute all or part of the steps of the methods described in the various embodiments of the present application. The aforementioned storage media include: U disk, mobile hard disk, read-only memory (Read-Only Memory, ROM), random access memory (Random Access Memory, RAM), magnetic disk or optical disc and other media that can store program codes. .

Finally, it should be noted that: the above-described embodiments are only specific implementations of the application, used to illustrate the technical solutions of the application, rather than limiting it, and the scope of protection of the application is not limited thereto, although referring to the aforementioned The embodiment has described this application in detail, and those of ordinary skill in the art should understand that any person familiar with this technical field can still modify the technical solutions described in the foregoing embodiments within the technical scope disclosed in this application Changes can be easily imagined, or equivalent replacements can be made to some of the technical features; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application, and should be covered by this application. within the scope of protection. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

A method for synthesizing pictures of a light sheet imaging microscope, characterized in that the method comprises:

Acquiring local three-dimensional images of different regions of the sample, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheets of a light sheet imaging microscope;

performing relative reliability screening on the local 3D image to obtain splicing parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the local 3D image;

The three-dimensional images are stitched corresponding to different regions of the sample based on the stitching parameters to obtain a complete three-dimensional image of the sample.
The method according to claim 1, wherein the step of acquiring local three-dimensional images of different regions of the sample comprises:

Obtaining planar image information of the sample, the planar image information including time stamp label data and planar image;

Calculate the time interval between adjacent planar images based on the time stamp tag data;

Obtaining the physical position of the planar image according to the time interval and the displacement data of the sample displacement stage;

According to the time interval and the optical zoom data of the shooting device, the position of the beam waist area of the excitation light sheet is obtained;

The planar image is reconstructed into the local three-dimensional image based on the physical position of the planar image and the position of the target beam waist area of the excitation light sheet.
The method according to claim 2, wherein the step of acquiring local three-dimensional images of different regions of the sample further comprises:

calculating time intervals between all adjacent planar images based on the timestamp tag data;

Based on the time interval between all adjacent planar images, the support vector machine method is used to cluster the displacement of the optical zoom system of the shooting device and the displacement stage of the sample during the generation interval of adjacent pictures, and obtain all the physical features of the planar images. location and location of the beam waist region of all excitation beams;

The planar image is reconstructed into a local three-dimensional image based on all physical positions of the planar image and target beam waist region positions of all excitation light sheets.
The method according to claim 1, wherein the step of acquiring local three-dimensional images of different regions of the sample comprises:

dividing the planar image into at least two small areas according to the direction perpendicular to the light emitted by the optical zoom system of the shooting device;

Evaluating the definition of each small region by using a gradient function to obtain an evaluation result for each small region;

By comparing the evaluation results, the relative definition change trend of the planar image is obtained;

Analyzing the relative definition change trend of the planar image, obtaining the change direction and change period of the beam waist position of the excitation light object;

Obtain the physical position of the sample corresponding to the planar image according to the displacement direction of the sample displacement stage, the change direction and change period of the beam waist position of the excitation light target;

The planar image is three-dimensionally reconstructed based on the physical position of the sample.
The method according to claim 1, wherein the step of performing relative reliability screening on the partial three-dimensional image to obtain stitching parameters includes:

calculating relative reliability data of the local 3D image based on the brightness and/or resolution of the 3D image;

Selecting the highest value in the reliability data as the boundary of the preset key area, wherein the preset key area is an overlapping area between the local 3D image and adjacent 3D images;

Selecting a three-dimensional image with a relative reliability difference on both sides of the boundary within a preset range as the overlapping image in the overlapping area;

The boundary and the overlapping image are used as the stitching parameters.
The method according to claim 5, wherein the step of calculating the relative reliability data of the local 3D image based on the brightness of the 3D image comprises:

Obtaining all effective pixels of the three-dimensional image, and obtaining the brightness ratio of all effective pixels of beam waist regions of different purposes under excitation light irradiation;

performing normalization processing on the brightness ratios of all effective pixels of the three-dimensional image, and fitting the excitation light energy distribution of different images by using a normal distribution with a negative exponential power term to obtain a two-dimensional normal distribution;

Then, the variance of the excitation light energy density of each effective pixel point is calculated according to the characteristic that the excitation light energy density obeys the two-dimensional normal distribution;

The variance is used as the relative reliability data.
The method according to claim 5, wherein the step of calculating the relative reliability data of the local 3D image based on the resolution of the 3D image comprises:

Using a gradient function to compare the image resolutions of different three-dimensional images at the same position in a direction perpendicular to the optical axis of the excitation light;

The resolution is taken as the relative reliability data.
The method according to claim 5, wherein the step of stitching the target three-dimensional image based on different areas of the sample corresponding to the stitching parameters includes:

synthesizing overlapping three-dimensional images by interpolation based on the boundaries and the overlapping images;

The three-dimensional images are stitched based on the overlapping three-dimensional images.
A picture synthesis device of a light sheet imaging microscope, characterized in that the device comprises:

The reconstruction module is used to obtain local three-dimensional images of different regions of the sample, wherein the local three-dimensional images are taken under excitation light sources with different beam waist positions of several light sheet objects of the light-sheet imaging microscope;

A screening module, configured to perform relative reliability screening on the partial three-dimensional image to obtain splicing parameters, wherein the relative reliability is calculated from the brightness and/or resolution of the partial three-dimensional image;

A stitching module, configured to stitch the target three-dimensional image based on different regions of the sample corresponding to the stitching parameters, to obtain a complete three-dimensional image of the sample.
An electronic device, characterized in that it includes: a processor, a storage medium and a bus, the storage medium stores machine-readable instructions executable by the processor, and when the electronic device is running, the processor and the The storage media communicate with each other through a bus, and the processor executes the machine-readable instructions to execute the steps of the image synthesis method for a light-sheet imaging microscope according to any one of claims 1 to 8.
A computer-readable storage medium, characterized in that a computer program is stored on the computer-readable storage medium, and when the computer program is run by a processor, the light-sheet imaging microscope according to any one of claims 1 to 8 is executed. The steps of the image synthesis method.