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CN-122018135-A - Cross-slide microscopic imaging rapid automatic focusing method

CN122018135ACN 122018135 ACN122018135 ACN 122018135ACN-122018135-A

Abstract

The invention discloses a rapid automatic focusing method for cross-slide microscopic imaging, which relates to the technical field of microscopic imaging, and realizes rapid automatic focusing in the same slide and among the cross-slides by introducing global focal plane matrix record of the focal position information and focal plane position of the focused crawling, and when the focal is continuously performed in the same slide, the focal plane position of the adjacent focused crawling above or on the left side is preferably used for setting a narrow focusing range, so that the focusing interval is obviously shortened, the focusing speed is improved, the rapid focusing is also realized by referring to the focal position information of the crawling after focusing is performed on the adjacent slide during the cross-slide focusing, and in addition, the repeated experiments of the same batch and the same specification can directly utilize the focal plane matrix data after archiving by virtue of the data archiving and multiplexing functions, the initial focusing step is skipped, and the focusing efficiency of batch experiments is further improved.

Inventors

  • DU WENTAO
  • WANG YING
  • ZHANG MENG

Assignees

  • 宁波力显智能科技有限公司

Dates

Publication Date
20260512
Application Date
20260311

Claims (10)

  1. 1. A rapid auto-focusing method for cross-slide microscopic imaging is characterized by comprising the following specific steps: s1, initial focusing, namely executing automatic focusing on a first slide of a first slide at the beginning of an experiment by adopting a Z-axis focusing range preset by a user to acquire the focal plane position of the slide; s2, after focusing of a single climbing sheet is completed, recording the focal plane position of the climbing sheet, and synchronously updating a global focal plane matrix, wherein the focal plane matrix records the position information of all the climbing sheets which are completed in focusing and the corresponding focal plane positions; S3, continuously focusing in the same slide, namely preferentially selecting the focal plane positions of the adjacent to-be-focused slide plates above the to-be-focused slide plates except the first slide plate in the same slide plate, setting a narrow focusing range and executing automatic focusing, if the adjacent slide plates above are not focused, selecting the focal plane positions of the adjacent to-be-focused slide plates on the left side to set the narrow focusing range and executing automatic focusing, and if the adjacent slide plates above and on the left are not focused, executing automatic focusing by adopting a Z-axis focusing range preset by a user; S4, focusing across the slide glass, namely, after switching to a new slide glass, preferentially selecting the focal plane position of the adjacent slide glass to be focused, which is above the slide glass and is finished with the focused slide glass, setting a narrow focusing range to execute automatic focusing; S5, data archiving, namely archiving the global focal plane matrix in a preset format after focusing and imaging of all the slide sheets are completed.
  2. 2. The rapid auto-focusing method for cross-slide microscopic imaging according to claim 1 is characterized in that the initial focusing process of the step S1 is specifically implemented according to the following steps, firstly, after the optical path calibration of a slide loading and microscopic imaging system is completed by a user before the experiment starts, the initial focusing process is firstly positioned to the central area of the first slide through a preview interface, the biological sample area on the slide is ensured to be completely covered by the preview image, interference of the slide edge, a slide blank area and a solution bubble area is eliminated, secondly, the corresponding Z-axis initial focusing range is set by the user according to the objective magnification, the sample type and the slide thickness used in the experiment, meanwhile, Z-axis stepping precision and single-step image acquisition parameters in the focusing process are set, thirdly, after the initial focusing is started, the system acquires images point by point in the initial focusing range according to the preset stepping precision, the sharpness evaluation value of each frame of images is calculated, the Z-axis coordinate corresponding to the positioning sharpness evaluation value peak value is used as an initial focal plane position, and after the initial focal plane positioning is completed, the system automatically carries out secondary focal plane position on the slide edge, the corresponding to the focal plane position is calibrated to the focal plane position in the initial focusing range, the step position is more than 10 mu m, the focal plane position is calculated to be more stable, and the focal plane position is adjusted to be more accurate and more stable than the focal plane position is acquired in the initial focusing range.
  3. 3. The rapid auto-focusing method of cross-slide microscopic imaging according to claim 1, wherein in the focal plane recording and matrix updating process of step S2, the system assigns a corresponding credibility weight to the focal plane data of each slide, and completes weight assignment by a focal plane credibility weight calculation formula, and synchronously stores the weight value into a global focal plane matrix, wherein the focal plane credibility weight calculation formula is as follows: wherein Is the first On the first slide Line 1 Focal plane data credibility weight of the column climbing slices; Contributing coefficients to sharpness; The normalized definition evaluation value is the normalized definition evaluation value when the focusing of the corresponding climbing film is completed; contributing coefficients to the focus range; The normalized credibility value of the focusing range of the corresponding climbing sheet is obtained; Contributing coefficients to position continuity; Normalized for position continuity of the corresponding crawling sheet.
  4. 4. The rapid auto-focusing method for cross-slide microscopic imaging according to claim 1, wherein the continuous focusing process in the same slide in step S3 is specifically implemented according to the following steps that firstly, the system plans scanning paths of all the crawl sheets in the same slide in advance, adopts a progressive scanning mode with line priority, sequentially completes the focusing of the crawl sheets from left to right in the same line from a first column position of a first line where a first crawl sheet is located, switches to the next line, and completes the focusing of all the crawl sheets in the line from left to right; the system comprises a first step of collecting the focal plane matrix of the current climbing plate to be focused, a second step of collecting the focal plane matrix of the current climbing plate to be focused, inquiring the focal plane data and the focal plane completion state of the same row of the adjacent climbing plate above the climbing plate, if the climbing plate is completed to focus, directly taking the focal plane position of the climbing plate as a focusing reference, setting a corresponding narrow focusing range to execute automatic focusing, a third step of collecting the focal plane data and the focal plane completion state of the previous row of the adjacent climbing plate on the left side of the climbing plate if the upper adjacent climbing plate is not completed to focus, taking the focal plane position of the climbing plate as the focusing reference, setting the corresponding narrow focusing range to execute automatic focusing, a fourth step of automatically collecting the initial focusing range preset by a user if the upper adjacent climbing plate is not completed to focus, executing automatic focusing operation of the full range, marking the climbing plate as the initial reference climbing plate of the row or the row after the focusing is completed, a fifth step of checking the focal plane position obtained by the system to effectively focus the climbing plate after the focusing is completed, and if the preset focal plane position exceeds the reference position deviation of the reference table, the focus range is automatically enlarged and the focus is re-performed.
  5. 5. The rapid auto-focusing method of cross-slide microscopic imaging according to claim 1, wherein in the step S3 and the step S4, the setting of the narrow focusing range is calculated by adopting a dynamic adaptive adjustment formula, the focusing range of the to-be-focused crawling is adjusted in real time according to the focal plane data of the focused crawling, the focusing range is reduced to the greatest extent while the focusing success rate is ensured, and the dynamic calculation formula of the narrow focusing range half-range is as follows: wherein A half-width Z-axis interval value of a narrow focusing range of the climbing sheet to be focused; A basic half-width focusing interval is adopted; Is a deviation response coefficient; the Z-axis coordinate value of the focal plane of the adjacent focused climbing plate cited by the focusing; the arithmetic average value of the Z-axis coordinates of the focal plane of all the climbing slices which are focused in the same slide; Is the maximum focus plane deviation allowed in the same batch experiment.
  6. 6. The rapid auto-focusing method for cross-slide microscopic imaging according to claim 1, wherein the cross-slide focusing process of step S4 is specifically implemented according to the following steps that, after the system completes focusing and imaging of all the slide sheets of the previous slide, the displacement stage is driven to complete slide switching action, a new slide is moved to the range of view shot by the objective lens of the microscopic imaging system, and meanwhile, coordinate calibration of the slide position is completed; the system firstly searches a global focal plane matrix for the first to-be-focused climbing plate on a new slide, inquires the focusing completion state and focal plane data of the same row of the climbing plates above the climbing plates, if the climbing plates are completed to be focused, directly takes the focal plane positions of the climbing plates as focusing references, sets corresponding narrow focusing ranges to execute automatic focusing, if the upper adjacent climbing plates are not focused, the system inquires the focusing completion state and focal plane data of the adjacent climbing plates at the positions of the current to-be-focused climbing plates on the previous slide adjacent to the left side, if the climbing plates are completed to be focused, the focal plane positions of the climbing plates are taken as focusing references, sets corresponding narrow focusing ranges to execute automatic focusing, and if the climbing plates at the two adjacent positions are not focused, the system automatically adjusts the initial focusing ranges preset by a user, executes automatic focusing operation of the full ranges, marks the climbing plates as initial reference plates of the new slide, and finally completes the focusing of the new climbing plates according to the following steps of the focal plane data of the climbing plates, and the following step S3 is executed to continuously, the focal plane matrix is updated after the focusing of each climbing plate is focused, meanwhile, the system can compare the focal plane data of the new slide and the focal plane deviation of the slide at the same position as the previous slide in real time, and if the deviation exceeds a preset threshold value, the focusing range of the subsequent slide is automatically adjusted adaptively.
  7. 7. A rapid auto-focusing method for cross-slide microscopic imaging according to claim 1 is characterized in that the data archiving and multiplexing process of step S5 is implemented according to the following steps, namely, firstly, after focusing and microscopic imaging operations of all the slide sheets are completed by the system, firstly, integrity checking is carried out on all data in a global focal plane matrix, checking whether fields such as slide serial numbers, row and column positions, focal plane Z-axis coordinates, focusing completion states and credibility weight values of each slide sheet are complete or not, invalid focusing failure data are removed, secondly, the system converts the calibrated global focal plane matrix into a file in a CSV format, focusing data of all the slide sheets are orderly arranged in the file according to the sequence of the slide serial numbers as a first index, the slide sheet row numbers as a second index and the slide sheet row numbers as a third index, meanwhile, experimental time, objective lens multiplying power, slide number, initial focusing range and maximum allowable focal plane deviation experimental basic parameters are recorded at the position of the table head of the CSV file, thirdly, the system stores the focal plane matrix file in the same time, and stores the focal plane data in the same time, and performs the same time, and records the focal plane deviation statistics according to the experimental data of the first and the focal plane matrix, and directly stores the focal plane data according to the same time, and the focal plane deviation is recorded by the same time, and the focal plane data is recorded and the focal plane data is directly stored in the same time, and the focal plane matrix is recorded according to the method.
  8. 8. A rapid auto-focusing method for cross-slide microscopic imaging according to claim 1 is characterized in that the auto-focusing operation in the steps S1, S3 and S4 is carried out by adopting a definition evaluation and misfocus identification mechanism optimized for a slide biological sample, specifically according to the following steps, firstly, when an auto-focusing acquisition image is executed, a system firstly extracts a region of interest from each frame of acquired image, automatically identifies a slide biological sample effective region in the image, shields invalid regions such as a glass region, a glass blank region, a solution bubble region and an impurity shielding region at the edge of the slide, calculates a definition evaluation value only for the effective sample region, secondly, calculates an image definition evaluation value in the effective region by adopting a gradient variance method, optimizes the gradient calculated convolution kernel size for the microscopic image characteristics of a cell biological sample, and preferentially identifies the gradient change of the cell nucleus and cytoskeletal sample characteristics, thirdly, after the Z-axis coordinates corresponding to the definition evaluation value peak are located, the system automatically carries out the misfocus identification verification, firstly compares the peak position with a reference focal plane position, and then the focal plane position is well-defined, and the focal plane position is well-defined after the focal plane position is well-defined, and the focal plane position is well-being well-known.
  9. 9. The rapid auto-focusing method for cross-slide microscopic imaging according to claim 1, further comprising an emergency treatment and closed-loop optimization mechanism for focusing failure, wherein the mechanism is implemented through all focusing processes of steps S1 to S4, and specifically comprises the following steps that firstly, when the auto-focusing operation of each slide is executed, the system monitors the state of the focusing process in real time, and if no effective definition evaluation value peak value is detected in a preset focusing range, or the definition evaluation value peak value is lower than a preset effective threshold value, or the misfocus identification check is not passed, the system determines that the focusing fails at the present time; the third step, if the first retry still fails to focus, the system automatically calls the global focus plane matrix, selects the focus plane position of the finished focus crawling plate with highest credibility weight value from 8 adjacent positions around the current crawling plate to be focused as a new reference standard, resets the focus range to execute the automatic focusing operation, if no crawling plate with finished focusing is arranged around, the system executes the full-range focusing by adopting the initial focus range preset by the user, and fourth step, if the three focusing operations are failed, the system automatically records the position information and the focusing failure state of the crawling plate, stores the focusing and imaging operation of the crawling plate, and continues to execute the focusing flow of the follow-up crawling plate, and the fifth step, after completing the focusing flow of all crawling plates, the system automatically generates a focusing failure statistics report to mark the position of all crawling plates with failed focusing, meanwhile, the success rate of focusing in the experiment is counted, the reason of focusing failure is analyzed, and focusing strategies and focusing range settings of the subsequent experiments of the same type are automatically optimized for the area with continuously occurring focusing failure, so that closed loop optimization is formed.
  10. 10. The rapid auto-focusing method for cross-slide microscopic imaging is characterized in that the rapid auto-focusing method is adaptive to imaging scenes of objective lenses with different multiplying powers, and specific adaptation logic is that before an experiment starts, a system automatically matches focusing parameters such as corresponding Z-axis stepping precision, initial focusing range, basic narrow focusing range, definition evaluation convolution kernel size, focal plane deviation threshold and the like according to objective lens multiplying powers selected by a user, the system reduces the basic narrow focusing range for high-multiplying power objective lens scenes, improves stepping precision of focal plane secondary verification, optimizes scanning paths for low-multiplying power objective lens scenes, enlarges focal plane reference ranges of adjacent climbing lenses, automatically completes focal plane coordinate conversion between different multiplying powers for scenes with the same batch of objective lens multiplying powers to be switched, does not need to re-execute full-range initial focusing, and synchronously records focusing experiment data under different objective lens multiplying powers to continuously optimize focusing parameters corresponding to the multiplying powers.

Description

Cross-slide microscopic imaging rapid automatic focusing method Technical Field The invention relates to the technical field of microscopic imaging, in particular to a rapid automatic focusing method for cross-slide microscopic imaging. Background In the field of microscopic imaging, particularly biomedical research, rapid and accurate autofocus of biological samples on slides is a critical step in obtaining high quality microscopic images. With the continued development of microscopic imaging techniques, researchers often need to process a large number of slide samples and perform multiple imaging under different experimental conditions. The traditional microscopic imaging automatic focusing method has several remarkable defects when processing a cross-slide and multi-slide experiment. Firstly, the traditional method often needs to perform independent full-range focusing search on each climbing sheet, which not only takes longer time, but also is easily influenced by factors such as uneven sample distribution, glass slide thickness difference and the like, so that a focusing result is unstable. Secondly, the traditional method lacks effective utilization of the position information of the successfully focused slide, and can not realize quick and intelligent focusing reference in the same slide or between slides, so that the improvement of focusing speed is limited. In addition, the traditional method often lacks an effective misfocus identification and emergency treatment mechanism in the focusing process, and once focusing fails, manual intervention is needed or the focusing process is restarted, so that the experimental efficiency is further reduced. Aiming at the defects of long time consumption, poor stability, lack of intelligent reference, insufficient misfocus processing capability and the like in the process of cross-slide and multi-slide experiments of the traditional microscopic imaging automatic focusing method, the invention provides a rapid automatic focusing method for cross-slide microscopic imaging. Disclosure of Invention The invention aims to make up the defects of the prior art and provides a rapid auto-focusing method for cross-slide microscopic imaging. The invention provides a rapid auto-focusing method for cross-slide microscopic imaging, which aims to solve the technical problems and comprises the following specific steps: s1, initial focusing, namely executing automatic focusing on a first slide of a first slide at the beginning of an experiment by adopting a Z-axis focusing range preset by a user to acquire the focal plane position of the slide; s2, after focusing of a single climbing sheet is completed, recording the focal plane position of the climbing sheet, and synchronously updating a global focal plane matrix, wherein the focal plane matrix records the position information of all the climbing sheets which are completed in focusing and the corresponding focal plane positions; S3, continuously focusing in the same slide, namely preferentially selecting the focal plane positions of the adjacent to-be-focused slide plates above the to-be-focused slide plates except the first slide plate in the same slide plate, setting a narrow focusing range and executing automatic focusing, if the adjacent slide plates above are not focused, selecting the focal plane positions of the adjacent to-be-focused slide plates on the left side to set the narrow focusing range and executing automatic focusing, and if the adjacent slide plates above and on the left are not focused, executing automatic focusing by adopting a Z-axis focusing range preset by a user; S4, focusing across the slide glass, namely, after switching to a new slide glass, preferentially selecting the focal plane position of the adjacent slide glass to be focused, which is above the slide glass and is finished with the focused slide glass, setting a narrow focusing range to execute automatic focusing; S5, data archiving, namely archiving the global focal plane matrix in a preset format after focusing and imaging of all the slide sheets are completed. The initial focusing process of the step S1 is implemented according to the following steps, namely, the first step is implemented according to the following steps, after the optical path calibration of a slide loading and microscopic imaging system is finished by a user, the user is positioned to the central area of the first slide through a preview interface, the preview image completely covers a biological sample area on the slide, interference of the slide edge, a slide blank area and a solution bubble area is eliminated, the second step is implemented according to the multiplying power of an objective lens, the sample type and the thickness of the slide used in the experiment, the corresponding Z-axis initial focusing range is set, meanwhile, Z-axis stepping precision and single-step image acquisition parameters in the focusing process are set, all possible focal plane positions o