CN109085695B - Method for quickly focusing and photographing plane sample - Google Patents
Method for quickly focusing and photographing plane sample Download PDFInfo
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- CN109085695B CN109085695B CN201810898671.2A CN201810898671A CN109085695B CN 109085695 B CN109085695 B CN 109085695B CN 201810898671 A CN201810898671 A CN 201810898671A CN 109085695 B CN109085695 B CN 109085695B
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Abstract
The invention discloses a method for quickly focusing and photographing a planar sample, which belongs to the technical field of sample imaging, and is characterized in that a photographing plane to be focused of the planar sample is equally divided into a plurality of unit areas, preferably three discrete points, the focusing distance of each discrete point is respectively calculated, then the focusing distance of each unit area is calculated by an interpolation method, and then the focusing distance of each unit area is combined to control a lens to move quickly, so that the quick focusing of the lens and the sample area is effectively realized, and the accurate photographing of each unit area can be completed without adjusting the focal length of the lens every time. The method for rapidly focusing and photographing the plane sample has the advantages of effectively reducing the focusing time of the lens and the sample area, improving the focusing and photographing efficiency of the plane sample, ensuring the focusing and photographing definition of the plane sample, reducing the focusing and photographing cost of the sample, along with simple steps and convenient operation, and has good popularization and application values.
Description
Technical Field
The invention belongs to the technical field of sample imaging, and particularly relates to a method for quickly focusing and photographing a plane sample.
Background
In the field of biological medicine, various samples are often required to be subjected to micro-scanning imaging, such as unicellular organisms, small algae, bacteria culture solution, loose tissues of animals and plants, spermary, anthers, blood, pus, skin tissue fluid, pharyngeal wall and the like; various samples are usually fixed on a medical glass slide firstly, then the glass slide is correspondingly placed and fixed on an object stage of a microscopic scanning imaging device, the object stage moves along the directions of an x axis and a y axis on a scanning imaging plane, so that the samples sequentially pass through a focusing plane of a lens, each point on the samples is imaged through the lens respectively, and then the images of each point are spliced to form the image of the whole medical sample.
At present, when a microscopic scanning imaging device is used to scan a sample, a sample to be scanned on a glass slide is often divided into a plurality of equally divided areas, for example, m × n areas shown in fig. 5, that is, the length direction and the width direction of the sample scanning plane are equally divided into n equal parts and m equal parts respectively, so as to form m × n scanning areas with the same size, and then each scanning area is photographed, and finally, the photographing results of the scanning areas are sequentially spliced to form a final scanning image of the sample.
In the prior art, in order to ensure that a scanned imaging picture can meet the requirements of software identification, the definition of the scanned imaging picture is often required to be high, the scanning time of a sample cannot be too long, and the photographing of the whole sample plane must be completed within a certain specified time. However, when the existing microscopic scanning imaging method is used to realize high-definition imaging of a sample, the lens of the device is often required to automatically and accurately focus each area divided by a sample plane, and then corresponding photographing of each area is completed, but the automatic and accurate focusing of the lens on each area at present often requires at least 3 seconds, and m × n areas are often not less than 800, that is, the time for focusing the sample plane is not less than 40 minutes, which causes the sample scanning time to be too long, and even if the lens works for 8 hours a day, only about 12 samples can be scanned and imaged, which is lower than the efficiency of manual operation (about 25 samples/day), greatly restricts the efficiency of scanning and imaging the sample, improves the scanning cost of high-definition scanning of the sample, restricts the rapid scanning and imaging of the sample, and cannot fully meet the requirements of rapid and accurate imaging of the sample, has great application limitation.
Disclosure of Invention
In view of the above-mentioned drawbacks and needs of the prior art, the present invention provides a method for fast focus photographing of a planar sample, the method comprises the steps of equally dividing a photographing plane to be focused of a plane sample into a plurality of unit areas, preferably selecting three discrete points on the photographing plane to be focused, then accurately calculating the focus distance of each discrete point, calculating the focus distance of each unit region by interpolation, and then controlling the lens to move rapidly by combining the focus distances of each unit region, thereby effectively realizing rapid focusing of the lens and the sample region, therefore, the accurate photographing of each unit area can be completed without adjusting the focal length of the lens every time, the focusing speed in the imaging process of the plane sample is greatly improved, the time for scanning and imaging the sample is shortened, the efficiency and the accuracy for scanning and imaging the sample are improved, and the requirements for quick and accurate imaging of the plane sample are fully met.
In order to achieve the above object, the present invention provides a method for fast focusing photographing of a planar sample, comprising the following steps:
s1: dividing a photographing region to be focused of the planar sample into n equal parts and m equal parts along the length direction and the width direction respectively to form m multiplied by n unit regions;
s2: a, B, C three discrete points are selected in the photographed area to be focused, namely a plane formed by A, B, C three discrete points is a sample plane where the plane sample is located;
s3: keeping the focal length of the lens unchanged, respectively adjusting the distance between the lens and the three discrete points, and sequentially realizing the focusing of the lens on each discrete point to obtain the focusing distance of each discrete point;
s4: calculating the focus distance of each unit area by adopting an interpolation method according to the focus distance of each discrete point;
s5: and sequentially finishing focusing and photographing of each unit area according to the focusing distance of each unit area, thereby finishing quick focusing and photographing of the plane sample.
As a further improvement of the present invention, step S4 further includes a step of correcting the focal distance of each of the unit regions, which includes the following steps:
and through comparing definition values of the two superposed areas adjacent to the unit areas, when the difference between the definition values is greater than a set threshold value, the lens refocuses and photographs the unit area with a low definition value by adopting the focusing distance of the unit area with a high definition value.
As a further refinement of the invention, the focal distance of each of said discrete points is determined by:
s31: controlling the lens to align with any discrete point, wherein the distance between the lens and any discrete point is H1And calculating to obtain the definition value f at the moment according to the definition calculation function1；
S32: controlling the lens to move a distance h along the axial direction relative to the discrete point, and calculating to obtain a definition value f at the moment2Comparison of f2And f1Magnitude of value, when f2Greater than f1If so, indicating that the moving direction of the lens is the focusing direction, and otherwise, indicating that the opposite direction of the moving direction of the lens is the focusing direction;
s33: controlling the lens to focus along the same sequenceMoving the direction for i times by a distance h, wherein i is an integer and is not less than 2, and respectively calculating to obtain a definition value after each movement until the definition value of the ith time is less than the definition value of the (i-1) th time, namely fiIs less than fi-1Completing the rough focusing of the lens;
s34: controlling the lens to move for j times by a distance l along the direction opposite to the focusing direction, j being an integer not less than 2 and l being not less than 0.001h and not more than 0.1h, respectively calculating to obtain a definition value after each movement until the definition value of the j time is less than that of the j-1 time, namely fjIs less than fj-1And then the lens stops moving after retreating by a distance l to finish the fine focusing of the lens, namely the distance between the lens and the corresponding discrete point is the focusing distance of the discrete point.
As a further improvement of the invention, the interpolation method is one or more of a linear interpolation method, a parabolic interpolation method and a spline interpolation method.
As a further improvement of the present invention, the cell region is a square region.
As a further improvement of the present invention, the step of sequentially focusing and photographing each of the unit regions in step S5 is as follows:
and controlling the lens to move from one end to the other end along the length direction of the plane sample to finish focusing and photographing of the unit areas in one row, then controlling the lens to move to an adjacent row along the width direction, and finishing focusing and photographing of each unit area in the row in sequence along the length direction of the plane sample, and so on, and finishing focusing and imaging of m multiplied by n unit areas in sequence.
As a further improvement of the present invention, the step of sequentially focusing and photographing each of the unit regions in step S5 is as follows:
and controlling the lens to move from one end to the other end along the width direction of the plane sample to finish focusing and photographing of the unit areas in one row, then controlling the lens to move to an adjacent row along the length direction, and finishing focusing and photographing of the unit areas in the row in sequence along the width direction of the plane sample, and so on, and finishing focusing and imaging of the m multiplied by n unit areas in sequence.
As a further refinement of the invention, the value of m is equal to the value of n, and the cell area is square.
As a further development of the invention, h is equal to 100. mu.m.
As a further development of the invention, l is equal to 1 μm.
Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
(1) the method for quickly focusing and photographing the plane sample comprises the steps of equally dividing a to-be-focused photographing plane of the plane sample into a plurality of unit areas, preferably selecting three discrete points on the to-be-focused photographing plane, further accurately calculating the focusing distance of each discrete point, calculating the focusing distance of each unit area by an interpolation method, and then controlling the lens to move quickly by combining the focusing distances of each unit area, so that the quick focusing of the lens and the sample area is effectively realized, the accurate photographing of each unit area can be completed without adjusting the focal length of the lens every time, the focusing speed in the imaging process of the plane sample is greatly improved, the time for scanning and imaging the sample is shortened, the efficiency and the accuracy for scanning and imaging the sample are improved, and the requirements for quick and accurate imaging of the plane sample are fully met;
(2) according to the method for rapidly focusing and photographing the plane sample, accurate acquisition of the focusing distance of each discrete point is correspondingly completed through the step of testing the focusing distance, the accuracy of focusing distance testing can be effectively guaranteed through the matching adjustment of rough focusing and fine focusing, and the efficiency of acquiring the focusing distance and the focusing and photographing definition are improved;
(3) according to the method for quickly focusing and photographing the plane sample, the trimming step of correspondingly setting the focusing distance is adopted, so that the focusing distance error caused when the flatness of two adjacent unit areas has a certain difference is effectively avoided, and the focusing and photographing definition of each position of the plane sample is fully ensured;
(4) according to the method for rapidly focusing and photographing the plane sample, the focusing distance of each discrete point is calculated, the interpolation method is used for calculating the focusing distance of each unit area, the focusing distance is determined conveniently and reliably, the focusing and photographing efficiency of the plane sample can be further improved, and the focusing and photographing time of the plane sample is shortened;
(5) the method for quickly focusing and photographing the plane sample has simple steps and simple and convenient operation, and can realize quick focusing of each unit area of the plane sample through quick movement of the lens, thereby realizing focusing and photographing of the corresponding area of the plane sample without adjusting the focal length of the lens every time, effectively reducing the focusing time of the lens, improving the focusing and photographing efficiency of the plane sample, ensuring the focusing and photographing definition of the plane sample, reducing the focusing and photographing cost of the sample, and having better popularization and application values.
Drawings
FIG. 1 is a schematic diagram illustrating steps of a method for fast focusing photographing of a planar sample according to an embodiment of the present invention;
FIG. 2 is a schematic diagram illustrating steps of a discrete point focusing method in the method for rapid focusing photographing of a planar sample according to an embodiment of the present invention;
FIG. 3 is a schematic diagram illustrating the location of discrete point A, B, C in an embodiment of the present invention;
FIG. 4 is a schematic diagram of the position of the lens and the sample plane of the micro-scanning imaging device in the embodiment of the invention;
FIG. 5 is a schematic diagram of dividing and photographing routes of a to-be-focused photographing plane of a planar sample according to an embodiment of the present invention;
in all the figures, the same reference numerals denote the same features, in particular: m. the number of equal divisions of the sample plane in the width direction; n. the number of sample planes equally divided along the length direction; a. a sample plane; b. a lens; H. the distance of the lens from the sample plane; A. discrete points; B. discrete points; C. discrete points.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The steps of the method for fast focusing photographing of a planar sample in the preferred embodiment of the present invention are shown in fig. 1 and fig. 2, and the fast focusing photographing of each photographing region to be focused of the sample is completed by a microscopic imaging device; specifically, in the preferred embodiment, the fast focus photographing of the plane sample on the sample plane a by the lens b is completed by the following steps:
s1: as shown in fig. 5, the photographed area to be focused on the planar sample is divided into n equal parts and m equal parts along the length direction and the width direction, respectively, so as to form m × n identical unit areas, where m and n in the preferred embodiment are preferably integers not less than 2, respectively; further, as shown in fig. 3, A, B, C three discrete points are selected from the divided regions, and then a plane formed by A, B, C three discrete points is a sample plane a where the planar sample is located; it is further preferable that A, B, C three discrete points are respectively disposed at the intersection points of four adjacent unit areas, and the corresponding connection lines of the three discrete points may form a triangle ABC as shown in fig. 3, and the plane where the triangle is located is the plane where the plane sample is located.
S2: keeping the focal length of the lens b unchanged, respectively adjusting the distance between a microscopic imaging device (such as a camera) and a point A, B, C, namely adjusting the height of the microscopic imaging device, and sequentially realizing focusing on a point A, B, C to obtain the focusing distances of the three points;
further, the focus of point A, B, C in the preferred embodiment is preferably determined by the following steps, as shown in FIG. 2.
S21: as shown in fig. 4, the lens b is controlled to move to a position vertically aligned with point A, B or point C, at which time the distance between the lens b and the corresponding point (e.g., discrete point a) is H1H of the reaction system1Namely the initial value of the focusing distance, and the definition value f at the moment is correspondingly calculated by using a definition calculation function1(ii) a It should be noted that the clarity in the preferred embodimentThe calculation function is a mature technology applied in the prior art, so that the detailed description is omitted, and the larger the definition value is, the closer the focus is to the correct position is, namely, the clearer the picture is;
s22: vertically moving the lens b by a smaller distance h along the axial direction to enable the lens b to be further close to a corresponding discrete point (such as a discrete point), namely, moving the lens b by a certain distance h downwards, and calculating by a definition calculation function to obtain a definition value f at the moment2When f is2Greater than f1When indicates f2Corresponding picture resolution higher than f1The corresponding picture definition, i.e. the focusing direction is downward; on the contrary, when f2Is less than f1When the lens b is in the focusing state, the focusing direction is upward, so that the focusing direction of the lens b is determined;
s23: controlling the lens b to move a distance h continuously along the focusing direction, and calculating by a definition calculation function to obtain a definition value f3When f is3Greater than f2Then, the lens b continues to move in the focusing direction by a distance h, and then a sharpness value f is obtained4Through f4And f3Making a comparison, and repeating the steps until f is reachediIs less than fi-1When the lens b moves, the coarse focusing of the lens b can be completed, and i described herein is preferably an integer not less than 2;
s24: controlling the lens b to move a distance l in a direction opposite to the focusing direction in the step S23, wherein l is more than or equal to 0.001h and less than or equal to 0.1h, and then obtaining a definition value k1Through k1And fiComparison, when k is1Greater than fiThen, the lens b continues to move the distance l to obtain the definition value k2Comparison k1And k is2When k is2Greater than k1When the lens b continues to move for the distance l, the process is repeated until k is reachedjLess than kj-1In this case, the lens b is moved back by the distance l and then stopped, so that the fine focusing of the lens b can be completed, and j described herein is preferably an integer not less than 2.
Through the adjustment of the steps, the vertical distance between the lens b and the A, B or the C point is H2H of the reaction system2I.e., the focus distance at point A, B or C.
S3: after the determination of the focal distances of the three discrete points A, B, C is completed respectively in step S2, the focal distances of the m × n unit areas in the sample plane a during photographing and imaging are respectively calculated by an interpolation method; and the interpolation method in the preferred embodiment may be further preferably one or more of a linear interpolation method, a parabolic interpolation method and a spline interpolation method, so as to obtain the focus distance when each unit region is photographed.
S4: and controlling the lens b to sequentially complete photographing and imaging of the m × n unit areas on the sample plane a according to a certain sequence, and before each photographing, quickly adjusting the height of the camera along the vertical direction according to the focusing distance obtained in the step S3, namely quickly adjusting the distance between the lens b and the sample plane a, so as to realize quick focusing photographing of the m × n unit areas.
Further preferably, the photographing sequence of the lens b in the step S4 is preferably as shown by the arrow direction in fig. 5, that is, photographing is performed sequentially along the longitudinal direction in the first row area on the left side, and then moving to the adjacent row horizontally and sequentially along the opposite direction; and then horizontally moving to the right to an adjacent row, and sequentially photographing in the opposite direction, and repeating the steps until all the unit areas are photographed.
In addition, since there is a certain deviation in the flatness of the sample plane a, the focus distance of the individual unit area may be slightly deviated; therefore, in order to improve the clarity of the picture, in a preferred embodiment, a repairing step of the focus distance is provided as follows:
s5: when the lens b takes a picture, the two pictures in the adjacent unit areas have coincident areas at the edges, the coincident areas of the two pictures are compared in definition value, and when the difference between the definition values of the two pictures is larger than a certain threshold value delta f, the camera takes a picture of refocusing the unit area with low definition value by adopting the focusing distance of the unit area with high definition value.
Through the steps of S1-S5, clear photographing of the plane sample on the glass slide can be quickly and accurately realized, the focusing time of a lens is shortened, and the scanning imaging efficiency of the sample is improved.
In one specific embodiment, the to-be-focused photographed area of the planar sample is a square plane of 10 × 10mm, and m ═ n ═ 10 in step S1, that is, the to-be-focused photographed area of the planar sample is divided into 100 unit areas with equal size and square shape; further, the distance h in the preferred embodiment is equal to 100 μm, and l is preferably equal to 1 μm. Of course, the values of m, n, h, l in the preferred embodiment are not limited to the above-mentioned preferred values, and they may be preferred according to actual needs, and are not limited herein.
The method for quickly focusing and photographing the planar sample in the preferred embodiment of the invention has simple steps and simple and convenient operation, the photographing plane to be focused of the planar sample is equally divided into a plurality of unit areas, three discrete points are preferably selected on the photographing plane to be focused, the focusing distance of each discrete point is respectively and accurately calculated, and the focusing distance of each unit area is calculated by an interpolation method, so that accurate photographing of each unit area can be completed without adjusting the focal length of a lens every time, the focusing speed in the imaging process of the sample is greatly improved, the time of scanning and imaging the sample is shortened, the efficiency and the accuracy of scanning and imaging the sample are improved, the requirements of quick and accurate imaging of the sample are fully met, the imaging definition of the planar sample is ensured, and the method has better application and popularization values.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method for fast focusing photographing of a plane sample comprises the following steps:
s1: dividing a photographing region to be focused of the planar sample into n equal parts and m equal parts along the length direction and the width direction respectively to form m multiplied by n unit regions;
s2: a, B, C three discrete points are selected in the photographed area to be focused, namely a plane formed by A, B, C three discrete points is a sample plane where the plane sample is located;
s3: keeping the focal length of the lens unchanged, respectively adjusting the distance between the lens and the three discrete points, and sequentially realizing the focusing of the lens on each discrete point to obtain the focusing distance of each discrete point;
s4: calculating the focus distance of each unit area by adopting an interpolation method according to the focus distance of each discrete point; and correcting the focus distance of each unit area in the following way:
respectively photographing and imaging two adjacent unit areas, and comparing definition values of the overlapped areas of the two pictures of the adjacent unit areas; when the difference between the two definition values is larger than a set threshold value, the lens adopts the focusing distance of the unit area with the high definition value to refocus and photograph the unit area with the low definition value;
s5: and sequentially finishing focusing and photographing of each unit area according to the focusing distance of each unit area, thereby finishing quick focusing and photographing of the plane sample.
2. The method for rapid focus photography of planar samples according to claim 1, wherein the focus distance of each of said discrete points is determined by:
s31: controlling the lens to align with any discrete point, wherein the distance between the lens and any discrete point is H1And calculating to obtain the definition value f at the moment according to the definition calculation function1；
S32: controlling the lens to move a distance h along the axial direction relative to the discrete point, and calculating to obtain a definition value f at the moment2Comparison of f2And f1Magnitude of value, when f2Greater than f1If so, indicating that the moving direction of the lens is the focusing direction, and otherwise, indicating that the opposite direction of the moving direction of the lens is the focusing direction;
s33: controlling the lens to move for i times along the focusing direction for a distance h, wherein i is an integer and is not less than 2, and respectively calculating to obtain a definition value after each movement until the definition value of the ith time is less than the definition value of the ith-1 time, namely fiIs less than fi-1Completing the rough pair of the lensCoke;
s34: controlling the lens to move j times along the opposite direction of the focusing direction in sequencelJ is an integer not less than 2 and is not more than 0.001hlNot more than 0.1h, and calculating respectively to obtain definition value after each movement until the definition value of the jth time is less than that of the jth-1 time, i.e. fjIs less than fj-1And then the lens is retracted by a distancelAnd then stopping moving to finish the fine focusing of the lens, namely the distance between the lens and the corresponding discrete point is the focusing distance of the discrete point.
3. The method for rapid focus photographing of a planar specimen according to claim 1, wherein the interpolation method is one or more of a linear interpolation method, a parabolic interpolation method and a spline interpolation method.
4. The method for rapid focus photographing of planar samples according to claim 1, wherein the unit area is a square area.
5. The method for fast focusing photographing of a planar sample according to claim 1, wherein the step of photographing in sequence of each unit area in step S5 is as follows:
and controlling the lens to move from one end to the other end along the length direction of the plane sample to finish focusing and photographing of the unit areas in one row, then controlling the lens to move to an adjacent row along the width direction, and finishing focusing and photographing of each unit area in the row in sequence along the length direction of the plane sample, and so on, and finishing focusing and imaging of m multiplied by n unit areas in sequence.
6. The method for fast focusing photographing of a planar sample according to claim 1, wherein the step of photographing in sequence of each unit area in step S5 is as follows:
and controlling the lens to move from one end to the other end along the width direction of the plane sample to finish focusing and photographing of the unit areas in one row, then controlling the lens to move to an adjacent row along the length direction, and finishing focusing and photographing of the unit areas in the row in sequence along the width direction of the plane sample, and so on, and finishing focusing and imaging of the m multiplied by n unit areas in sequence.
7. The method for fast focus photographing of planar samples according to claim 1, wherein the value of m is equal to the value of n.
8. The method for fast focus photographing of planar samples according to claim 2, wherein h is equal to 100 μm.
9. The method for rapid focus photographing of planar samples according to claim 2, wherein,lequal to 1 μm.
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| CN109085695A (en) | 2018-12-25 |
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