Target-free automatic focusing method, device, equipment and medium

Abstract

The invention relates to the technical field of optical imaging, and discloses a target-free automatic focusing method, device, equipment and medium, which are used for controlling a microscope to focus a target plane in a focusing space, and the focusing space comprises a first reference plane and a second reference plane which are parallel to the target plane and are located at the two sides of the target plane. Controlling the microscope to focus a preset pattern on the first reference plane to obtain a first focusing distance; controlling the microscope to focus a preset pattern on the second reference plane to obtain a second focusing distance; and controlling the microscope to focus to the target plane according to the first focusing distance, the second focusing distance and the relative distance relationship between the target plane and the first reference plane and the second reference plane. The beneficial effects are that the focusing positioning precision of the microscope is improved, and the efficiency and reliability of cell microscopic examination are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical imaging technology, and in particular to a target-free automatic focusing method, device, equipment and medium. BACKGROUND

[0002] Cell microscopy is one of the basic technologies in the fields of biology, medicine and inspection, which mainly magnifies the sample through a microscope and the like to enable the inspector to directly observe and evaluate the sample and determine whether the sample meets the inspection requirements. In some microscopy processes, the focusing operation of the microscope usually needs to be completed in advance before the sample is placed, so as to shorten the time length occupied by the focusing operation in the cell microscopy process, prolong the time length corresponding to the observation and detection process, and improve the accuracy of cell microscopy. However, in actual situations, since there is no focusing target in the observation plane where the sample is placed, the microscope is difficult to accurately focus on the observation plane, thereby affecting the clarity and accuracy during cell microscopy.

[0003] Therefore, it is urgent to propose a method capable of accurately focusing on the observation plane in the absence of a target, so as to improve the efficiency and reliability of the cell microscopy process. SUMMARY

[0004] The present application provides a target-free automatic focusing method, device, equipment and medium, which sets a first reference plane and a second reference plane with a known relative distance relationship between the focusing space and the target plane, and controls the microscope to focus on the target plane according to the first focusing distance of the first reference plane and the second focusing distance of the second reference plane, thereby improving the focusing positioning accuracy of the microscope and enhancing the efficiency and reliability of cell microscopy.

[0005] In order to achieve the above-mentioned purpose, the main technical solution adopted by the present application includes: In a first aspect, the present application provides a target-free automatic focusing method applied to control a microscope to focus on a target plane located in a focusing space, wherein the focusing space includes a first reference plane and a second reference plane parallel to the target plane, and the first reference plane and the second reference plane are respectively on both sides of the target plane; the method includes: controlling the microscope to focus on a preset pattern on the first reference plane to obtain a first focusing distance when the preset pattern on the first reference plane is focused; controlling the microscope to focus on a preset pattern on the second reference plane to obtain a second focusing distance when the preset pattern on the second reference plane is focused; controlling the microscope to focus on the target plane according to the first focusing distance and the second focusing distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane.

[0006] The target-free automatic focusing method provided in the embodiments of the present application sets a first reference plane and a second reference plane parallel to the target plane and having a known relative distance relationship in the focusing space where the target plane is located, and the first reference plane and the second reference plane are respectively provided with preset patterns for microscope focusing; the preset pattern on the first reference plane is focused by the microscope to obtain a first focusing distance corresponding to the first reference plane, and the preset pattern on the second reference plane is focused by the microscope to obtain a second focusing distance corresponding to the second reference plane; the microscope is controlled to focus on the target plane between the first reference plane and the second reference plane according to the first focusing distance, the second focusing distance and the relative distance relationship between the planes. Compared with the related art, the reference planes are set in the focusing space as the reference objects for focusing on the target plane, so that the target plane can be focused according to the relative distance relationship between the reference planes and the target plane, the time required for repeated focusing of the microscope in the focusing space is reduced, and the efficiency of cell microscopy is improved. In addition, the microscope is actively controlled to focus on the reference plane, a high-precision focusing distance is obtained as a reference, and the focusing accuracy in the target-free focusing condition is effectively improved.

[0007] Optionally, the control of the microscope to focus on the preset pattern on the first reference plane to obtain a first focusing distance when the microscope focuses on the preset pattern on the first reference plane comprises: controlling the microscope to focus and image the preset pattern on the first reference plane to obtain an initial imaging image of the first reference plane; performing annular difference verification on the initial imaging image to obtain a definition evaluation result of the initial imaging image; in a case where the definition evaluation result meets a preset definition requirement, taking a focusing distance between the microscope and the first reference plane as the first focusing distance.

[0008] Optionally, the annular difference verification on the initial imaging image to obtain a definition evaluation result of the initial imaging image comprises: performing response difference calculation on any pixel point in the initial imaging image according to an annular convolution kernel to obtain a convolution response value of the any pixel point; wherein the annular convolution kernel comprises a center convolution kernel and an annular convolution kernel surrounding the center convolution kernel, and the center convolution kernel and the annular convolution kernel correspond to different weights; after traversing all pixel points in the initial imaging image, performing response statistical processing on the convolution response values of all pixel points respectively to obtain the definition evaluation result.

[0009] Optionally, the controlling the microscope to focus image the preset pattern on the first reference plane to obtain the initial imaging image of the first reference plane comprises: From the initial position of the microscope, the microscope is moved step by step according to a set step, and at each position of the set step, the microscope is controlled to focus image the preset pattern on the first reference plane to obtain the initial imaging image of the first reference plane; The step-by-step moving and focusing imaging process is repeated until the microscope reaches the maximum moving range.

[0010] Optionally, the controlling the microscope to focus on the target plane according to the first focusing distance and the second focusing distance and the relative distance relationship between the target plane and the first reference plane and the second reference plane comprises: According to the first focusing distance and the second focusing distance and the relative distance relationship between the target plane and the first reference plane and the second reference plane, a focusing distance calculation is performed on the target plane to determine a target focusing distance when the microscope focuses on the target plane; According to the target focusing distance, the microscope is controlled to focus on the target plane.

[0011] Optionally, the method further comprises: Under a preset focusing depth of field setting, the microscope is controlled to focus on the preset pattern on the first reference plane to obtain the first focusing distance; Under the preset focusing depth of field setting, the microscope is controlled to focus on the preset pattern on the second reference plane to obtain the second focusing distance.

[0012] Optionally, the method further comprises: Under a preset observation depth of field setting, a target object on the target plane is observed to obtain a feature image of the target object; wherein the preset observation depth of field setting is different from the preset focusing depth of field setting.

[0013] In a second aspect, the embodiments of the present application provide a target-free automatic focusing device, which is applied to control a microscope to focus on a target plane located in a focusing space, the focusing space comprising a first reference plane and a second reference plane parallel to the target plane, and the first reference plane and the second reference plane are respectively on two sides of the target plane; the device comprises: A first focusing module is configured to control the microscope to focus on a preset pattern on the first reference plane to obtain a first focusing distance when focusing on the preset pattern on the first reference plane; a second focusing module, configured to control the microscope to focus on a preset pattern on the second reference plane to obtain a second focusing distance when the microscope is focused on the preset pattern on the second reference plane; a target focusing module, configured to control the microscope to focus on the target plane according to the first focusing distance and the second focusing distance, and a relative distance relationship between the target plane and the first reference plane and the second reference plane.

[0014] In a third aspect, an embodiment of the present application provides a computer device, including a memory and a processor, which are in communication connection with each other, and the memory stores computer instructions, and the processor executes the computer instructions to perform the method in any of the above embodiments.

[0015] In a fourth aspect, an embodiment of the present application provides a computer readable storage medium, which stores computer instructions, and the computer instructions are used to make a computer perform the method in any of the above embodiments.

[0016] In a fifth aspect, an embodiment of the present application provides a computer program product, which includes computer instructions, and the computer instructions are used to make a computer perform the method in any of the above embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the description of the embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0018] Figure 1 a step diagram of the target-free automatic focusing method provided by the embodiment of the present application; Figure 2a a structural schematic diagram of the microscope in the embodiment of the present application; Figure 2b a structural schematic diagram of the flow channel chip in the embodiment of the present application; Figure 3 a step diagram of focusing on the preset pattern on the first reference plane in the embodiment of the present application; Figure 4 a step diagram of the ring-shaped differential verification in the embodiment of the present application; Figure 5a a schematic diagram of the center convolution kernel in the embodiment of the present application; Figure 5b a schematic diagram of the ring-shaped convolution kernel in the embodiment of the present application; Figure 6a is an initial imaging image in a focus state in the embodiment of the present application; Figure 6b is a center response image in a focus state in the embodiment of the present application; Figure 6c is an annular response image in a focus state in the embodiment of the present application; Figure 6d is a response difference image in a focus state in the embodiment of the present application; Figure 7a is an initial imaging image in a defocus state in the embodiment of the present application; Figure 7b is a center response image in a defocus state in the embodiment of the present application; Figure 7c is an annular response image in a defocus state in the embodiment of the present application; Figure 7d is a response difference image in a defocus state in the embodiment of the present application; Figure 8 is a step diagram of iterative focusing in the embodiment of the present application; Figure 9 is a step diagram of controlling focusing of a microscope and a target plane in the embodiment of the present application; Figure 10 is a step diagram of focusing according to a preset depth of field setting in the embodiment of the present application; Figure 11 is a module diagram of a target-free automatic focusing device provided by the embodiment of the present application; Figure 12 is a structural schematic diagram of a computer device provided by the embodiment of the present application. DETAILED DESCRIPTION

[0019] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0020] Cell microscopy is one of the basic techniques in the fields of biology, medicine, and testing, etc. The sample is imaged by magnifying through a microscope and the like, so that the tester can directly observe and evaluate the sample, and determine whether the sample meets the testing requirements. In some microscopy processes, the focusing operation of the microscope usually needs to be completed in advance before the sample is placed, so as to shorten the time length occupied by the focusing operation in the cell microscopy process, prolong the time length corresponding to the observation and detection process, and improve the accuracy of the cell microscopy. However, in actual situations, since there is no focusing target in the observation plane where the sample is placed, the microscope is difficult to accurately focus on the observation plane, thereby affecting the clarity and accuracy of the cell microscopy. Therefore, it is urgent to propose a method capable of accurately focusing on the observation plane without a target, so as to improve the efficiency and accuracy of the cell microscopy process.

[0021] Based on the above problems, the present application provides a target-free automatic focusing method, device, equipment and medium, which is applied to control the microscope to focus on the target plane located in the focusing space. The focusing space includes the first reference plane and the second reference plane which are parallel to the target plane and on both sides of the target plane. The microscope is controlled to focus on the preset pattern on the first reference plane to obtain the first focusing distance. The microscope is controlled to focus on the preset pattern on the second reference plane to obtain the second focusing distance. According to the first focusing distance and the second focusing distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane, the microscope is controlled to focus on the target plane.

[0022] The target-free automatic focusing method provided by the present application sets the first reference plane and the second reference plane which are parallel to the target plane and have a known relative distance relationship in the focusing space of the target plane. The first reference plane and the second reference plane are respectively provided with a preset pattern for focusing of the microscope. The microscope is controlled to focus on the preset pattern on the first reference plane to obtain the first focusing distance corresponding to the first reference plane, and the microscope is controlled to focus on the preset pattern on the second reference plane to obtain the second focusing distance corresponding to the second reference plane. According to the first focusing distance and the second focusing distance, and the relative distance relationship between the planes, the microscope is controlled to focus on the target plane between the first reference plane and the second reference plane.

[0023] Compared with the related art, the present application sets the reference plane in the focusing space as a reference for focusing on the target plane, so that the target plane can be focused according to the relative distance relationship between the reference plane and the target plane, thereby reducing the time required for repeated focusing of the microscope in the focusing space, and improving the efficiency of the cell microscopy. In addition, by actively controlling the microscope to focus on the reference plane, a high-precision focusing distance is obtained as a reference, which effectively improves the focusing accuracy in the target-free focusing condition.

[0024] The targetless automatic focusing method provided by the specification can be applied to control a microscope to pre-focus on a target plane where cells to be inspected are located during cell microscopic examination. The cells to be inspected can be various animal cells including bovine milk somatic cells. It can be understood that, after adaptive modification, the application can also be used in the inspection process of different kinds of cells, or used to control other imaging devices such as cameras or camera heads to perform targetless focusing.

[0025] According to the embodiments of the application, a targetless automatic focusing method is provided. It should be noted that the steps shown in the flowchart of the accompanying drawings can be executed in a computer system such as a set of computer executable instructions, and although the logical order is shown in the flowchart, in some cases, the steps shown or described can be executed in an order different from that shown here.

[0026] Reference Figure 1 As shown in the accompanying drawings, a targetless automatic focusing method is provided in the embodiments. The method is applied to control a microscope to focus on a target plane located in a focusing space. The focusing space includes a first reference plane and a second reference plane parallel to the target plane, and the first reference plane and the second reference plane are respectively on both sides of the target plane. The method includes: S100. Control the microscope to focus on a preset pattern on the first reference plane to obtain a first focusing distance when the preset pattern on the first reference plane is focused.

[0027] S200. Control the microscope to focus on a preset pattern on the second reference plane to obtain a second focusing distance when the preset pattern on the second reference plane is focused.

[0028] S300. According to the first focusing distance and the second focusing distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane, control the microscope to focus on the target plane.

[0029] Specifically, when the target to be tested is inspected by the microscope, there is a focusing space corresponding to the imaging direction of the microscope. The focusing space can be obtained according to the field of view and focal length of the microscope, and any position in the focusing space can be clearly imaged by the microscope. The focusing space includes a target plane, a first reference plane and a second reference plane, which are all perpendicular to the imaging direction of the microscope. The target plane can be a plane for placing the target to be tested. The first reference plane and the second reference plane can be reference planes respectively provided with a preset pattern. The first reference plane and the second reference plane are both parallel to the target plane and are respectively on the two sides of the target plane, that is, the first reference plane can be closer to the microscope than the target plane, and the second reference plane can be farther away from the microscope than the target plane; or the second reference plane can be closer to the microscope than the target plane, and the first reference plane can be farther away from the microscope than the target plane. The relative distance relationship between the first reference plane and the target plane and the relative distance relationship between the second reference plane and the target plane are both known.

[0030] Referring to Figure 2a The plane formed by the x-axis and the y-axis is perpendicular to the paper. The microscope includes a camera, a variable aperture and a lens. The variable aperture is used to adjust the aperture size of the microscope to change the imaging depth of field of the microscope. The microscope is installed on a z-axis displacement table and moves in the z-axis direction by the z-axis displacement table to adjust the distance between the microscope and the focusing space. A flow channel chip is arranged in the focusing space corresponding to the microscope, which is used to pass the cell sample thereon so that the cell sample can be observed and inspected in the microscope.

[0031] The cross-sectional structure of the flow channel chip at the observation window position can refer to Figure 2b The flow channel chip is a back-shaped structure, and the outer layer structure is made of transparent material. A sample flow channel is formed in the center of the flow channel chip, so that the microscope can observe the cell sample to be tested in the sample flow channel through the outer layer structure. When the cell sample to be tested flows uniformly in the sample flow channel, the cell to be tested is usually located at the center position of the sample flow channel. Therefore, the center plane of the sample flow channel can be selected as the target plane, the upper surface of the sample flow channel can be selected as the first reference plane, and the lower surface of the sample flow channel can be selected as the second reference plane. The upper surface and the lower surface of the sample flow channel are both provided with a preset pattern. The preset pattern can be a process texture generated in the production process of the flow channel chip, or a specific texture pattern designed in advance. The preset patterns on the upper surface and the lower surface can be the same or different.

[0032] Further, the microscope is controlled to move along the z-axis, and a preset pattern on the first reference plane is imaged by the microscope to obtain a first focus image. The number of first focus images can be multiple, and the sharpness of each of the multiple first focus images is evaluated to obtain first sharpness data of each of the first focus images. A maximum value is selected from all the first sharpness data, and the microscope is controlled to move to a z-axis coordinate corresponding to the maximum value along the z-axis direction. At this time, the microscope is in focus with the preset pattern on the first reference plane, and the preset pattern on the first reference plane is clearly visible in the field of view of the microscope. It can be understood that, according to the coordinate of the microscope on the z-axis at this time, the first focus distance when the microscope is in focus with the preset pattern on the first reference plane can be obtained.

[0033] Similarly, the process of focusing on the preset pattern on the second reference plane can be the same as that of focusing on the preset pattern on the first reference plane. The microscope is controlled to move along the z-axis, and a preset pattern on the second reference plane is imaged by the microscope to obtain a second focus image. The number of second focus images can be multiple, and the sharpness of each of the multiple second focus images is evaluated to obtain second sharpness data of each of the second focus images. A maximum value is selected from all the second sharpness data, and the microscope is controlled to move to a z-axis coordinate corresponding to the maximum value along the z-axis direction. At this time, the microscope is in focus with the preset pattern on the second reference plane, and the preset pattern on the second reference plane is clearly visible in the field of view of the microscope. It can be understood that, according to the coordinate of the microscope on the z-axis at this time, the second focus distance when the microscope is in focus with the preset pattern on the second reference plane can be obtained.

[0034] Further, based on the relative distance relationship between the target plane and the first reference plane and the second reference plane, the focus distance when the microscope is in focus with the target plane is determined according to the first focus distance and the second focus distance, and the microscope is controlled to move along the z-axis direction according to the focus distance, so that the microscope can be in focus with the target plane. The position when the microscope is in focus with the preset pattern on the first reference plane is taken as the first focus position, and the position when the microscope is in focus with the preset pattern on the second reference plane is taken as the second focus position. It can be understood that, when the target plane is a central position between the first reference plane and the second reference plane, the microscope is controlled to move to a central position between the first focus position and the second focus position. At this time, the microscope is in focus with the target plane, and the microscope can be used to observe and inspect the cells flowing through the sample flow channel.

[0035] The target-free automatic focusing method provided by the embodiment sets a first reference plane and a second reference plane parallel to the target plane and having a known relative distance relationship in the focusing space where the target plane is located, and the first reference plane and the second reference plane are respectively provided with preset patterns for microscope focusing; the microscope is controlled to focus on the preset pattern on the first reference plane to obtain a first focusing distance corresponding to the first reference plane, and the microscope is controlled to focus on the preset pattern on the second reference plane to obtain a second focusing distance corresponding to the second reference plane; and the microscope is controlled to focus on the target plane between the first reference plane and the second reference plane according to the first focusing distance, the second focusing distance and the relative distance relationship between the planes.

[0036] Compared with the related art, the application can focus on the target plane according to the relative distance relationship between the reference plane and the target plane by setting the reference plane in the focusing space as a reference for focusing on the target plane, thereby reducing the time required for the microscope to repeatedly focus in the focusing space and improving the efficiency of cell microscopy. In addition, by actively controlling the microscope to focus on the reference plane, a high-precision focusing distance is obtained as a reference, effectively improving the focusing accuracy in the case of target-free focusing.

[0037] Reference Figure 3 As shown in FIG. 1, as an embodiment of the application, the microscope is controlled to focus on the preset pattern on the first reference plane to obtain a first focusing distance when the preset pattern on the first reference plane is focused, which includes: S110. The microscope is controlled to focus on the preset pattern on the first reference plane to obtain an initial imaging image of the first reference plane.

[0038] S120. The initial imaging image is subjected to annular difference verification to obtain a definition evaluation result of the initial imaging image.

[0039] S130. In the case where the definition evaluation result meets the preset definition requirement, the focusing distance between the microscope and the first reference plane is taken as the first focusing distance.

[0040] It should be noted that the contrast focusing method is usually used to control the microscope to focus in the related art. In the focusing process, the imaging image is subjected to contrast gradient calculation by means of Sobel or Laplacian operator or the like to determine the overall contrast gradient of the imaging image, so as to judge whether the microscope is accurately focused. When the overall contrast gradient of the imaging image reaches the maximum value, the microscope is accurately focused with the reference plane. However, in the actual situation, when the microscope approaches the actual focal point of the reference plane, the overall contrast gradient of the imaging image changes slowly, and there may be multiple pseudo-peak values, which makes it difficult to determine the maximum value of the overall contrast gradient, resulting in the back-and-forth movement of the microscope in the z-axis direction, and causing damage to the microscope and its control motor.

[0041] Based on the above factors, the present embodiment adopts the annular difference method to preform fluorescent dot embedding in the flow channel chip to form fluorescent dot array patterns at a set height above the upper surface of the sample flow channel and at the same height below the lower surface of the sample flow channel, respectively, as the preset patterns of the first reference plane and the second reference plane, respectively.

[0042] Specifically, before focusing, the fluorescent dot array pattern on the first reference plane emits point-like fluorescence by irradiating the fluorescent dot array pattern with an excitation light source. A plurality of initial imaging images containing the fluorescent dot array pattern are acquired by the microscope, and the plurality of initial imaging images are respectively subjected to annular difference verification, so as to evaluate the clarity of the initial imaging images by the response difference between the fluorescent dot array pattern and the surrounding background, and obtain the clarity evaluation results of the initial imaging images. It can be understood that the greater the response difference between the fluorescent dot array pattern and the surrounding background, the higher the clarity of the initial imaging images, and the better the focusing effect of the microscope on the first reference plane.

[0043] Further, the clarity evaluation results of all the initial imaging images are evaluated according to the preset clarity requirement, and the clarity evaluation result meeting the preset clarity requirement is determined, and the focusing distance between the microscope and the first reference plane at this time is taken as the first focusing distance. In some embodiments, the preset clarity requirement can be the maximum value in all the clarity evaluation results. After obtaining the respective clarity evaluation results of all the initial imaging images, the clarity evaluation results are sorted, and the maximum value of the clarity evaluation results is selected therefrom. According to the z-axis coordinate corresponding to the maximum value, the first focusing distance when the microscope is focused on the preset pattern on the first reference plane can be obtained.

[0044] It can be understood that the second focusing distance can be obtained in the same way.

[0045] Referring to Figure 4 As shown in FIG. 13, as an embodiment of the present application, annular difference verification is performed on the initial imaging image to obtain the clarity evaluation result of the initial imaging image, which includes: S122. According to the annular convolution kernel, the response difference of any pixel point in the initial imaging image is calculated to obtain the convolution response value of any pixel point; wherein the annular convolution kernel includes a center convolution kernel and an annular convolution kernel surrounding the center convolution kernel, and the center convolution kernel and the annular convolution kernel correspond to different weights.

[0046] S124. After traversing all the pixel points in the initial imaging image, the convolution response values of all the pixel points are subjected to response statistical processing to obtain the clarity evaluation result.

[0047] Specifically, for the initial imaging image containing the fluorescent dot array pattern, a corresponding ring-shaped convolution kernel is designed, and the ring-shaped convolution kernel is used to calculate the response difference of any pixel point in the initial imaging image, to obtain the convolution response value of the any pixel point and its surrounding pixel points.

[0048] Exemplarily, the ring-shaped convolution kernel includes a center convolution kernel and a ring convolution kernel surrounding the center convolution kernel, and the center convolution kernel and the ring convolution kernel correspond to different weights. The ring-shaped convolution kernel can refer to Figure 5a to Figure 5b As shown in the figure, the ring-shaped convolution kernel can be a Gaussian kernel with a standard deviation of 1.0, and the size of the ring-shaped convolution kernel can be determined according to the size of the fluorescent dot in the fluorescent dot array pattern. In the embodiment, the diameter of the fluorescent dot is 10 pixels, so the radius of the ring-shaped convolution kernel is 7 pixels, the inner diameter of the ring-shaped convolution kernel is 3 pixels, and the outer diameter of the ring-shaped convolution kernel is 7 pixels. The center convolution kernel corresponds to a negative weight, the inner ring of the ring convolution kernel corresponds to a negative weight, and the outer ring of the ring convolution kernel corresponds to a positive weight, and the sum of all weights is zero.

[0049] Further, in the case that the microscope is in focus with the fluorescent dot array pattern, the imaging result of the potential fluorescent dot in the fluorescent dot array pattern in the initial imaging image is as shown in the figure Figure 6a Exemplarily, the potential fluorescent dot follows a Gaussian distribution with a standard deviation of 1.5 in brightness. By performing convolution operation on any pixel point in the initial imaging image by using the ring-shaped convolution kernel, the center response of the image data of the any pixel point to the center convolution kernel and the ring-shaped response of the image data of the any pixel point to the ring convolution kernel are obtained, respectively, as shown in Figure 6b and Figure 6c According to the respective weights of the center convolution kernel and the ring convolution kernel, the center response and the ring-shaped response are weighted and fused to obtain the center-ring response difference of the any pixel point, as the convolution response value of the any pixel point, as shown in Figure 6d .

[0050] Similarly, in the case that the microscope is out of focus with the fluorescent dot array pattern, the imaging result of the potential fluorescent dot in the fluorescent dot array pattern in the initial imaging image is as shown in the figure Figure 7a Exemplarily, the potential fluorescent dot follows a Gaussian distribution with a standard deviation of 5 in brightness. By performing convolution operation on any pixel point in the initial imaging image by using the ring-shaped convolution kernel, the center response of the image data of the any pixel point to the center convolution kernel and the ring-shaped response of the image data of the any pixel point to the ring convolution kernel are obtained, respectively, as shown in Figure 7b and Figure 7c According to the respective weights of the center convolution kernel and the ring convolution kernel, the center response and the ring-shaped response are weighted and fused to obtain the center-ring response difference of the any pixel point, as the convolution response value of the any pixel point, as shown in Figure 7d .

[0051] Further, after traversing all the pixel points in the initial imaging image, the convolution response values corresponding to each pixel point in the initial imaging image are statistically processed to obtain the definition evaluation result of the initial imaging image. Exemplarily, the statistical processing can be an average calculation of the multiple response values, or an accumulation calculation of the multiple response values.

[0052] It should be noted that the annular difference method does not need to perform boundary padding on the image when evaluating the definition of the image, and the definition can be evaluated by the real pixel values of the image, which maximizes the use of the data of the image itself and effectively improves the accuracy of the definition evaluation result. In addition, the definition evaluation result obtained by the annular difference method can be intuitively understood as the brightness difference between the center region and the surrounding region of the pixel point, which is the same as the definition perception of the human eye, so that the definition evaluation result matches the observation result of the human eye, and the observation effect of the microscope imaging is improved.

[0053] Referring to Figure 8 As an embodiment of the present application, the microscope is controlled to focus image the preset pattern on the first reference plane to obtain an initial imaging image of the first reference plane, including: S112. From the initial position of the microscope, the microscope is moved step by step according to the set step size, and at each set step size position, the microscope is controlled to focus image the preset pattern on the first reference plane to obtain an initial imaging image of the first reference plane.

[0054] S114. Repeat the above step-by-step moving and focusing imaging process until the microscope reaches the maximum moving range.

[0055] Specifically, the maximum moving range of the microscope on the z-axis is set, the microscope is moved to the initial position on the z-axis, and the microscope is controlled to move along the z-axis direction according to the set step size. After each movement of the microscope, the preset pattern on the first reference plane is imaged by the microscope to obtain a first focus image. After the microscope moves to the preset maximum moving range, the movement of the microscope is stopped, and multiple initial imaging images of the first reference plane by the microscope are obtained.

[0056] Similarly, the maximum moving range of the microscope on the z-axis is set, the microscope is moved to the initial position on the z-axis, and the microscope is controlled to move along the z-axis direction according to the set step size. After each movement of the microscope, the preset pattern on the second reference plane is imaged by the microscope to obtain a second focus image. After the microscope moves to the preset maximum moving range, the movement of the microscope is stopped, and multiple initial imaging images of the second reference plane by the microscope are obtained.

[0057] It can be understood that when the initial imaging image of the first reference plane and the initial imaging image of the second reference plane are obtained, the initial position of the microscope, the maximum moving range and the set step length can be the same or different.

[0058] Referring to Figure 9 As shown in the figure, as an embodiment of the present application, according to the first focus distance and the second focus distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane, the microscope is controlled to focus on the target plane, comprising: S310. According to the first focus distance and the second focus distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane, the target focus distance is calculated, and the target focus distance when the microscope is focused on the target plane is determined.

[0059] S320. According to the target focus distance, the microscope is controlled to focus on the target plane.

[0060] Specifically, in the case where the relative distance relationship between the target plane and the first reference plane and the second reference plane is known, the target focus distance is calculated according to the first focus distance and the second focus distance, and the distance between the position when the microscope is focused on the target plane and the first focus position and the second focus position is determined, to obtain the target focus distance.

[0061] Exemplarily, for a flow channel chip, the target plane is at the center position between the first reference plane and the second reference plane, and at this time the target focus distance can be represented as: Wherein, z final is the target focus distance; z1 is the first focus distance; z2 is the second focus distance.

[0062] Referring to Figure 10 As an embodiment of the present application, the method further comprises: S410. Under the preset focus depth of field setting, the microscope is controlled to focus on the preset pattern on the first reference plane, to obtain the first focus distance.

[0063] S420. Under the preset focus depth of field setting, the microscope is controlled to focus on the preset pattern on the second reference plane, to obtain the second focus distance.

[0064] Specifically, when the microscope is controlled to focus on the preset pattern on the first reference plane, the variable aperture of the microscope is adjusted so that the microscope focuses on the preset pattern on the first reference plane with a preset focus depth setting. It should be noted that the preset focus depth setting can be a setting in which the depth of field range of the microscope is less than a preset depth threshold, and at this time the adjustable aperture size of the microscope is greater than a preset aperture threshold. Exemplarily, the relationship between the adjustable aperture size of the microscope and the depth of field range can be represented as: Wherein, ΔL1 is the front depth of field of the microscope; ΔL2 is the back depth of field of the microscope; F is the aperture value of the microscope; δ is the allowable diameter of the circle of confusion; L is the focusing distance; f is the focal length of the microscope. According to the front depth of field and the back depth of field, the depth of field range of the microscope can be obtained, which can be represented as: ΔL = ΔL1 + ΔL2 Wherein, ΔL is the depth of field range of the microscope.

[0065] It can be understood that in the case that the depth of field range of the microscope is large, the microscope can clearly image multiple planes within the depth of field range, resulting in an error between the actual first focus plane and the first reference plane when focusing on the preset pattern on the first reference plane. Similarly, when focusing on the preset pattern on the second reference plane, there is also an error between the actual second focus plane and the second reference plane.

[0066] The error between the actual focus plane and the first reference plane is denoted as a first error, and the error between the actual focus plane and the second reference plane is denoted as a second error; in the case that the first focus plane is closer to the microscope than the first reference plane, and the second focus plane is closer to the microscope than the second reference plane, or in the case that the first focus plane is farther away from the microscope than the first reference plane, and the second focus plane is farther away from the microscope than the second reference plane, the first error and the second error are same-direction errors, and at this time the error of the microscope when focusing on the first reference plane and the second reference plane is the smallest. Conversely, in the case that the first focus plane is closer to the microscope than the first reference plane, and the second focus plane is farther away from the microscope than the second reference plane, or in the case that the first focus plane is farther away from the microscope than the first reference plane, and the second focus plane is closer to the microscope than the second reference plane, the first error and the second error are opposite-direction errors, and at this time the error of the microscope when focusing on the first reference plane and the second reference plane is the largest.

[0067] It can be seen that when the microscope is focused on the first reference plane and the second reference plane, respectively, an unremovable focusing error will occur, and the size of the focusing error is related to the depth of field range of the microscope. The larger the depth of field range of the microscope, the greater the focusing error; the smaller the depth of field range, the smaller the focusing error. Therefore, when controlling the microscope to focus on the first reference plane and the second reference plane, by reducing the depth of field range of the microscope, the focusing error of the microscope can be effectively reduced, the focusing accuracy between the microscope and the first reference plane and the second reference plane can be improved, and then the focusing accuracy between the microscope and the target plane can be improved.

[0068] As an embodiment of the present application, the method further comprises: S430. Under a preset observation depth of field setting, observing the target object on the target plane to obtain a feature image of the target object; wherein the preset observation depth of field setting is different from the preset focusing depth of field setting.

[0069] Specifically, after controlling the microscope to focus on the target plane, the variable aperture of the microscope is adjusted so that the microscope observes and inspects the cell sample under test on the target plane under a preset observation depth of field setting. It should be noted that the preset focusing depth of field setting can be a setting in which the depth of field range of the microscope is greater than a preset depth threshold, and in this case, the size of the adjustable aperture of the microscope can be smaller than a preset aperture threshold.

[0070] It can be understood that in the case where the depth of field range of the microscope is large, the microscope can clearly image multiple planes within the depth of field range, so that the microscope can clearly image the cell under test at multiple levels, thereby improving the observation efficiency and inspection accuracy of the cell sample under test.

[0071] Correspondingly, please refer to Figure 11 The embodiment of the present application provides an automatic focusing device without target, which is applied to control a microscope to focus on a target plane located in a focusing space. The focusing space comprises a first reference plane and a second reference plane parallel to the target plane, and the first reference plane and the second reference plane are respectively on both sides of the target plane. The device comprises: A first focusing module 1110 is configured to control the microscope to focus on a preset pattern on the first reference plane to obtain a first focusing distance when the preset pattern on the first reference plane is focused.

[0072] A second focusing module 1120 is configured to control the microscope to focus on a preset pattern on the second reference plane to obtain a second focusing distance when the preset pattern on the second reference plane is focused.

[0073] The target focusing module 1130 is configured to control the microscope to focus on the target plane according to the first focusing distance, the second focusing distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane.

[0074] In some optional embodiments, the first focusing module 1110 includes: The initial focusing imaging unit is configured to control the microscope to perform focusing imaging on the preset pattern on the first reference plane to obtain an initial imaging image of the first reference plane.

[0075] The annular differential verification unit is configured to perform annular differential verification on the initial imaging image to obtain a definition evaluation result of the initial imaging image.

[0076] The focusing distance acquisition unit is configured to, in a case where the definition evaluation result meets a preset definition requirement, acquire the focusing distance between the microscope and the first reference plane as the first focusing distance.

[0077] In some optional embodiments, the annular differential verification unit includes: The response difference calculation subunit is configured to perform response difference calculation on any pixel point in the initial imaging image according to an annular convolution kernel to obtain a convolution response value of the any pixel point; the annular convolution kernel includes a center convolution kernel and an annular convolution kernel surrounding the center convolution kernel, and the center convolution kernel and the annular convolution kernel correspond to different weights.

[0078] The response statistical processing subunit is configured to, after traversing all the pixel points in the initial imaging image, perform response statistical processing on the convolution response values of all the pixel points respectively to obtain the definition evaluation result.

[0079] In some optional embodiments, the initial focusing imaging unit includes: The moving imaging subunit is configured to move the microscope step by step from an initial position of the microscope according to a set step size, and control the microscope to perform focusing imaging on the preset pattern on the first reference plane at each set step size position to obtain the initial imaging image of the first reference plane.

[0080] The repeated imaging subunit is configured to repeat the above-mentioned step-by-step moving and focusing imaging process until the microscope reaches a maximum moving range.

[0081] In some optional embodiments, the target focusing module 1130 includes: The focusing distance calculation unit is configured to perform focusing distance calculation on the target plane according to the first focusing distance, the second focusing distance, and the relative distance relationship between the target plane and the first reference plane and the second reference plane to determine the target focusing distance when the microscope focuses on the target plane.

[0082] A target plane focusing unit is configured to control the microscope to focus on the target plane according to the target focusing distance.

[0083] In some optional embodiments, the device further comprises a depth of field setting adjustment module, comprising: A preset first focusing unit is configured to control the microscope to focus on the preset pattern on the first reference plane under the preset focusing depth of field setting, to obtain the first focusing distance.

[0084] A preset second focusing unit is configured to control the microscope to focus on the preset pattern on the second reference plane under the preset focusing depth of field setting, to obtain the second focusing distance.

[0085] In some optional embodiments, the depth of field setting adjustment module further comprises: A preset target observation unit is configured to observe the target object on the target plane under the preset observation depth of field setting, to obtain the feature image of the target object; wherein the preset observation depth of field setting is different from the preset focusing depth of field setting.

[0086] Further function descriptions of the above-mentioned various modules and units are the same as those of the above-mentioned corresponding embodiments, and will not be described here again.

[0087] The target-free automatic focusing device in the embodiment is presented in the form of a functional unit. The unit herein refers to an ASIC (Application Specific Integrated Circuit) circuit, a processor and a memory executing one or more software or fixed programs, and / or other devices that can provide the above-mentioned functions.

[0088] Please refer to Figure 12 , Figure 12 is a structural schematic diagram of a computer device provided by the embodiment of the present application. As shown in the figure, the computer device comprises one or more processors 10, a memory 20, and an interface for connecting various components, including a high-speed interface and a low-speed interface. Various components communicate and connect with each other by using different buses, and can be installed on a common mainboard or in other ways as needed. The processor can process instructions executed in the computer device, including instructions stored in the memory or the memory to display GUI graphical information on an external input / output device (such as a display device coupled to the interface). In some optional embodiments, if necessary, multiple processors and / or multiple buses can be used with multiple memories and multiple memories. Similarly, multiple computer devices can be connected, each device providing part of the necessary operations (for example, as a server array, a group of blade servers, or a multi-processor system). Figure 12 In the figure, the processor 10 is taken as an example.

[0089] The processor 10 can be a central processing unit, a network processing unit, or a combination thereof. The processor 10 can further include a hardware chip. The hardware chip can be an application specific integrated circuit, a programmable logic device, or a combination thereof. The programmable logic device can be a complex programmable logic device, a field programmable logic device, a generic array logic, or any combination thereof.

[0090] The memory 20 stores instructions executable by the at least one processor 10 for performing the methods illustrated in the above embodiments.

[0091] The memory 20 can include a program storage area and a data storage area. The program storage area can store an operating system, application programs required by at least one function, and the like. The data storage area can store data created according to the use of the computer device, and the like. In addition, the memory 20 can include a high-speed random access memory, and can further include a non-transitory memory such as at least one of a magnetic disk storage device, a flash memory device, or other non-transitory solid state memory device. In some alternative embodiments, the memory 20 can optionally include a memory disposed remotely from the processor 10, and these remote memories can be connected to the computer device through a network. Examples of the network include, but are not limited to, the Internet, an intranet, a local area network, a mobile communication network, and a combination thereof.

[0092] The memory 20 can include a volatile memory such as a random access memory, and can also include a non-volatile memory such as a flash memory, a hard disk, or a solid state disk. The memory 20 can further include a combination of the above-mentioned types of memories.

[0093] The computer device further includes a communication interface 30 for communication of the computer device with other devices or communication networks.

[0094] The embodiments of the present application further provide a computer readable storage medium, and the method according to the embodiments of the present application can be implemented in hardware, firmware, or recorded in a storage medium, or be implemented as computer code originally stored in a remote storage medium or a non-transitory machine readable storage medium and downloaded to a local storage medium, so that the method described herein can be processed by such software on a storage medium using a general purpose computer, a special purpose processor, or programmable or special hardware. The storage medium can be a disk, an optical disk, a read-only memory, a random access memory, a flash memory, a hard disk, or a solid state disk, etc. Further, the storage medium can also include a combination of the above-mentioned memories. It can be understood that the computer, the processor, the microprocessor controller, or the programmable hardware includes a storage component that can store or receive software or computer code, when the software or computer code is accessed and executed by the computer, the processor, or the hardware, the method shown in the above embodiments is implemented.

[0095] The embodiments of the present application provide a computer program product, which includes computer instructions stored in a computer readable storage medium. A processor of a computer device reads the computer instructions from the computer readable storage medium, and the processor executes the computer instructions, so that the computer device executes the method of any of the embodiments of the present application.

[0096] Although the embodiments of the present application are described with reference to the drawings, various modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the present application, and such modifications and changes fall within the scope defined by the appended claims.

[0097] The system, device, module or unit illustrated in the above embodiments can be specifically implemented by a computer chip or entity, or by a product with certain function. A typical implementation device is a computer. Specifically, the computer can be, for example, a personal computer, a laptop computer, a cellular phone, a camera phone, a smart phone, a personal digital assistant, a media player, a navigation device, an email device, a game console, a tablet computer, a wearable device, or a combination of any of these devices.

[0098] For the convenience of description, the above device is described as various units respectively described in functions. Of course, the functions of each unit can be implemented in the same or more software and / or hardware in the implementation of the present application.

[0099] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program products. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can 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, etc.) containing computer-usable program code.

[0100] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can 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, generate instructions for implementing the flowchart... Figure One One or more processes and / or boxes Figure One A device that provides the functions specified in one or more boxes.

[0101] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure One One or more processes and / or boxes Figure One The function specified in one or more boxes.

[0102] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure One One or more processes and / or boxes Figure One The steps of the function specified in one or more boxes.

[0103] It should also be noted that the terms "comprises" and / or "comprising", or "includes" and / or "including" when used in this specification, specify the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof. In the context of the specification, the term "and / or" means "and" or "or", and the term "or" means "and" or "or". In the context of the specification, the term "exemplary" means "example" or "an example of".

[0104] Each of the embodiments in the specification is described in a progressive manner, and the same or similar parts between the embodiments can be referred to each other. Each of the embodiments focuses on the difference from other embodiments. In particular, for the system embodiments, since they are basically similar to the method embodiments, the description is relatively simple, and the relevant parts can be referred to the part of the method embodiments.

[0105] The above only describes the embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the scope of the claims of the present application.

[0106] Although the embodiments of the present application are described in conjunction with the drawings, those skilled in the art can make various modifications and changes without departing from the spirit and scope of the present application, and such modifications and changes fall within the scope defined by the appended claims.

Claims

1. A targetless auto-focusing method, characterized by, The application is applied to control a microscope to focus on a target plane located in a focusing space, the focusing space comprising a first reference plane and a second reference plane parallel to the target plane, the first reference plane and the second reference plane being respectively on two sides of the target plane; The method comprises: controlling the microscope to focus on a preset pattern on the first reference plane to obtain a first focusing distance when the microscope focuses on the preset pattern on the first reference plane; controlling the microscope to focus on a preset pattern on the second reference plane to obtain a second focusing distance when the microscope focuses on the preset pattern on the second reference plane; controlling the microscope to focus on the target plane according to the first focusing distance and the second focusing distance and the relative distance relationship between the target plane and the first reference plane and the second reference plane.

2. The method of claim 1, wherein, The control of the microscope to focus on the preset pattern on the first reference plane to obtain the first focusing distance when the microscope focuses on the preset pattern on the first reference plane comprises: controlling the microscope to focus on the preset pattern on the first reference plane to obtain an initial imaging image of the first reference plane; performing annular difference verification on the initial imaging image to obtain a definition evaluation result of the initial imaging image; in the case that the definition evaluation result meets preset definition requirements, taking the focusing distance between the microscope and the first reference plane as the first focusing distance.

3. The method of claim 2, wherein, The annular difference verification on the initial imaging image to obtain the definition evaluation result of the initial imaging image comprises: performing response difference calculation on any pixel point in the initial imaging image according to an annular convolution kernel to obtain a convolution response value of the any pixel point; wherein the annular convolution kernel comprises a center convolution kernel and an annular convolution kernel surrounding the center convolution kernel, and the center convolution kernel and the annular convolution kernel correspond to different weights; after traversing all pixel points in the initial imaging image, performing response statistical processing on the convolution response values of all pixel points respectively to obtain the definition evaluation result.

4. The method of claim 2, wherein, The control of the microscope to focus on the preset pattern on the first reference plane to obtain the initial imaging image of the first reference plane comprises: gradually moving the microscope according to a set step length from an initial position of the microscope, and controlling the microscope to focus on the preset pattern on the first reference plane to obtain the initial imaging image of the first reference plane at each set step length position; repeating the above gradual movement and focusing imaging process until the microscope reaches a maximum movement range.

5. The method of claim 1, wherein, The control of the microscope to focus on the target plane according to the first focusing distance and the second focusing distance and the relative distance relationship between the target plane and the first reference plane and the second reference plane comprises: According to the first focusing distance and the second focusing distance, and a relative distance relationship between the target plane and the first reference plane and the second reference plane, a target focusing distance when the microscope is focused on the target plane is calculated. According to the target focusing distance, the microscope is controlled to be focused on the target plane.

6. The method according to any one of claims 1 to 5, characterized in that, The method further comprises: Under a preset focusing depth of field setting, a preset pattern on the first reference plane is focused by the microscope, and the first focusing distance is obtained. Under the preset focusing depth of field setting, a preset pattern on the second reference plane is focused by the microscope, and the second focusing distance is obtained.

7. The method of claim 6, wherein, The method further comprises: Under a preset observation depth of field setting, a target object on the target plane is observed, and a feature image of the target object is obtained; wherein the preset observation depth of field setting is different from the preset focusing depth of field setting.

8. A targetless auto-focusing device, characterized by, The application is applied to control a microscope to focus on a target plane located in a focusing space, the focusing space comprising a first reference plane and a second reference plane parallel to the target plane, and the first reference plane and the second reference plane are respectively on two sides of the target plane. The device comprises: A first focusing module is configured to control the microscope to focus on a preset pattern on the first reference plane, and obtain a first focusing distance when the preset pattern on the first reference plane is focused. A second focusing module is configured to control the microscope to focus on a preset pattern on the second reference plane, and obtain a second focusing distance when the preset pattern on the second reference plane is focused. A target focusing module is configured to control the microscope to focus on the target plane according to the first focusing distance and the second focusing distance, and a relative distance relationship between the target plane and the first reference plane and the second reference plane.

9. A computer device, comprising: The memory and the processor are connected with each other in communication, and the memory stores computer instructions, and the processor executes the computer instructions to perform the method in any one of claims 1 to 7. The computer readable storage medium stores computer instructions, and the computer instructions are used to make a computer execute the method in any one of claims 1 to 7.

10. A computer-readable storage medium, characterized in that, ​

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