CN114040111B - Sequencer imaging focusing method and device, equipment and storage medium - Google Patents
Sequencer imaging focusing method and device, equipment and storage medium Download PDFInfo
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
- H04N23/60—Control of cameras or camera modules
- H04N23/67—Focus control based on electronic image sensor signals
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
- H04N23/60—Control of cameras or camera modules
- H04N23/64—Computer-aided capture of images, e.g. transfer from script file into camera, check of taken image quality, advice or proposal for image composition or decision on when to take image
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
- H04N23/60—Control of cameras or camera modules
- H04N23/67—Focus control based on electronic image sensor signals
- H04N23/671—Focus control based on electronic image sensor signals in combination with active ranging signals, e.g. using light or sound signals emitted toward objects
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02D—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN INFORMATION AND COMMUNICATION TECHNOLOGIES [ICT], I.E. INFORMATION AND COMMUNICATION TECHNOLOGIES AIMING AT THE REDUCTION OF THEIR OWN ENERGY USE
- Y02D10/00—Energy efficient computing, e.g. low power processors, power management or thermal management
Abstract
The invention belongs to the technical field of gene sequencing, and discloses a sequencer imaging focusing method, a sequencer imaging focusing device, equipment and a storage medium, wherein after an initial focusing process is carried out on an objective lens of a sequencer to determine a target position, when the objective lens shoots at the target position to obtain a target image, the target image is subjected to definition grading, if the definition grading of the target image is smaller than a grading threshold value, a refocusing process is carried out on the objective lens to redetermine the target position, and the step of shooting the target image to carry out definition grading is repeatedly carried out until the definition grading of the target image is larger than or equal to the grading threshold value; wherein the refocusing process includes all or part of the steps of the initial focusing process; real-time automatic focusing can be realized, even in the process of sequencing the same sequencing sample, clear images can be ensured to be always captured, bases can be more accurately identified, and the working efficiency and accuracy of the sequencer can be improved.
Description
Technical Field
The invention belongs to the technical field of gene sequencing, and particularly relates to a sequencer imaging focusing method, a sequencer imaging focusing device, equipment and a storage medium.
Background
Working distance refers to the distance of the front edge of the objective lens from the sample surface when the focus is aligned. Theoretical working distance refers to the working distance noted in the product manual when the objective lens is described.
In the application field of gene sequencer, base identification is a key link of high-throughput gene sequencing, and is mainly to obtain gene sequence information from a fluorescence dot diagram containing base information. At present, a fluorescent dot diagram is captured through a camera, but the position of the fluorescent dot diagram is changed when shooting each time, and a clear image can be captured only by carrying out micro distance adjustment and real-time judgment on imaging quality. The manual focusing mode ensures that the working efficiency and the accuracy of the sequencer are low.
Disclosure of Invention
The invention aims to provide a sequencer imaging focusing method, a sequencer imaging focusing device, sequencer imaging focusing equipment and a storage medium, which can realize automatic focusing, capture clear images of different samples in real time and improve the working efficiency and the accuracy of sequencers.
The first aspect of the embodiment of the invention discloses a sequencer imaging focusing method, which comprises the following steps:
performing an initial focusing process on an objective lens of the sequencer to determine a target position;
controlling the objective lens to shoot the sequencing sample at the target position to obtain a target image;
determining a plurality of fluorescent sample points in the target image;
dividing the plurality of fluorescent sample points into a clear sample point, a fuzzy sample point and an error sample point;
the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point are used as input data and are input into a pre-constructed definition scoring model to obtain definition scores of the target images;
if the definition score of the target image is smaller than a score threshold, performing refocusing process on the objective lens to redefine the target position, and repeatedly performing the step of controlling the objective lens to shoot the sequencing sample at the target position to obtain the target image until the definition score of the target image is larger than or equal to the score threshold; wherein the refocusing process includes all or part of the initial focusing process.
The second aspect of the embodiment of the invention discloses a sequencer imaging focusing device, which comprises:
the initial focusing unit is used for executing an initial focusing process on an objective lens of the sequencer so as to determine a target position;
the shooting unit is used for controlling the objective lens to shoot the sequencing sample at the target position to obtain a target image;
a determining unit for determining a plurality of fluorescent sample points in the target image;
the dividing unit is used for dividing the plurality of fluorescent sample points into clear sample points, fuzzy sample points and error sample points;
the scoring unit is used for taking the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point as input data, and inputting the input data into a pre-constructed definition scoring model to obtain definition scores of the target images;
a refocusing unit, configured to trigger the initial focusing unit to perform a refocusing process on the objective lens when the sharpness score of the target image is smaller than a score threshold, so as to redetermine a target position until the sharpness score of the target image is greater than or equal to the score threshold; wherein the refocusing process includes all or part of the initial focusing process.
A third aspect of an embodiment of the invention discloses an electronic device comprising a memory storing executable program code and a processor coupled to the memory; the processor invokes the executable program code stored in the memory for performing the sequencer imaging focusing method disclosed in the first aspect.
A fourth aspect of the embodiment of the present invention discloses a computer-readable storage medium storing a computer program, wherein the computer program causes a computer to execute the sequencer imaging focusing method disclosed in the first aspect.
The imaging focusing method and device of the sequencer, the equipment and the storage medium have the advantages that after an initial focusing process is carried out on an objective lens of the sequencer to determine a target position, the objective lens is controlled to sequence a sequencing sample at the target position, in the sequencing process, when the objective lens shoots to obtain a target image, the target image is subjected to definition grading, if the definition grading of the target image is smaller than a grading threshold value, a refocusing process is carried out on the objective lens to redetermine the target position, and the step of shooting the target image to carry out definition grading is repeatedly carried out until the definition grading of the target image is larger than or equal to the grading threshold value; wherein the refocusing process includes all or part of the steps of the initial focusing process; based on the method, real-time automatic focusing can be realized, even in the process of sequencing the same sequencing sample, clear images can be ensured to be captured all the time, and bases can be identified more accurately, so that the working efficiency and accuracy of the sequencer can be improved.
In addition, by constructing a definition scoring model in advance, each time the definition scoring is carried out on the target image, only a plurality of fluorescent sample points in the target image are required to be determined, and the fluorescent sample points are divided into clear sample points, fuzzy sample points and error sample points; and then, the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point are used as input data and are input into a definition scoring model, so that the definition score of a target image can be rapidly calculated and obtained, and the automatic focusing speed and accuracy are further improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles and effects of the invention.
Unless specifically stated or otherwise defined, the same reference numerals in different drawings denote the same or similar technical features, and different reference numerals may be used for the same or similar technical features.
FIG. 1 is a flow chart of a sequencer imaging focusing method disclosed in an embodiment of the invention;
FIG. 2 is a schematic illustration of projection data of fluorescent sample points disclosed in an embodiment of the present invention;
FIG. 3 is a schematic diagram of a sequencer imaging focusing device according to an embodiment of the present invention;
fig. 4 is a schematic structural diagram of an electronic device according to an embodiment of the present invention.
Reference numerals illustrate:
301. an initial focusing unit; 302. a photographing unit; 303. a determination unit; 304. dividing units; 305. a scoring unit; 306. refocusing the unit; 401. a memory; 402. a processor.
Detailed Description
In order that the invention may be readily understood, a more particular description of specific embodiments thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings.
Unless defined otherwise or otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the context of a realistic scenario in connection with the technical solution of the invention, all technical and scientific terms used herein may also have meanings corresponding to the purpose of the technical solution of the invention. The terms "first and second …" are used herein merely for distinguishing between names and not for describing a particular number or order. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items. As used herein, "said," "the" or "the" are the features or aspects of the technology previously mentioned or described in the corresponding locations, which may be the same or similar to the features or aspects of the technology mentioned. Clearly, technical contents or features that are contrary to the object of the present invention or that are clearly contradictory should be excluded.
As shown in fig. 1, an embodiment of the present invention discloses a sequencer imaging focusing method, including:
s1, performing an initial focusing process on an objective lens of the sequencer to determine a target position.
In step S1, an initial focusing process is required to be performed before each new sequencing sample is sequenced, so as to determine a target position, which is the optimal shooting position. The initial focusing process may include a coarse focusing step, a medium focusing step, and a fine focusing step; wherein, the liquid crystal display device comprises a liquid crystal display device,
s1.1, coarse focusing step comprises: determining a theoretical position of the objective lens meeting a theoretical working distance, starting from a first starting point positioned above the theoretical position, controlling the objective lens to move downwards once in a first designated step length, shooting to obtain a sample image, and carrying out definition scoring on the sample image until the definition scoring and coarse focusing ideal scoring S of the sample image A The proximity measurement value of (2) meets a preset condition; and determining the first position of the objective lens with the highest time sharing in the coarse focusing process according to the definition scores of the plurality of sample images obtained in the coarse focusing process.
Wherein the first starting point and the theoretical positionThe distance of (2) is a first distance, which may be X 1 Millimeter; the first designated step size may be Y 1 A micron; the proximity metric value refers to the degree of proximity between the sharpness score of the sample image and the coarse focus ideal score, and when the proximity metric value meets the preset condition, the two values are close enough. Sharpness scoring S of a sample image may be employed Sample Ideal score S of coarse focus A The ratio or difference between the two refers to the proximity metric; if S is adopted A /S Sample To refer to the proximity metric, a specified threshold may be determined as S A 0.95 when S A /S Sample Less than or equal to S A When the value of the proximity measurement value meets 0.95, judging that the value of the proximity measurement value meets a preset condition; if S is adopted Sample /S A To refer to the proximity metric, a specified threshold value of 0.95/S may be determined A When S Sample /S A Greater than or equal to 0.95/S A When the proximity measurement value meets the preset condition, judging; for example, assume S A ＝1，S Sample = 0.96,0.96 is greater than 0.95, so the proximity metric satisfies a preset condition.
S1.2, the middle focusing step comprises the following steps: starting from a second starting point above the first position, controlling the objective lens to move downwards by a second designated step length, shooting to obtain a sample image, and carrying out definition scoring on the sample image until the definition scoring S of the sample image Sample Ideal grading S of middle focusing B The proximity measurement value of (2) meets a preset condition; and determining a second position of the objective lens with the highest time sharing in the middle focusing process according to the definition scores of the plurality of sample images obtained in the middle focusing process.
Wherein the second specified step size may be Y 2 Micron, Y 2 ＜Y 1 The distance between the second starting point and the first position is a second distance, and the second distance can be X 2 And micron, less than the first distance, so that finer mid-focus than coarse focus may be performed.
S1.3, fine focusing comprises the following steps: starting from a third starting point located above the second position, controlling the objective lens to move downwards every time by a third designated step length, shooting to obtain a sample image, and aiming at the sample imageThe sample image is subjected to definition scoring until the definition scoring S of the sample image Sample Ideal score S with fine focus adjustment C The proximity measurement value of (2) meets a preset condition; determining a third position of the objective lens with the highest time sharing in the fine focusing process according to the definition scores of the plurality of sample images obtained in the fine focusing process; determining the third location as the target location; the distance between the third starting point and the second position is a third distance.
Wherein the third specified step size may be Y 3 Micron, Y 3 ＜Y 2 The third distance may be X 3 And microns, less than the second distance.
Wherein S is A ＜S B ＜S C Judgment S Sample Ideal grading S of middle focusing B Ideal grading S of fine focus C The approach of whether the proximity metric value satisfies the preset condition can be determined by referring to the above Sample Ideal score S of coarse focus A The manner of whether the proximity metric value satisfies the preset condition is not described herein.
After the above initial focusing process is performed, a scoring threshold to be used in the sequencing process may be further determined according to sharpness scores of a plurality of sample images obtained in the coarse focusing process, the medium focusing process, and the fine focusing process. For example, the minimum definition score in the three processes of coarse focusing, medium focusing and fine focusing can be determined as the scoring threshold S 0 The method comprises the steps of carrying out a first treatment on the surface of the The average definition of the three processes of coarse focusing, medium focusing and fine focusing can be determined as a scoring threshold S 0 The method comprises the steps of carrying out a first treatment on the surface of the The highest definition in the three processes of coarse focusing, medium focusing and fine focusing can also be determined; taking the product of the highest definition and the specified coefficient as a scoring threshold S 0 The method comprises the steps of carrying out a first treatment on the surface of the Wherein the specified coefficient is less than 1, e.g. 0.8. In the subsequent sequencing process, the evaluation can be performed according to the scoring threshold, if the sharpness score of the shot target image is greater than or equal to S 0 Refocusing is not required; if the sharpness score is less than S 0 Refocusing is required.
S2, controlling an objective lens to shoot the sequencing sample at a target position, and obtaining a target image.
The target image may be a fluorescence spot diagram in a sequencing process, and in the sequencing process, a plurality of times of shooting are generally required to be performed on a sequencing sample so as to capture the fluorescence spot diagram. In practice it has been found that the image becomes unclear after several shots, requiring focusing. Therefore, in order to track whether the shot fluorescent dot diagram is clear or not in real time so as to focus in time, the definition of each shot target image can be scored. The manner of scoring the sharpness of the target image may include the following steps S3-S5.
S3, determining a plurality of fluorescent sample points in the target image.
In step S3, the gray value of each pixel of the target image may be calculated first; determining a pixel point with the gray value reaching a gray threshold value as a fluorescent pixel point; then judging whether the number of fluorescent pixel points in the square grid with the fluorescent pixel points as the center is at least two; if yes, judging that all fluorescent pixel points included in the nine-grid are continuous and belong to the same fluorescent sample point; if not, judging the fluorescent pixel point as the boundary point of the fluorescent sample point; after traversing each fluorescent pixel point of the target image, a plurality of fluorescent sample points are obtained.
The gray value refers to the color depth of the pixel point in the black-and-white image, and the larger the energy of fluorescence is in the shot fluorescence dot diagram, the higher the gray value is. The gray values typically range from 0 to 255, with white being 255 and black being 0. The gray threshold, which refers to the gray limit that distinguishes between fluorescence and background, may be preset by a developer. If the gray value of any pixel point is greater than or equal to the gray threshold value, judging that the pixel point is a fluorescent pixel point; if the gray value of any pixel point is smaller than the gray threshold value, the pixel point is judged to be a background pixel point, filtering can be carried out, only the fluorescent pixel point is analyzed, and the fluorescent sample point is identified. If at least 1 of 8 adjacent fluorescent pixel points is a fluorescent pixel point, the fluorescent pixel points are determined to be continuous and belong to the same fluorescent sample point, otherwise, the fluorescent pixel point is a boundary point of the fluorescent sample point, and after all the fluorescent pixel points are traversed, a plurality of fluorescent sample points can be identified.
For example, regarding the fluorescent pixel point a as the center, if the point b is recognized as the fluorescent pixel point among 8 pixel points adjacent to the point a, the point a and the point b are judged to be continuous, the point a and the point b belong to the same fluorescent sample point, and the point b is continuously recognized as the center, and if the point c is recognized to be continuous with the point b, the point a, the point b and the point c belong to the same fluorescent sample point, and the point c is continuously recognized. However, if no consecutive spots are identified at the time of spot b, this indicates that the fluorescent sample spot has been identified.
S4, dividing the plurality of fluorescent sample points into clear sample points, fuzzy sample points and error sample points.
In step S4, each fluorescent sample point may be projected to the X-axis and the Y-axis, so as to obtain projection data of each fluorescent sample point; and then determining the number of the fluorescent pixel points on the side with the largest fluorescent pixel points as the side length of the fluorescent sample points according to the projection data. The schematic diagram of projection data of fluorescent sample points can be shown in fig. 2, and the side lengths of fluorescent sample points in each column from left to right in the diagram are respectively 1, 2, 3 and 4.
Further, a sharpness threshold and a blur threshold may be set to classify fluorescent sample points. Wherein the clear threshold value refers to the threshold value of the number of the edge length pixel points of the fluorescent point of the clear sample; the blurring threshold refers to the threshold of the number of edge length pixel points of the blurring sample fluorescent spot.
Based on the above, if the side length of any fluorescent sample point is smaller than or equal to the clear threshold value, judging the fluorescent sample point as a clear sample point; if the side length of any fluorescent sample point is larger than the clear threshold and smaller than or equal to the fuzzy threshold, judging that the fluorescent sample point is a fuzzy sample point; if the side length of any fluorescent sample point is larger than the fuzzy threshold value, judging the fluorescent sample point as an error sample point.
S5, inputting the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point as input data to a pre-built definition scoring model to obtain the definition score of the target image.
After the step S4 is executed, the number of fluorescent sample points of different types can be counted, namely the number of error sample points, clear sample points and fuzzy sample points are counted respectively, and the number of clear sample points, the number of fuzzy sample points and the number of error sample points are obtained; and calculating the gray value and the side length of each fluorescent sample point.
When the definition scoring model is built in advance, the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point of the sample fluorescent image are obtained by processing the sample fluorescent image of the training set and serve as input data, and the definition scoring model is built and trained.
In step S4, specifically, based on the definition scoring model, an effective sample score is calculated according to the number of clear sample points, the number of fuzzy sample points and the number of error sample points, an effective sample focusing score is calculated according to the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the side lengths of each fluorescent sample point and the average gray value, and a gray score is calculated according to the highest gray value and the average gray value of the fluorescent sample points; and multiplying the effective sample fraction, the effective sample focusing fraction and the gray scale fraction to obtain the definition score of the target image. Therefore, the accuracy of the definition scoring can be improved by classifying the fluorescent sample points and carrying out the definition scoring in combination with the number of clear sample points, the number of fuzzy sample points and the number of error sample points.
And S6, judging whether the definition score of the target image is smaller than a score threshold value. If yes, executing the step S7, and turning to the step S2; otherwise, step S8 is executed, and the present flow is ended.
Wherein, the definition score of the target image is recorded as S, if S is more than or equal to S 0 The refocusing process need not be performed; if S is less than S 0 A refocusing process needs to be performed.
S7, performing refocusing process on the objective lens to redetermine the target position; wherein the refocusing process includes all or part of the steps of the initial focusing process.
In step S7, if S is less than S 0 To the objective lensA line refocusing process comprising:
if the sharpness score is less than the score threshold and greater than the fine focus ideal score, S C ＜S＜S 0 The objective lens is controlled to move up a third distance (X 3 Micron) and performing a fine focus step;
if the sharpness score is smaller than or equal to the fine focusing ideal score and larger than the medium focusing ideal score, namely S B ＜S≤S C The objective lens is controlled to move upward a second distance (X 2 Micron), and performing a middle focusing step and a fine focusing step;
if the sharpness score is smaller than or equal to the ideal score of the middle focusing, namely S is less than or equal to S B The objective lens is controlled to move upward a first distance (X 1 Millimeter), a coarse focus adjustment process, a medium focus adjustment step, and a fine focus adjustment step are performed.
In the embodiment of the invention, X 1 、X 2 、X 3 、Y 1 、Y 2 、Y 3 Taken from values of 0-20, respectively.
S8, a refocusing process is not required to be executed.
By implementing the embodiment of the invention, a software algorithm is adopted to identify the mode of combining software and hardware which is mainly used and is used for adjusting the working distance of the Z-axis motor to be auxiliary, the cost is low, the identification accuracy is high, the method is applicable to cover slips (Flow Cell) with different thicknesses, dyes with different luminous efficiencies and magnetic beads with different sizes are applicable, and even the identification algorithm can be designed according to occasion systems, so that the method can be applicable to different application occasions without changing hardware. Because the optimal shooting position is obtained through image definition scoring comparison, factors such as interference of ambient light, dye difference, device performance change generated by long-term running of an instrument and the like can not influence a final result, and the most clear focal plane can be automatically and accurately found.
As shown in fig. 3, an embodiment of the present invention discloses a sequencer imaging focusing device, which includes an initial focusing unit 301, a shooting unit 302, a determining unit 303, a dividing unit 304, a scoring unit 305, and a refocusing unit 306; wherein, the liquid crystal display device comprises a liquid crystal display device,
an initial focusing unit 301, configured to perform an initial focusing process on an objective lens of the sequencer to determine a target position;
the shooting unit 302 is used for controlling the objective lens to shoot the sequencing sample at the target position to obtain a target image;
a determining unit 303 for determining a plurality of fluorescent sample points in the target image;
a dividing unit 304 for dividing the plurality of fluorescent sample points into clear sample points, blurred sample points and error sample points;
the scoring unit 305 is configured to input the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point as input data to a pre-constructed definition scoring model, so as to obtain a definition score of the target image;
a refocusing unit 306, configured to trigger the initial focusing unit 301 to perform a refocusing process on the objective lens when the sharpness score of the target image is less than the score threshold, so as to redetermine the target position, until the sharpness score of the target image is greater than or equal to the score threshold; wherein the refocusing process includes all or part of the steps of the initial focusing process.
In some embodiments, the determining unit 303 may include the following sub-units, not shown:
a calculating subunit, configured to calculate a gray value of each pixel point of the target image;
a determining subunit, configured to determine, as a fluorescent pixel, a pixel where the gray value reaches the gray threshold;
the judging subunit is used for judging whether the number of the fluorescent pixel points in the nine-square grid taking the fluorescent pixel point as the center is at least two; after traversing each fluorescent pixel point of the target image, obtaining a plurality of fluorescent sample points;
the judging subunit is used for judging that all the fluorescent pixel points in the nine-square grid are continuous and belong to the same fluorescent sample point when the judging subunit judges that the number of the fluorescent pixel points in the nine-square grid with the fluorescent pixel point as the center is at least two; otherwise, the fluorescent pixel point is judged to be the boundary point of the fluorescent sample point.
In some embodiments, the above-described dividing unit 304 may include the following sub-units, not shown:
the projection subunit is used for projecting each fluorescent sample point to the X axis and the Y axis to obtain projection data of each fluorescent sample point;
the side length determining subunit is used for determining the number of the fluorescent pixel points on the side with the largest fluorescent pixel points as the side length of the fluorescent sample points according to the projection data;
the dividing unit is used for judging that any fluorescent sample point is a clear sample point when the side length of the fluorescent sample point is smaller than or equal to the clear threshold value; and when the side length of any fluorescent sample point is larger than the clear threshold and smaller than or equal to the fuzzy threshold, judging that the fluorescent sample point is a fuzzy sample point; and when the side length of any fluorescent sample point is larger than the fuzzy threshold value, judging that the fluorescent sample point is an error sample point.
In some embodiments, the initial focusing unit 301 may include the following sub-units, not shown:
a coarse focusing subunit for performing a coarse focusing step; specifically, determining a theoretical position of the objective lens meeting a theoretical working distance, starting from a first starting point positioned above the theoretical position, controlling the objective lens to move downwards once in a first designated step length, shooting to obtain a sample image, and carrying out definition scoring on the sample image until a proximity measurement value of the definition scoring of the sample image and an ideal rough focusing scoring meets a preset condition; determining a first position of an objective lens with the highest time sharing in the coarse focusing process according to definition scores of a plurality of sample images obtained in the coarse focusing process; wherein the distance between the first starting point and the theoretical position is a first distance;
a middle focusing subunit for executing a middle focusing step; specifically, starting from a second starting point positioned above the first position, controlling the objective lens to move downwards once at a second designated step length, shooting to obtain a sample image, and scoring the definition of the sample image until the similarity measurement value of the definition score of the sample image and the ideal middle focusing score meets a preset condition; determining a second position of the objective lens with the highest time sharing in the middle focusing process according to the definition scores of the plurality of sample images obtained in the middle focusing process; the second designated step length is smaller than the first designated step length, the distance between the second starting point and the first position is a second distance, and the second distance is smaller than the first distance;
a fine focus subunit for performing a fine focus adjustment step; specifically, starting from a third starting point positioned above the second position, controlling the objective lens to move downwards once at a third designated step length, shooting to obtain a sample image, and carrying out definition scoring on the sample image until the proximity measurement value of the definition scoring of the sample image and the fine focusing ideal scoring meets a preset condition; determining a third position of the objective lens with the highest time sharing in the fine focusing process according to the definition scores of the plurality of sample images obtained in the fine focusing process; determining the third location as the target location; the third designated step length is smaller than the second designated step length, the distance between the third starting point and the second position is a third distance, and the third distance is smaller than the second distance.
In some embodiments, the above refocusing unit 306 is configured to trigger the initial focusing unit 301 to perform the refocusing process on the objective lens when the sharpness score of the target image is smaller than the score threshold, that is, trigger the initial focusing unit 301 to perform all or part of the steps specifically:
the refocusing unit 306 is configured to control the objective lens to move upward from the current position by a third distance when the sharpness score of the target image is smaller than the score threshold and greater than the fine focus ideal score, and trigger the fine focus subunit to execute the fine focus adjustment step; the method comprises the steps of,
when the definition score of the target image is smaller than or equal to the fine focusing ideal score and larger than the middle focusing ideal score, controlling the objective lens to move upwards from the current position for a second distance, and triggering the middle focusing subunit to execute a middle focusing step and the fine focusing subunit to execute a fine focusing step; the method comprises the steps of,
when the sharpness score of the target image is smaller than or equal to the ideal middle focusing score, the objective lens is controlled to move upwards from the current position by a first distance, and the coarse focusing subunit is triggered to execute a coarse focusing step, the middle focusing subunit executes a middle focusing step and the fine focusing subunit executes a fine focusing step.
In some embodiments, the imaging focusing device of the sequencer further comprises a threshold determining unit, configured to determine a scoring threshold according to sharpness scores of a plurality of sample images obtained in three processes of coarse focusing, medium focusing and fine focusing after the coarse focusing step is performed by the coarse focusing subunit, the medium focusing step is performed by the medium focusing subunit, and the fine focusing step is performed by the fine focusing subunit.
Further, the threshold determining unit may be specifically configured to determine, as a scoring threshold, a lowest score of sharpness according to sharpness scores of a plurality of sample images obtained in three processes of coarse focusing, medium focusing and fine focusing; or determining the average definition score as a scoring threshold according to definition scores of a plurality of sample images obtained in the three processes of coarse focusing, medium focusing and fine focusing; or determining the highest definition score according to definition scores of a plurality of sample images obtained in the three processes of coarse focusing, medium focusing and fine focusing; and taking the product of the definition highest value and the appointed coefficient as a scoring threshold value, wherein the appointed coefficient is smaller than one.
As shown in fig. 4, an embodiment of the present invention discloses an electronic device including a memory 401 storing executable program codes and a processor 402 coupled with the memory 401;
the processor 402 invokes executable program codes stored in the memory 401, and executes the sequencer imaging focusing method described in the above embodiments.
The embodiment of the invention also discloses a computer readable storage medium storing a computer program, wherein the computer program causes a computer to execute the imaging focusing method of the sequencer described in each embodiment.
The foregoing embodiments are provided for the purpose of exemplary reproduction and deduction of the technical solution of the present invention, and are used for fully describing the technical solution, the purpose and the effects of the present invention, and are used for enabling the public to understand the disclosure of the present invention more thoroughly and comprehensively, and are not used for limiting the protection scope of the present invention.
The above examples are also not an exhaustive list based on the invention, and there may be a number of other embodiments not listed. Any substitutions and modifications made without departing from the spirit of the invention are within the scope of the invention.
Claims (9)
1. The imaging focusing method of the sequencer is characterized by comprising the following steps of:
performing an initial focusing process on an objective lens of the sequencer to determine a target position;
controlling the objective lens to shoot the sequencing sample at the target position to obtain a target image;
calculating the gray value of each pixel point of the target image;
determining the pixel point of which the gray value reaches a gray threshold value as a fluorescent pixel point;
judging whether the number of fluorescent pixel points in a square grid taking the fluorescent pixel point as the center is at least two;
if yes, judging that all the fluorescent pixel points in the nine-square grid are continuous and belong to the same fluorescent sample point;
if not, judging the fluorescent pixel points to be boundary points of fluorescent sample points;
after traversing each fluorescent pixel point of the target image, obtaining a plurality of fluorescent sample points;
dividing the plurality of fluorescent sample points into a clear sample point, a fuzzy sample point and an error sample point;
the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point are used as input data and are input into a pre-constructed definition scoring model to obtain definition scores of the target images; the side length of each fluorescent sample point is determined according to projection data obtained by projecting each fluorescent sample point to an X axis and a Y axis, and the side length of each fluorescent sample point is the number of fluorescent pixel points on the side with the largest fluorescent pixel points;
if the definition score of the target image is smaller than a score threshold, performing refocusing process on the objective lens to redefine the target position, and repeatedly performing the step of controlling the objective lens to shoot the sequencing sample at the target position to obtain the target image until the definition score of the target image is larger than or equal to the score threshold; wherein the refocusing process includes all or part of the initial focusing process.
2. The sequencer imaging focusing method of claim 1, wherein said dividing said plurality of fluorescent sample points into clear sample points, blurred sample points and error sample points comprises:
projecting each fluorescent sample point to an X axis and a Y axis to obtain projection data of each fluorescent sample point;
according to the projection data, determining the number of fluorescent pixel points on the side with the most fluorescent pixel points as the side length of the fluorescent sample points;
if the side length of any fluorescent sample point is smaller than or equal to the clear threshold value, judging the fluorescent sample point as a clear sample point;
if the side length of any fluorescent sample point is larger than the clear threshold and smaller than or equal to the fuzzy threshold, judging that the fluorescent sample point is a fuzzy sample point;
and if the side length of any fluorescent sample point is larger than the fuzzy threshold value, judging that the fluorescent sample point is an error sample point.
3. The sequencer imaging focusing method according to claim 1 or 2, wherein the initial focusing process includes a coarse focusing step, a medium focusing step, and a fine focusing step; wherein, the liquid crystal display device comprises a liquid crystal display device,
the coarse focusing step comprises the following steps: determining a theoretical position of the objective lens meeting a theoretical working distance, starting from a first starting point positioned above the theoretical position, controlling the objective lens to move downwards once at a first designated step length, shooting to obtain a sample image, and carrying out definition grading on the sample image until a proximity measurement value of the definition grading of the sample image and an ideal rough focusing grading meets a preset condition; determining a first position of the objective lens when the coarse focusing process is highest according to definition scores of a plurality of sample images obtained in the coarse focusing process; wherein the distance between the first starting point and the theoretical position is a first distance;
the middle focusing step comprises the following steps: starting from a second starting point above the first position, controlling the objective lens to move downwards once in a second designated step length, shooting to obtain a sample image, and scoring the definition of the sample image until the similarity measurement value of the definition score of the sample image and the ideal middle focusing score meets the preset condition; determining a second position of the objective lens when the middle focusing process is highest according to definition scores of a plurality of sample images obtained in the middle focusing process; the second designated step length is smaller than the first designated step length, and the distance between the second starting point and the first position is a second distance which is smaller than the first distance;
the fine-tuning step includes: starting from a third starting point above the second position, controlling the objective lens to move downwards once at a third designated step length, shooting to obtain a sample image, and scoring the sample image in definition until the proximity measurement value of the definition score and the fine focusing ideal score of the sample image meets the preset condition; determining a third position of the objective lens when the fine focusing process is highest according to definition scores of a plurality of sample images obtained in the fine focusing process; determining the third location as a target location; the third designated step length is smaller than the second designated step length, and the distance between the third starting point and the second position is a third distance which is smaller than the second distance.
4. A sequencer imaging focusing method according to claim 3, wherein said performing a refocusing process on said objective lens comprises:
if the sharpness score of the target image is greater than the fine focus ideal score, controlling the objective lens to move upwards a third distance from the current position, and executing the fine focus Jiao Buzhou;
if the sharpness score of the target image is smaller than or equal to the fine focus ideal score and larger than the middle focus ideal score, controlling the objective lens to move upwards from the current position by the second distance, and executing the middle focus step and the fine focus Jiao Buzhou;
and if the definition score of the target image is smaller than or equal to the middle focusing ideal score, controlling the objective lens to move upwards from the current position by the first distance, and executing the coarse focusing process, the middle focusing step and the fine focusing step.
5. The sequencer imaging focusing method of claim 3, further comprising:
and determining the scoring threshold according to the definition scores of the plurality of sample images obtained in the three processes of coarse focusing, medium focusing and fine focusing.
6. The sequencer imaging focus method of claim 5, wherein said determining said scoring threshold based on sharpness scores for a plurality of sample images obtained from three processes of coarse focus, medium focus, and fine focus, comprises:
determining the lowest definition score as the scoring threshold according to definition scores of a plurality of sample images obtained in the three processes of coarse focusing, medium focusing and fine focusing; or alternatively, the process may be performed,
determining average definition score as the scoring threshold according to definition scores of a plurality of sample images obtained in three processes of coarse focusing, medium focusing and fine focusing; or alternatively, the process may be performed,
determining the highest definition score according to definition scores of a plurality of sample images obtained in the three processes of coarse focusing, medium focusing and fine focusing; and taking the product of the definition highest value and a specified coefficient as the scoring threshold value, wherein the specified coefficient is smaller than one.
7. Sequencer formation of image focusing device, its characterized in that includes:
the initial focusing unit is used for executing an initial focusing process on an objective lens of the sequencer so as to determine a target position;
the shooting unit is used for controlling the objective lens to shoot the sequencing sample at the target position to obtain a target image;
a determining unit for determining a plurality of fluorescent sample points in the target image;
the dividing unit is used for dividing the plurality of fluorescent sample points into clear sample points, fuzzy sample points and error sample points;
the scoring unit is used for taking the number of clear sample points, the number of fuzzy sample points, the number of error sample points, the gray value and the side length of each fluorescent sample point as input data, and inputting the input data into a pre-constructed definition scoring model to obtain definition scores of the target images; the side length of each fluorescent sample point is determined according to projection data obtained by projecting each fluorescent sample point to an X axis and a Y axis, and the side length of each fluorescent sample point is the number of fluorescent pixel points on the side with the largest fluorescent pixel points;
a refocusing unit, configured to trigger the initial focusing unit to perform a refocusing process on the objective lens when the sharpness score of the target image is smaller than a score threshold, so as to redetermine a target position until the sharpness score of the target image is greater than or equal to the score threshold; wherein the refocusing process includes all or part of the initial focusing process;
wherein the determining unit includes:
a calculating subunit, configured to calculate a gray value of each pixel point of the target image;
a determining subunit, configured to determine, as a fluorescent pixel, a pixel where the gray value reaches a gray threshold;
the judging subunit is used for judging whether the number of the fluorescent pixel points in the square grid taking the fluorescent pixel point as the center is at least two; after traversing each fluorescent pixel point of the target image, obtaining a plurality of fluorescent sample points;
the judging subunit is used for judging that all the fluorescent pixel points in the nine-square grid are continuous and belong to the same fluorescent sample point when the judging subunit judges that the number of the fluorescent pixel points in the nine-square grid with the fluorescent pixel point as the center is at least two; otherwise, the fluorescent pixel point is judged to be the boundary point of the fluorescent sample point.
8. An electronic device comprising a memory storing executable program code and a processor coupled to the memory; the processor invokes the executable program code stored in the memory for performing the sequencer imaging focusing method of any one of claims 1 to 6.
9. A computer-readable storage medium storing a computer program, wherein the computer program causes a computer to execute the sequencer imaging focusing method according to any one of claims 1 to 6.
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| CN115278089B (en) * | 2022-09-26 | 2022-12-02 | 合肥岭雁科技有限公司 | Face fuzzy image focusing correction method, device, equipment and storage medium |
| CN116112795B (en) * | 2023-04-13 | 2023-06-23 | 北京城建智控科技股份有限公司 | Adaptive focusing control method, camera and storage medium |
| CN116528047B (en) * | 2023-07-03 | 2023-09-08 | 深圳赛陆医疗科技有限公司 | Focusing method, focusing device, gene sequencer and storage medium |
| CN116643372B (en) * | 2023-07-27 | 2023-10-13 | 深圳赛陆医疗科技有限公司 | Adjustment method and device, gene sequencer and storage medium |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106791663A (en) * | 2016-12-28 | 2017-05-31 | 天津天地伟业生产力促进有限公司 | A kind of tracking focusing algorithm based on background modeling |
| CN207411295U (en) * | 2017-07-21 | 2018-05-29 | 胡华梅 | Buoy packing tube with fluorescent paint bottle base |
| CN109946911A (en) * | 2019-03-20 | 2019-06-28 | 深圳市橙子数字科技有限公司 | Focus adjustment method, device, computer equipment and storage medium |
| CN110531484A (en) * | 2019-07-24 | 2019-12-03 | 中国地质大学（武汉） | A kind of microscope Atomatic focusing method that focus process model can be set |
| CN112351196A (en) * | 2020-09-22 | 2021-02-09 | 北京迈格威科技有限公司 | Image definition determining method, image focusing method and device |
| CN112468721A (en) * | 2020-11-19 | 2021-03-09 | 江汉大学 | Visual acquisition method and device with automatic focusing function |
| CN112672039A (en) * | 2020-12-07 | 2021-04-16 | 北京集光通达科技股份有限公司 | Multi-zone-based active focusing conversion method and equipment |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007233247A (en) * | 2006-03-03 | 2007-09-13 | Fujifilm Corp | Focus adjusting amount decision device, method and program and imaging apparatus |
-
2021
- 2021-11-25 CN CN202111415728.7A patent/CN114040111B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106791663A (en) * | 2016-12-28 | 2017-05-31 | 天津天地伟业生产力促进有限公司 | A kind of tracking focusing algorithm based on background modeling |
| CN207411295U (en) * | 2017-07-21 | 2018-05-29 | 胡华梅 | Buoy packing tube with fluorescent paint bottle base |
| CN109946911A (en) * | 2019-03-20 | 2019-06-28 | 深圳市橙子数字科技有限公司 | Focus adjustment method, device, computer equipment and storage medium |
| CN110531484A (en) * | 2019-07-24 | 2019-12-03 | 中国地质大学（武汉） | A kind of microscope Atomatic focusing method that focus process model can be set |
| CN112351196A (en) * | 2020-09-22 | 2021-02-09 | 北京迈格威科技有限公司 | Image definition determining method, image focusing method and device |
| CN112468721A (en) * | 2020-11-19 | 2021-03-09 | 江汉大学 | Visual acquisition method and device with automatic focusing function |
| CN112672039A (en) * | 2020-12-07 | 2021-04-16 | 北京集光通达科技股份有限公司 | Multi-zone-based active focusing conversion method and equipment |
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