CN114578537A - Microscopic scanning platform focal plane determination method based on regional multipoint plane determination method - Google Patents
Microscopic scanning platform focal plane determination method based on regional multipoint plane determination method Download PDFInfo
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Abstract
The invention discloses a microscopic scanning platform focal plane determination method based on a regional multipoint fixed surface method, which combines a focal plane equation to control an objective table to vertically move so as to quickly realize the field of view positioning focusing of an objective lens, avoids 'same slide multi-field repeated focusing', and calculates a Z coordinate value by substituting an x coordinate value and a y coordinate value of a point into a focal plane equation model of the region according to which region the collected field of view is positioned, so that a clear slide image can be observed in a lens by only adjusting the Z coordinate value from the Z axis direction at the point, the working efficiency is improved, the working difficulty and the intensity of technical personnel are reduced, the efficiency and the precision are ensured, the problems of poor accuracy and low imaging quality caused by single focusing of a slide are solved, and the problems of time consumption, low power and low efficiency caused by focusing of each field of view on the slide are solved, is a method for quickly positioning and focusing when automatically scanning a tissue pathological slide.
Description
Technical Field
The invention relates to the technical field of medical information acquisition and detection and clinical medicine, in particular to a method for determining a focal plane of a micro-scanning platform based on a regional multipoint fixed-plane method.
Background
With the development of science and technology, the traditional microscope has complicated operation and low efficiency, and is gradually replaced by a microscopic vision automatic scanning platform. Along with the gradual maturity of digital image processing technology, the microscopic vision automatic scanning platform has greatly promoted efficiency and pathological judgment accuracy compared with manual microscopy. Compared with the traditional microscope, the microscopic automatic scanning platform has the characteristics of convenience, accuracy and high efficiency, and is widely applied in various aspects, for example, due to the rapid development of optical imaging, the result can be obtained through microscopic imaging in histopathological examination, and therefore the diagnosis accuracy is improved. Furthermore, by means of medical histopathological examination, the cancer diagnosis, disease condition determination, tumor screening and prognosis treatment all play important roles.
When the tissue pathology slide is scanned and focused, the ideal state is that the slide plane is coincident with the focal plane, if the objective lens is regarded as a mass point, the slide is placed in a completely horizontal state, and any point on the slide plane observed by the objective lens is clearly focused. However, no matter the traditional microscope examination or the microscope vision automatic scanning platform is used for scanning, the ideal state cannot be achieved, and the focusing problem exists, namely, the plane where the slide is located and the reference plane of the lens have a certain inclination degree, so that the plane where the slide is located and the focal plane are not coincident. The reasons for this are due to errors in the mechanical structure during processing and assembly, and the fact that the slide is inclined in its own plane after the slide is produced. Therefore, when only one point on the slide is focused, the imaging blur in the field of view may occur in moving-field observation due to the unevenness of the surface caused by the slide manufacturing process or an error caused by the mounting process.
In the microscopic scanning process, in order to ensure that the clearest image is observed in the field of view, a common method is to perform refocusing every time the lens moves to a new observation field point. When only one visual field point is focused in a common focusing method, the focusing accuracy and the imaging quality are poor, and the influence of the position relation between a slide plane and a focal plane is large; if each view point is focused, the focusing times are too many, and the process is time-consuming and inefficient.
Disclosure of Invention
Aiming at the technical problems, the invention provides a microscopic scanning platform focal plane determining method based on a region multipoint plane fixing method, which is characterized in that the flatness of a slide is classified according to the manufacturing process of the slide, a plurality of regions are divided according to the flatness of the slide and the number of points needing to be focused is determined according to the needs, and further a focal plane equation in each grid region is determined, so that the complete focal plane distribution of the slide is obtained, the efficiency and the accuracy are considered, and the imaging quality is guaranteed to be clear and readable.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a microscopic scanning platform focal plane determination method based on a regional multipoint surface determination method, which comprises the following steps:
s1: firstly, judging the type of a slide, and determining a strategy by applying different focal planes according to different types;
s2: performing grid division by taking the whole cover glass as a complete area, and determining a focal plane one by one according to the divided grid area, wherein each grid is provided with at least three marking points so as to determine the number of the marking points of the whole glass;
s3: establishing a space rectangular coordinate system for a Z axis upwards perpendicular to the plane of the slide by taking the upper left corner of the slide as a coordinate origin O, the long side of the slide as an X axis, the short side of the slide as a Y axis and the long side of the slide as a Z axis;
s4: performing focusing operation by using a reciprocating focusing method at each mark point to obtain a vertical projection of the point on a focal plane thereof as (x)i，yi，zi)；
S5: in each grid area, vertical projection points (x) of three Mark points in each grid area on a focal planei，yi，zi) Simultaneously establishing an equation set to obtain a focal plane equation of the grid region i;
s6: and selecting any position point, judging which area the position point is located in, substituting the x and y coordinate values of the position point into the focal plane equation of the area to solve, and obtaining the coordinate of the point on the focal plane, namely only moving the point to adjust to the z coordinate value of the required coordinate.
Further, the slide types in step S1 include a slice-like slide and a smear-like slide.
Further, in step S2, for the slide of slice type, three defined mark points in the four top corners of the cover glass are directly selected, i.e. one divided grid region is selected.
Further, in step S2, for smear-like slides, the entire coverslip where the sample smear region is located is divided into grids as a complete region, and focal planes are determined one by one according to the divided grid regions, where each grid has at least three marker points, so as to determine the number of marker points of the entire slide.
Further, in the division of the grid, the long side and the short side of the slide glass are equally divided, respectively.
Further, the vertex of each grid is taken as a mark point.
Further, after dividing the slide into a grid with a grid number of m × n, at least (m +1) · (n +1) - (2| m +1) · (2| n +1) · marker points are required for the slide, wherein "|" is an integer division symbol, and the algorithm divides the former number by the latter number to get a quotient, and at most (m +1) · (n +1) · marker points are required.
Further, the reciprocal focusing method in step S4 is specifically: firstly, completing a focusing operation from top to bottom, and obtaining the clearest image according to an image evaluation function so as to obtain the vertical height of a lens from a focal plane of a slide during focusing; then completing the focusing operation again from bottom to top to obtain the vertical height of the lens from the focal plane of the slide during the focusing; comparing the vertical height obtained in the first focusing, if the front-back difference is smaller than the depth of field of the lens, then taking the average value of the height of the focal plane from the vertical height of the lens to the vertical height of the lens, if the front-back difference is larger than the depth of field of the lens, then carrying out the top-down focusing operation on the slide again, comparing the heights obtained by the three focusing operations, if the front-back difference is smaller than the depth of field of the lens, then taking the average value of the three, and if the difference is still larger, repeating the above operations, thus obtaining the optimal vertical height.
Further, when each mark point is focused back and forth, a frame extraction evaluation focusing method is adopted.
Further, the frame extraction evaluation focusing method specifically comprises the following steps: when aiming at a visual field point to be focused, the point is subjected to stepping shooting, the frame rate is synchronized, a video file is stored, video frame extraction is carried out on the video file to analyze each frame image, a function value of each frame image is calculated by applying an evaluation function, an image corresponding to a peak value of the function is obtained and is the clearest frame image, and an index corresponding to the peak value corresponds to the position of stepping shooting, namely the position of a focal plane.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a microscopic scanning platform focal plane determining method based on a regional multipoint fixed surface method, which avoids multi-view multi-focusing operation when a same slide is subjected to microscopic examination of a plurality of views, can effectively improve the efficiency of an automatic scanning process, and reduces the difficulty in realizing control of automatic focusing, is a method for quickly positioning and focusing when a tissue pathological slide is subjected to microscopic automatic scanning, provides a new thought for the microscopic automatic focusing method of the tissue pathological slide, and has wide application prospects in the microscopic visual automatic detection industry of medical pathological slides. Its main advantage is as follows:
(1) the working difficulty and the intensity of technicians are reduced, and the efficiency and the precision are ensured;
(2) the objective lens visual field positioning focusing is quickly realized by combining the 'focal plane equation' to control the objective table to vertically move, the 'same slide multi-visual field repeated focusing' is avoided, and according to which region the collected visual field point is located, the x and y coordinate values of the point are substituted into the focal plane equation model of the region to be calculated to obtain the Z coordinate value, so that a clear slide image can be observed through the lens by only adjusting the Z axis direction to the Z coordinate value at the point, and the working efficiency is improved;
(3) the problems of poor accuracy and low imaging quality caused by single focusing of the slide are solved, and the problems of time consumption, labor consumption and low efficiency caused by focusing of each visual field point on the slide are solved.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
Fig. 1 is a diagram illustrating a non-overlapping correspondence relationship between a grid area and a focal plane area of a glass plane in an actual test according to an embodiment of the present invention.
Fig. 2 is a schematic flow chart of a method for determining a focal plane of a micro-scanning platform based on a region multi-point surface fixing method according to an embodiment of the present invention.
Fig. 3 is a diagram of a process of frame extraction evaluation focusing of a slide designated Mark point provided by an embodiment of the invention.
Fig. 4 is a diagram illustrating a method for determining a focal plane based on three Mark points, which is provided by an embodiment of the present invention and is exemplified by a slide-type slide.
Fig. 5 is a diagram illustrating a multi-Mark point-based focal plane determination method, which is provided by the embodiment of the invention and takes a smear-like glass as an example.
Fig. 6 is a schematic flow chart of a method for determining a focal plane of a micro-scanning platform based on a region multi-point surface fixing method according to an embodiment of the present invention.
Fig. 7 is a guidance diagram of the relationship between the number of the glass slide area division grids and the number of Mark points according to the embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. The embodiments of the present invention, and all other embodiments obtained by those skilled in the art without any inventive step, belong to the protection scope of the present invention.
When the automatic focusing is carried out on a slide sample detected on a microscopic scanning platform, the position of a focal plane needs to be determined so as to adjust the slide plane to be coincident with the focal plane as much as possible, and the position of the focal plane must be determined firstly. However, the types of slides are various, and the slides can be divided into slice-type slides and smear-type slides. Due to the differences in the manufacturing process, the surface flatness of the sections is better than that of the smears. The three points which are not collinear can determine a plane, namely the focal plane of the section can be determined by selecting three points of four top angles of the cover glass as marking points and establishing a space coordinate system by taking the plane of the glass slide as a reference surface. When a smear is produced, a sample is unevenly distributed on a slide, so that the difference between a focal plane determined by 3 marking points and the actual focal plane of the slide is large, and at the moment, when one large plane cannot meet the actual requirement, the large plane is divided into a plurality of small planes. The focal plane of each small plane area is determined by at least three marking points, and the focal plane in the corresponding small plane area can be obtained by solving the corresponding plane equation in the small plane area. As shown in fig. 1, the I, II, III, IV regions are small grid regions divided on the slide plane, and the I ', II', III ', IV' small plane regions are focal planes corresponding to the I, II, III, IV small grid regions. When the method is used for automatic focusing of a microscopic scanning platform, the multipoint method is an effective method for determining the designated focal plane, and the designated focal plane in the corresponding area can be quickly determined.
Based on the above analysis, the present invention provides a method for determining a focal plane of a micro-scanning platform based on a region multi-point surface determination method, as shown in fig. 2, which specifically includes the following steps:
the method comprises the following steps: firstly, judging the type of the slide, and grading the surface flatness of the slide according to the characteristics of the slides of the slices and the smears, wherein the surface flatness of the slices is better than that of the smears, so that different focal planes are applied to determine strategies.
Step two: for the glass slide with better surface evenness, such as the smoother glass slide in the slice type glass slide, Mark points can be directly selected and marked by three definitions in four vertex angles of the cover glass, namely one divided grid area is selected; for a glass slide with slightly poor surface flatness, such as a smear type glass slide, the whole cover glass where a sample smear region is located is divided into grids as a complete region, then a focal plane is determined one by one according to the divided grid regions, and each grid has at least three Mark points, so that the number of the Mark points of the whole glass slide is determined.
Step three: no matter which kind of slide, the upper left corner of the slide is taken as the origin of coordinates O, the long side of the slide is taken as the X axis, the short side of the slide is taken as the Y axis, and the vertical slide plane is upwards taken as the Z axis to establish a rectangular space coordinate system.
Step four: performing focusing operation by using a reciprocating focusing method at each Mark point to obtain a vertical projection of the point on a focal plane of the point as (x)i，yi，zi)。
Step five: in each grid area, vertical projection points (x) of three Mark points in each grid area on a focal planei，yi，zi) And simultaneously establishing an equation set to obtain a focal plane equation of the grid region i.
Step six: and selecting any position point, judging which area the position point is located in, substituting the x and y coordinate values of the position point into the focal plane equation of the area to solve, and obtaining the coordinate of the point on the focal plane, namely only moving the point to adjust to the z coordinate value of the required coordinate.
The method carries out focusing operation on a plurality of Mark points specified on the glass slide to obtain the vertical projection of the Mark points on the focal plane of the glass slide. In the focusing process, a 'reciprocating focusing' scheme is adopted, namely, a focusing operation is finished from top to bottom, and the clearest image is obtained according to an image evaluation function, so that the vertical height of the lens from the focal plane of the slide is obtained during the focusing; then completing another focusing operation from bottom to top to obtain the vertical height of the lens from the focal plane of the slide during the focusing; comparing the height difference with the vertical height obtained in the first focusing, if the height difference between the two heights is not very large (the front-back difference is smaller than the depth of field of the lens), then taking the average value of the height between the focal plane and the vertical height of the lens, if the difference between the two heights is larger (the front-back difference is larger than the depth of field of the lens), then carrying out the top-down focusing operation on the slide again, comparing the heights obtained by the three focusing operations, if the front-back difference is smaller than the depth of field of the lens, then taking the average value of the three, and if the difference is still larger, repeating the above operations, thereby obtaining the optimal vertical height.
When each Mark point is focused in a reciprocating way, a frame extraction evaluation focusing method is adopted, the evaluation focusing is a focusing process of rapidly storing a video at a high frame rate and performing frame extraction evaluation on the video to find out an evaluation function extreme value when an image evaluation function is applied to step shooting of a slide sample image, the core idea of the method is that when a visual field point to be focused is aimed at, a video file is synchronously stored at the frame rate when the point is shot in a stepping way, video extraction is performed on the video file to analyze each frame image, the evaluation function is applied to calculate a function value of each frame image, an image corresponding to the peak value of the function is obtained and is the clearest frame image, an index corresponding to the peak value corresponds to the position of step shooting, namely the position of a focal plane, and the flow is roughly as shown in figure 3.
Since the surface of the slice is more planar than the surface of the smear, the focal plane of the slice will be more readily available than the focal plane of the smear. If the section type slide is taken as an example, three of the four top corners of the cover glass are respectively selected as Mark points, as shown in fig. 4. And establishing a spatial rectangular coordinate system as shown in the figure by taking the upper left corner of the slide as a coordinate origin O, the long side of the slide as an X axis, the short side of the slide as a Y axis and the Z axis in the vertical direction. Performing focusing operation by using a reciprocating focusing method at the Mark point 1 to obtain a vertical projection of the point on a focal plane of the point as (x)1，y1，z1) The vertical projection of the Mark point 2 on the focal plane can be obtained as (x) in the same way2，y2，z2) The vertical projection of Mark point 3 on its focal plane is (x)3，y3，z3). The focal plane equation of the slide with one plane determined by three points is as follows:
aX+bY+cZ+d＝0 (c≠0)
wherein a, b, c and d all relate to xi，yi，zi(i ═ 1, 2, 3) is constant. Thus, at any position on the slide, i.e. at the X-coordinate value X 'and the Y-coordinate value Y', only the Z-coordinate has to be moved to:
z′＝a′x′+b′y′+d′
where a ', a/c, b', b/c, d ', d/c, the sharpest definition of the field of view at this point can be achieved, i.e. the coordinates of this point in the focal plane are (x', y ', z').
Taking the smear as an example, since the distribution of the sample on the slide is not uniform during the smear production, the focal plane determined by the 3 marker points is greatly different from the focal plane of the slide, and thus the focal plane is determined as follows. The whole cover glass in which the sample smear region is located is divided into complete regions by grids, and as shown in fig. 5, the regions are equally divided into 4 regions: region I, region II, region III, and region IV. As a plane is determined by the three non-collinear points, each grid needs 3 marking points to carry out focusing operation so as to accurately obtain the position of the focal plane of the slide. Therefore, 5 mark points are selected as shown in fig. 5, and the 5 mark points are focused by a reciprocal focusing method, so as to obtain a vertical projection of each point on the focal plane. According to the principle of determining a focal plane by slicing a slide, the upper left corner of the slide is taken as an origin of coordinates O, the long side of the slide is taken as an X axis, the short side of the slide is taken as a Y axis, a rectangular spatial coordinate system as shown in the figure is established for the Z axis which is vertical to the plane of the slide, in an area I, an equation set is established for coordinates of vertical projection points of a Mark point 1, a Mark point 2 and a Mark point 4 on the focal plane respectively in a simultaneous mode, so that a focal plane equation of the area I can be obtained, namely when a visual field is obtained at any position point in the area I, the position point X and Y coordinates are substituted into the focal plane equation set of the area I to obtain a Z value, and then the clearest image of the point can be obtained only by adjusting the position to the Z value at the point. Focal planes of the area II, the area III and the area IV are determined to be obtained in the same manner, and only the z coordinate value needs to be adjusted in the same manner when the visual fields are acquired at any positions of the area II, the area III and the area IV. In short, in the smear glass slide, the focal plane is determined region by region according to the number of divided regions, and the z coordinate value to be adjusted can be obtained by acquiring the visual field at any position of the smear glass slide only by judging which region the position point is located in first and substituting the position point into the focal plane equation of the region to solve.
In the smear-like slide, a grid can be made according to the desired field of view, as shown in fig. 6. The number of the divided grids determines the number of the required marking points. When the grids are divided, the long sides and the short sides of the glass slide are generally equal respectively, so that the accuracy of the focal plane obtained by each grid in the direction of the glass slide is realized.
And dividing the area of the slide needing microscopic examination into grids, wherein the vertex of each grid can be used as a marking point. For a slide with a grid number of 1, there are 4 markers, and a focusing operation needs to be performed on at least 3 markers to obtain the focal plane of the entire slide, as shown in fig. 7. The larger the number of grids, the more accurate the determined focal plane. Similarly, after dividing the glass slide into grids with the grid number of m × n, at least (m +1) · (n +1) - (2| m +1) · (2| n +1) · (where "|" is an integer division symbol, and the algorithm is to divide the former number by the latter number and then take the quotient), and at most (m +1) · (n +1) · mark points are needed to perform the focusing operation to obtain the accurate focal plane.
The method for determining the focal plane of the microscopic scanning platform based on the regional multipoint surface fixing method adopts a strategy of determining the focal plane by multiple points in a divided region, performs grid division on a slide sample region to define marking points, constructs a coordinate system based on a slide plane as a reference surface to solve a corresponding focal plane equation in each grid region, provides a new idea for the method for automatically focusing the microscope on the tissue pathological slide, avoids the operation of multi-view multi-focusing when performing microscopic examination on the same slide, can effectively improve the efficiency of an automatic scanning process, reduces the difficulty in realizing the control of the automatic focusing, and has wide application prospect in the field of automatically detecting the microscopic vision of the medical pathological slide.
Through practical application, the results show that: when the focal plane determining method based on the regional multipoint fixed surface is oriented to the automatic focusing requirement of the automatically scanned medical pathological slide, the image focusing rate is high, the rapid focusing and image acquisition requirement of the microscopic visual inspection of the medical pathological slide sample can be met, and the use scene of the microscopic automatic focusing of most medical pathological slides can also be met.
The above-mentioned embodiments are only specific embodiments of the present application, and are used for illustrating the technical solutions of the present application, but not limiting the same, and the scope of the present application is not limited thereto, and although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: those skilled in the art can still make modifications or easily conceive of changes to the technical solutions described in the foregoing embodiments, or make equivalents to some of them, within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the present disclosure, which should be construed in light of the above teachings. Are intended to be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (10)
1. A microscopic scanning platform focal plane determining method based on a region multipoint surface fixing method is characterized by comprising the following steps:
s1: firstly, judging the type of a slide, and determining a strategy by applying different focal planes according to different types;
s2: performing grid division by taking the whole cover glass as a complete area, and determining a focal plane one by one according to the divided grid area, wherein each grid is provided with at least three marking points so as to determine the number of the marking points of the whole glass;
s3: establishing a space rectangular coordinate system for a Z axis upwards perpendicular to the plane of the slide by taking the upper left corner of the slide as a coordinate origin O, the long side of the slide as an X axis, the short side of the slide as a Y axis and the long side of the slide as a Z axis;
s4: performing focusing operation by using a reciprocating focusing method at each mark point to obtain a vertical projection of the point on a focal plane thereof as (x)i，yi，zi)；
S5: in each grid area, vertical projection points (x) of three Mark points in each grid area on a focal planei，yi，zi) Simultaneously establishing an equation set to obtain a focal plane equation of the grid region i;
s6: and selecting any position point, judging which area the position point is located in, substituting the x and y coordinate values of the position point into the focal plane equation of the area to solve, and obtaining the coordinate of the point on the focal plane, namely only moving the point to adjust to the z coordinate value of the required coordinate.
2. The method for determining the focal plane of a micro-scanning platform based on the area multi-spot faceting method of claim 1, wherein the slide types in step S1 include slice-like slides and smear-like slides.
3. The method for determining the focal plane of the micro-scanning platform based on the area multi-point surface method as claimed in claim 2, wherein in step S2, for the slide of slice type, three defined marking points at four top corners of the cover glass are directly selected, i.e. one divided grid area is selected.
4. The method for determining the focal plane of the micro-scanning platform based on the area multi-spot mapping method as claimed in claim 2, wherein in step S2, the smear-like slide is divided into grids by using the whole coverslip where the smear area of the sample is located as the whole area, and the focal plane is determined one by one according to the divided grid areas, wherein each grid has at least three marker points, so as to determine the number of marker points of the whole slide.
5. The method for determining the focal plane of the micro-scanning platform based on the area multi-point surface positioning method as claimed in claim 4, wherein the long side and the short side of the slide are equally divided when the grid is divided.
6. The method for determining the focal plane of the micro-scanning platform based on the area multi-point surface positioning method as claimed in claim 4, wherein the vertex of each grid is used as a mark point.
7. The method for determining the focal plane of a micro-scanning platform based on the area multi-point surface definition method according to claim 4, wherein after dividing the slide into grids with the grid number of m × n, at least (m +1) · (n +1) - (2| m +1) · (2| n +1) · (m +1) · marker points are needed for the slide, wherein "|" is an integer division symbol, and the algorithm is that the latter integer divides the former integer and then the quotient is taken, and at most (m +1) · (n +1) ·.
8. The method for determining the focal plane of the micro-scanning platform based on the area multi-point surface positioning method according to claim 1, wherein the reciprocating focusing method in the step S4 specifically comprises the following steps: firstly, completing a focusing operation from top to bottom, and obtaining the clearest image according to an image evaluation function so as to obtain the vertical height of a lens from a focal plane of a slide during focusing; then completing the focusing operation again from bottom to top to obtain the vertical height of the lens from the focal plane of the slide during the focusing; comparing the vertical height obtained in the first focusing, if the front-back difference is smaller than the depth of field of the lens, then taking the average value of the height of the focal plane from the vertical height of the lens to the vertical height of the lens, if the front-back difference is larger than the depth of field of the lens, then carrying out the top-down focusing operation on the slide again, comparing the heights obtained by the three focusing operations, if the front-back difference is smaller than the depth of field of the lens, then taking the average value of the three, and if the difference is still larger, repeating the above operations, thus obtaining the optimal vertical height.
9. The method for determining the focal plane of the micro-scanning platform based on the area multi-point surface method according to claim 8, wherein a frame extraction evaluation focusing method is adopted when each marking point is focused back and forth.
10. The microscopic scanning platform focal plane determination method based on the area multi-point surface determination method according to claim 9, characterized in that the frame extraction evaluation focusing method specifically comprises: when aiming at a visual field point to be focused, the point is subjected to stepping shooting, the frame rate is synchronized, a video file is stored, video frame extraction is carried out on the video file to analyze each frame image, a function value of each frame image is calculated by applying an evaluation function, an image corresponding to a peak value of the function is obtained and is the clearest frame image, and an index corresponding to the peak value corresponds to the position of stepping shooting, namely the position of a focal plane.
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