CN115308129A - Method and device for automatically determining focusing position of fluorescent dark field camera - Google Patents
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Abstract
The invention provides a method and a device for automatically determining a focusing position of a fluorescent dark field camera, wherein the method comprises the following steps: obtaining immunofluorescence pictures of different focusing positions of the glass slide; carrying out gray processing on the immunofluorescence picture to form a first information set of the immunofluorescence picture; carrying out fuzzy processing and gray processing on the immunofluorescence picture to form a second information set of the immunofluorescence picture; and calculating the MSE value of the immunofluorescence picture based on the first information set and the second information set, wherein the MSE value is used as the definition value of the immunofluorescence picture. The method for automatically determining the focusing position of the fluorescent dark field camera can effectively and quickly determine the focusing position of the fluorescent dark field camera, has high focusing speed and high efficiency, and is convenient for automatic operation.
Description
Technical Field
The invention relates to the technical field of fluorescent dark field detection, in particular to a method and a device for automatically determining a focusing position of a fluorescent dark field camera.
Background
The immunofluorescence staining picture is generally formed by collecting fluorescence of a specific wave band by a fluorescence dark field camera so as to find the position and the quantity of the focus cells or focus bacteria.
The traditional fluorescent dark field detection device has the following flow of obtaining an immunofluorescence staining picture: the distance between the microscope objective and the glass slide (namely the focusing position of the fluorescence dark field camera) is manually adjusted, when clear focus cells or focus bacteria are observed, the adjustment of the distance between the microscope objective and the glass slide is stopped, and the fluorescence dark field camera shoots the clear focus cells or focus bacteria observed by the microscope to obtain an immunofluorescence picture. In the traditional process of obtaining the immunofluorescence picture, the distance between the microscope objective and the glass slide needs to be manually adjusted (namely, the focusing position of the fluorescence dark field camera is manually adjusted), the adjusting speed is low, the efficiency is low, and automatic operation is not used.
Disclosure of Invention
Therefore, the technical problem to be solved by the present invention is to overcome the problems of slow speed and low efficiency of obtaining a clear immunofluorescence picture in the prior art, so as to provide a method and an apparatus for automatically determining a focusing position of a fluorescent dark field camera.
In one aspect of the present invention, there is provided a method for automatically determining a focus position of a fluorescent dark field camera, comprising the steps of:
obtaining immunofluorescence pictures of different focusing positions of the glass slide;
carrying out gray level processing on the immunofluorescence picture, and then calculating horizontal information, vertical information and diagonal information of the immunofluorescence picture by using two-dimensional first-level wavelet change to form a first information set of the immunofluorescence picture;
fuzzy processing and gray processing are carried out on the immunofluorescence picture, and then the horizontal information, the vertical information and the diagonal information of the immunofluorescence picture are calculated through haar wavelets to form a second information set of the immunofluorescence picture;
and calculating the MSE value of the immunofluorescence picture based on the first information set and the second information set, wherein the MSE value is used as the definition value of the fluorescence picture, and the focusing plane position or the focusing position is determined according to the definition value of the immunofluorescence picture.
Preferably, the two-dimensional one-level wavelet transform is one of Daubechies wavelet and haar wavelet.
Preferably, the blurring process is a gaussian blurring process or a laplacian blurring process.
Further, the MSE value is calculated using the following calculation formula:
wherein, y i Represents the values in v1, h1, c1,
represents the value in v2, h2, c2, w i Equal to 1,SSE as a total difference; n is the number of pixel points of the immunofluorescence picture; and m is the number of pixel points of the immunofluorescence picture.
Another technical problem to be solved by the present invention is to overcome the defect of inaccurate focusing position of a fluorescent dark field camera in the prior art, and thereby provide an apparatus for automatically determining the focusing position of the fluorescent dark field camera, comprising:
the acquisition module is used for acquiring immunofluorescence pictures of different focusing positions of the glass slide;
the first information set acquisition module is used for carrying out gray processing on the immunofluorescence picture, and then calculating first horizontal information, first vertical information and first diagonal information of the immunofluorescence picture by utilizing two-dimensional one-level wavelet change to form a first information set of the immunofluorescence picture;
the second information set acquisition module is used for carrying out fuzzy processing and gray processing on the immunofluorescence picture, and then calculating second horizontal information, second vertical information and second diagonal information of the immunofluorescence picture by using haar wavelets to form a second information set of the immunofluorescence picture;
and the immunofluorescence picture definition value calculating module is configured to calculate an MSE value of the immunofluorescence picture based on the first information set and the second information set, the MSE value serves as the definition value of the immunofluorescence picture, and the focusing position is judged according to the definition value.
In another aspect of the present invention, an electronic device is further provided, which includes a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the above method for automatically determining the focus position of an immunofluorescence dark field camera.
In a further aspect of the invention, a non-transitory computer-readable storage medium is provided, on which a computer program is stored which, when being executed by a processor, carries out the method for automatically determining a focus position of a fluorescent dark-field camera.
The technical scheme of the invention has the following advantages:
1. according to the method for automatically determining the focusing position of the immunofluorescence dark field camera, the first information set is calculated after the immunofluorescence picture is subjected to gray scale processing, the second information set is calculated after the immunofluorescence picture is subjected to fuzzy processing and gray scale processing, the MSE value of the immunofluorescence picture is calculated through the first information set and the second information set, and whether the focusing position of the immunofluorescence dark field camera is at the optimal position or not is judged according to the MSE value.
2. The method for automatically determining the focusing position of the immunofluorescence dark field camera can be realized by adopting various two-dimensional one-level wavelet transforms, preferably adopts Daubechies wavelets or haar wavelets, has simple algorithms and can effectively reduce the hardware load.
3. The method for automatically determining the focusing position of the immunofluorescence dark field camera can be realized by adopting various fuzzy processing, preferably, the fuzzy processing adopts Gaussian fuzzy processing or Laplace fuzzy processing, and the two processing methods can effectively reduce hardware load.
4. According to the method for automatically determining the focusing position of the immunofluorescence dark field camera, provided by the invention, the MSE value is simple in algorithm, the hardware load can be effectively reduced, and the efficiency is improved.
5. According to the device for automatically determining the focusing position of the immunofluorescence dark field camera, the immunofluorescence picture is obtained through the obtaining module, the first information set is calculated after the grayscale processing is carried out on the immunofluorescence picture through the first information set obtaining module, the second information set is calculated after the fuzzy processing and the grayscale processing are carried out on the immunofluorescence picture through the second information set obtaining module, the MSE value of the immunofluorescence picture is calculated through the first information set and the second information set, the focusing position of the immunofluorescence dark field camera is judged according to the MSE value, the focusing speed is high, the efficiency is high, and the automatic operation is facilitated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a flowchart of a method for automatically determining a focus position of a fluorescent dark field camera in embodiment 1 of the present invention;
FIG. 2 is a schematic view of a focusing path for obtaining an immunofluorescent-stained image in example 1 of the present invention;
FIG. 3 is a first photograph of immunofluorescent staining taken in example 1;
FIG. 4 is a schematic view of an immunofluorescent staining picture processed by Haler wavelet in example 1;
FIG. 5 is 9 pictures after the treatment of example 1;
FIG. 6 is an apparatus for automatically determining a focus position of a fluorescent dark field camera in embodiment 3;
fig. 7 is a schematic block diagram of an electronic device in embodiment 5 of the present invention.
Reference numerals:
1-cover glass; 2-air bubbles; 3-normal saline; 4-target sample; 5-glass slide.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; the two elements may be directly connected or indirectly connected through an intermediate medium, or may be communicated with each other inside the two elements, or may be wirelessly connected or wired connected. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
The embodiment provides a method for automatically determining the focusing position of a fluorescent dark field camera, as shown in fig. 1, comprising the following steps:
step 101, obtaining immunofluorescence pictures of different focusing positions of the slide, as shown in fig. 2, collecting the immunofluorescence pictures of different focusing positions of the slide from top to bottom by a fluorescence dark field camera (for example, a CCD camera), wherein a focusing path is in a stepping manner. For ease of understanding, the present embodiment assumes that a total of 9 images are acquired from top to bottom, and since the focus path is stepped, each image has a z-axis distance corresponding thereto.
The sharpness evaluation was performed on 9 images. The clarity evaluation was started for 9 pictures in the order of acquisition from top to bottom, and the clarity evaluation procedure for each picture was as follows:
102, performing gray processing on a first immunofluorescence picture (shown in fig. 3), and then calculating horizontal information h1, vertical information v1 and diagonal information c1 of the immunofluorescence picture by using two-dimensional first-level wavelet change (haar wavelet is adopted in the embodiment) to form a first information set of the immunofluorescence picture;
step 104, calculating an MSE value of the first immunofluorescence picture based on the first information set and the second information set, where the MSE value is used as a sharpness value of the first fluorescence picture, where a calculation method of the MES value is not limited, and the MSE value is calculated by using the following calculation formula in this embodiment:
wherein, y i Represents the values in v1, h1, c1,
represents the values in v2, h2, c 2. And w i Then isEqual to 1.
The above-described sharpness evaluation process was repeated to obtain sharpness evaluation values of 9 immunofluorescence pictures, as shown in fig. 5. The picture in fig. 4 is named by using a method of Img + sequence number + sharpness value, that is: the first number after Img is the serial number and the second number is the sharpness value the sharpness estimate for the first picture shown in fig. 4 is: 0.000348; where the sharpness evaluation value of image Img _5_0.0473679.Jpg of serial number 5 is the largest, the z-axis position corresponding to the picture of serial number 5 is the ideal focus position.
The principle of evaluating whether the focusing position is accurate by using the image definition value in the embodiment of the invention is as follows: the clearer the image is, the larger the information entropy (representing that the picture contains more information); after Gaussian blur processing, the larger the information entropy loss, and the information entropy loss difference (MSE value) before and after blur is used as the definition value of the immunofluorescence picture. The more fuzzy the picture is, the less information entropy loss is before and after the Gaussian blur processing; the clearer the picture is, the greater the information entropy loss before and after gaussian blurring processing.
According to the method for automatically determining the focusing position of the immunofluorescence dark field camera, the first information set is calculated after the immunofluorescence picture is subjected to gray scale processing, the second information set is calculated after the immunofluorescence picture is subjected to fuzzy processing and gray scale processing, the MSE value of the immunofluorescence picture is calculated through the first information set and the second information set, the focusing position of the immunofluorescence dark field camera is judged according to the MSE value, the focusing speed is high, the efficiency is high, and automatic operation is facilitated.
Example 2
The present embodiment is different from embodiment 1 in that: in step 102, the two-dimensional one-level wavelet change adopts a Daubechies wavelet; in step 103, laplace blur processing is used for the blur processing of the immunofluorescence picture.
The pictures are processed by using Daubechies wavelets and Laplace fuzzy algorithm, so that on one hand, a more diversified processing method is provided for picture processing, and the universality of the technical scheme of the invention is effectively improved; on the other hand, the two algorithms are simple in structure, the calculation efficiency can be effectively improved, and the hardware load is reduced.
Example 3
The embodiment provides an apparatus for automatically determining a focus position of a fluorescent dark field camera, as shown in fig. 6, comprising:
an acquisition module 201 for acquiring fluorescence pictures of different focusing positions of the slide glass;
the first information set acquisition module 202 is used for performing gray level processing on the immunofluorescence picture, and then calculating first low latitude information, first horizontal information, first vertical information and diagonal information of the immunofluorescence picture by using two-dimensional one-level wavelet change to form a first information set of the immunofluorescence picture;
the second information set acquisition module 203 is used for performing fuzzy processing and gray processing on the immunofluorescence picture, and then calculating second low latitude information, second horizontal information, second vertical information and second diagonal information of the immunofluorescence picture by using haar wavelets to form a second information set of the immunofluorescence picture;
an immunofluorescence picture sharpness value calculation module 204 configured to calculate MSE values of the immunofluorescence picture based on the first set of information and the second set of information, the MSE values being used as sharpness values for evaluating the immunofluorescence picture.
According to the device for automatically determining the focusing position of the immunofluorescence dark field camera, the immunofluorescence picture is obtained through the obtaining module, the first information set is calculated after the grayscale processing is carried out on the immunofluorescence picture through the first information set obtaining module, the second information set is calculated after the fuzzy processing and the grayscale processing are carried out on the immunofluorescence picture through the second information set obtaining module, the MSE value of the immunofluorescence picture is calculated through the first information set and the second information set, the accuracy of the focusing position of the immunofluorescence dark field camera is judged according to the MSE value, the focusing speed is high, the efficiency is high, and the automatic operation is facilitated.
Example 4
Based on the same inventive concept, the present embodiment provides an electronic device, see fig. 7, including a memory 302, a processor 301, a communication interface 303, a communication bus 304, and a computer program stored in the memory and running on the processor, wherein the processor 301, the communication interface 303, and the memory 302 complete communication with each other through the communication bus 304; the communication interface 303 is used for realizing information transmission between related devices such as modeling software, an intelligent equipment module library and the like; the processor 301 is configured to call the computer program in the memory 302, and when the processor executes the computer program, the processor implements the method provided by the above method embodiments, for example, when the processor executes the computer program, the processor performs the following operations:
obtaining immunofluorescence pictures of different focusing positions of the glass slide;
carrying out gray level processing on the immunofluorescence picture, and then calculating horizontal information, vertical information and diagonal information of the immunofluorescence picture by using two-dimensional first-level wavelet change to form a first information set of the immunofluorescence picture;
fuzzy processing and gray processing are carried out on the immunofluorescence picture, and then horizontal information, vertical information and diagonal information of the immunofluorescence picture are calculated by using haar wavelets to form a second information set of the immunofluorescence picture;
and calculating an MSE value of the immunofluorescence picture based on the first information set and the second information set, wherein the MSE value is used as a definition value of the immunofluorescence picture, and the immunofluorescence camera focusing position is determined according to the definition value of the immunofluorescence picture.
Example 5
The present embodiments provide a non-transitory computer readable storage medium having stored thereon a computer program that, when executed by a processor, performs the following:
obtaining immunofluorescence pictures of different focusing positions of the glass slide;
carrying out gray level processing on the immunofluorescence picture, and then calculating horizontal information, vertical information and diagonal information of the immunofluorescence picture by using two-dimensional first-level wavelet change to form a first information set of the immunofluorescence picture;
fuzzy processing and gray processing are carried out on the immunofluorescence picture, and then horizontal information, vertical information and diagonal information of the immunofluorescence picture are calculated by using haar wavelets to form a second information set of the immunofluorescence picture;
and calculating the MSE value of the immunofluorescence picture based on the first information set and the second information set, wherein the MSE value is used as the definition value of the immunofluorescence picture, and the focusing plane position or the focusing position is determined according to the definition value of the immunofluorescence picture.
As will be appreciated by one skilled in the art, embodiments of the present invention may be provided as a method, system, or computer program product. Accordingly, the present invention may take the form of an entirely hardware embodiment, an entirely software embodiment or an embodiment combining software and hardware aspects. Furthermore, the present invention may take the form of a computer program product embodied on one or more computer-usable storage media (including, but not limited to, disk storage, CD-ROM, optical storage, and the like) having computer-usable program code embodied therein.
The present invention is described with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It will be understood that each flow and/or block of the flowchart illustrations and/or block diagrams, and combinations of flows and/or blocks in the flowchart illustrations and/or block diagrams, can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.
These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means which implement the function specified in the flowchart flow or flows and/or block diagram block or blocks.
These computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions specified in the flowchart flow or flows and/or block diagram block or blocks.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.
Claims (7)
1. A method for automatically determining a focus position of a fluorescent dark field camera, comprising the steps of:
obtaining immune immunofluorescence pictures of different focusing positions of the glass slide;
carrying out gray level processing on the immunofluorescence picture, and then calculating horizontal information, vertical information and diagonal information of the immunofluorescence picture by using two-dimensional first-level wavelet change to form a first information set of the immunofluorescence picture;
fuzzy processing and gray processing are carried out on the immunofluorescence picture, and then horizontal information, vertical information and diagonal information of the immunofluorescence picture are calculated by using a haar wavelet to form a second information set of the immunofluorescence picture;
and calculating the MSE value of the immunofluorescence picture based on the first information set and the second information set, wherein the MSE value is used as the definition value of the immunofluorescence picture.
2. The method of claim 1, wherein the two-dimensional one-level wavelet transform is a Daubechies wavelet or a haar wavelet.
3. The method of claim 1, wherein the blurring process is a gaussian blurring process or a laplacian blurring process.
4. The method of claim 1, wherein the MSE value is calculated using the following equation:
wherein, y i Represents the values in v1, h1, c1,
represents the value in v2, h2, c2, w i Equal to 1,SSE as the total difference; n is the number of pixel points of the immunofluorescence picture; and m is the number of pixel points of the immunofluorescence picture.
5. An apparatus for automatically determining a focal position of a fluorescent dark field camera, comprising:
the acquisition module is used for acquiring immunofluorescence pictures of different focusing positions of the glass slide;
the first information set acquisition module is used for carrying out gray processing on the immunofluorescence picture, and then calculating first horizontal information, first vertical information and diagonal information of the immunofluorescence picture by utilizing two-dimensional one-level wavelet change to form a first information set of the immunofluorescence picture;
the second information set acquisition module is used for carrying out fuzzy processing and gray processing on the immunofluorescence picture, and then calculating second horizontal information, second vertical information and second diagonal information of the immunofluorescence picture by using a haar wavelet to form a second information set of the immunofluorescence picture;
and the immunofluorescence picture definition value calculating module is configured to calculate the MSE value of the fluorescence picture based on the first information set and the second information set, and the MSE value is used as the definition value of the immunofluorescence picture.
6. An electronic device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor when executing the program implements the method for automatically determining a focus position of a fluorescent dark field camera according to any one of claims 1 to 4.
7. A non-transitory computer readable storage medium having stored thereon a computer program, which when executed by a processor, performs the method of automatically determining a focus position of a fluorescent dark field camera according to any one of claims 1 to 4.
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