CN110865453A - Automatic focusing method of automatic microscope - Google Patents
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- CN110865453A CN110865453A CN201910921045.5A CN201910921045A CN110865453A CN 110865453 A CN110865453 A CN 110865453A CN 201910921045 A CN201910921045 A CN 201910921045A CN 110865453 A CN110865453 A CN 110865453A
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Abstract
The invention discloses an automatic focusing method of an automatic microscope, which comprises the following steps: a preprocessing step, namely moving a focusing device on the automatic microscope onto a slice to obtain an image of the current position, calculating the definition of the image, setting the definition and the position of the current position as initial definition and position, and setting a preset step length and an initial direction; searching, namely searching a peak value by a preset step length to obtain a rising edge and a falling edge; and fitting the peak value, the rising edge and the falling edge to find out the optimal focal plane position, and moving a focusing device on the automatic microscope to the position to complete the focusing process. According to the method, through foreground region extraction, peak value, rising edge and falling edge measurement, parabola fitting and four-point unimodal fitting, the defects of insufficient focusing speed and accuracy of a traditional automatic focusing method are solved, and the problem that the accuracy is lower when the field of view with extremely low contrast or extremely little foreground content is focused is solved.
Description
Technical Field
The invention relates to the technical field of microscope focusing, in particular to a main camera focusing method, and specifically relates to an automatic microscope focusing method aiming at improving the speed and accuracy of automatic focusing in digital slice scanning, especially the accuracy of focusing on an extremely low-contrast or extremely low-foreground target.
Background
Pathological diagnosis is an important clinical diagnosis basis, and the accuracy of pathological diagnosis directly affects the health and fate of patients. In the conventional pathological diagnosis, a doctor observes and analyzes cells or tissues in pathological sections one by one under a microscope to make a diagnosis suggestion. The process is time-consuming and labor-consuming, so that the body, especially eyes, of a doctor are easy to fatigue, and missed diagnosis or misdiagnosis is caused; for some difficult cases, it is also not advantageous to seek third party consultation (especially remote consultation) because of the possible risks of delay, breakage and loss during transportation. The 'digital pathology' provides a perfect solution to these problems, and it acquires digital images of each visual field area in the glass slice by means of scanning and splices them into digital slices, and then presents the digital slices on a display terminal by means of software for a doctor to browse and analyze. The digitalization mode relieves the pathologist from the heavy under-mirror film reading, provides natural support for third-party consultation, particularly remote consultation and provides possibility for better quantitative analysis.
As can be seen from the above, the basis of digital pathology is the digitization of glass sections. Two of the most important requirements for this process: firstly, the quality of the digital slice, the most key measurement index is the definition of the image; the second is the speed at which the digital slices are generated. The former is determined by the focusing method during scanning, and the latter is also largely affected by the focusing method. Focusing comprises automatic focusing, active focusing, passive focusing, auxiliary camera focusing and main camera focusing, as the focusing mode essential in digital slice scanning, the main camera focusing method has good performance in speed, precision and adaptive range, and in consideration of quality, most of the existing digital slice scanning systems adopt the passive focusing mode for focusing, however, the existing methods have the following problems:
1. the existing method needs to reduce the step length when turning back in the searching process, although the total searching distance is shorter and shorter, the searching times in the previous turning back process can not be reduced a lot;
2. the method has the advantages that the anti-noise capability is poor, the local extreme value is easy to fall into in the searching process, focusing is not accurate, the foldback judgment of the existing method is only based on the definition comparison between the current position and a plurality of continuous positions in front of the current position, the judgment is easy to meet in the range containing a smaller local extreme value, and the judgment is especially suitable for the visual field area with low contrast or less prospect;
3. focusing on a low-contrast or low-foreground view is prone to errors, all pixels in a calculation area are viewed equally when the definition is calculated by the existing method, so that when the definition is calculated on the low-contrast or low-foreground view, the contribution of the pixels belonging to the background to the result is possibly close to or even larger than the contribution of the pixels belonging to the foreground to the result, the calculation result cannot reflect the real definition, or the obtained definition evaluation curve has poor unimodal performance, and finally the focusing fails.
In view of the above, the present invention provides an automatic focusing method for an automatic microscope, which is fast, has good noise immunity, and is suitable for both high and low contrast ratios and high and low prospects.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an automatic focusing method of an automatic microscope, which solves the problems of insufficient focusing speed and accuracy of the traditional automatic focusing method, and lower accuracy when focusing the visual field with extremely low contrast or extremely little foreground content.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: an automatic microscope auto-focus method, the method comprising the steps of:
a pretreatment step: moving a focusing device on the automatic microscope onto a slice to obtain an image of the current position, calculating the definition of the image, setting the definition and the position of the current position as the initial definition and the position, and setting a preset step length and an initial direction;
a searching step: searching a peak value according to a preset step length, and obtaining a rising edge and a falling edge of the peak value;
fitting: and fitting the peak value, the rising edge and the falling edge to find out the optimal focal plane position, and moving a focusing device on the automatic microscope to the position to finish the focusing process.
Preferably, the method for calculating the sharpness of the image in the preprocessing step includes:
a. extracting a foreground area of the image;
b. and taking the foreground area as a calculation area to perform definition calculation.
Preferably, the step of preprocessing sets the definition and the position of the current position as the initial definition and position, and specifically includes the following steps:
a. initializing definition peak values and position settings thereof in a current position area;
b. initializing the left endpoint position and definition setting searched in the current position area;
c. and initializing the position and definition setting of the right end point searched in the current position area.
Preferably, the specific step of searching for the peak, the rising edge and the falling edge with a preset step length in the searching step includes;
s1: judging whether the whole stroke of the focusing device is searched, if so, determining that the focusing is failed and quitting, and if not, entering the next step;
s2: moving the focusing device;
s3: taking a picture and calculating definition;
s4: updating the left endpoint and the right endpoint with the current position and the definition;
s5: judging whether the current definition is greater than the peak value, if so, entering the next step, otherwise, turning to S9;
s6: updating the peak value and the corresponding position by using the current definition and the position;
s7: updating the rising edge or the falling edge, if the current moving direction is positive (namely increasing), searching a position with the difference between the definition and the peak value larger than a given threshold value and the position smaller than and closest to the peak value in the searched position, if the current moving direction is negative (namely decreasing), searching a position with the difference between the definition and the peak value larger than the given threshold value and the position larger than and closest to the peak value in the searched position as the rising edge of the current position, if the current moving direction is negative (namely decreasing), and if the current moving direction is negative, searching the position with the difference between the definition and the peak value larger than the given threshold value and the position larger than and closest to the peak value as the falling edge;
s8: judging whether the rising edge and the falling edge are found, if so, entering a fitting step, otherwise, turning to S1;
s9: judging whether the difference between the current definition and the peak value is larger than a given threshold value, if so, entering the next step, otherwise, turning to S13;
s10: updating the rising edge or the falling edge, if the current moving direction is positive (namely increased), taking the position as the rising edge of the current position, otherwise, taking the position as the falling edge of the current position;
s11: judging whether the rising edge and the falling edge are found, if so, entering a fitting step, otherwise, entering the next step;
s12: reversing the moving direction, moving the focusing device to the left end point or the right end point, and then turning to S1;
s13: the number of positions successively smaller than the sharpness peak in the forward direction is updated, and if the number is larger than a given threshold, the routine goes to S12, otherwise, the routine goes to S1.
Preferably, the step of fitting the peak value and the rising edge and the falling edge in the step of fitting includes:
s1: judging whether the difference between the definition values corresponding to the rising edge and the falling edge is smaller than a given threshold value, if so, moving the focusing device to the middle position of the two, and exiting the device after the focusing is considered to be successful, otherwise, entering the next step;
s2: carrying out parabolic fitting by using the rising edge, the peak value and the falling edge to obtain the extreme value position of a parabola;
s3: moving the focusing device to the maximum position, then taking the image and calculating the definition;
s4: judging whether the difference between the definition and the peak value at the maximum value is smaller than a given threshold value, if so, moving the focusing device to the middle position of the two, and exiting the focusing device after the focusing is considered to be successful, otherwise, entering the next step;
s5: fitting a unimodal symmetric curve by using four points of a rising edge, a peak value, a maximum value and a falling edge to obtain the maximum value position of the fitted curve;
s6: and moving the focusing device to the maximum position to exit successfully.
Preferably, the unimodal symmetric curve in the fitting step S5 may be set to be one of a parabola, a lorentz curve, a gaussian curve, and the like.
(III) advantageous effects
The invention provides an automatic focusing method of an automatic microscope, which has the following beneficial effects:
(1) the invention has high focusing speed, and only one step length is used in the searching process to obtain a preliminary peak value, a rising edge and a falling edge so as to solve the problem of excessive searching times.
(2) The method has good noise immunity, the foreground region is adopted for definition calculation during calculation, in order to avoid trapping in a local extreme value, the current value is compared with the peak value so far in the turn-back judgment of search, and the non-current value is compared with the definition values of the previous times continuously.
(3) The method has good adaptability, can be used for focusing high-contrast or high-foreground targets and can also be used for focusing low-contrast and low-foreground targets, only the definition threshold value needs to be adjusted, the focusing precision is high, parabolic fitting and four-point unimodal fitting are sequentially carried out on the found peak value and the rising edge and the falling edge of the peak value after the searching is finished, and the final fitting result is used as the final focal plane position.
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FIG. 1 is a schematic block diagram of the present invention;
FIG. 2 is a flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1-2, the present invention provides a technical solution: an automatic focusing method of an automatic microscope, comprising the following steps:
a pretreatment step: moving a focusing device on the automatic microscope onto a slice to obtain an image of a current position, calculating the definition of the image, setting the definition and the position of the current position as an initialization definition and a position, and setting a preset step length and an initial direction, wherein the definition calculating method of the image comprises the following steps: extracting a foreground area of the image; taking the foreground area as a calculation area to calculate the definition, and setting the definition and the position of the current position as the initial definition and position, wherein the method specifically comprises the following steps: initializing definition peak values and position settings thereof in a current position area; initializing the left endpoint position and definition setting searched in the current position area; initializing the position and definition setting of the right end point searched in the current position area;
a searching step: searching a peak value according to a preset step length, and obtaining a rising edge and a falling edge of the peak value;
the method comprises the following specific steps of;
s1: judging whether the whole stroke of the focusing device is searched, if so, determining that the focusing is failed and quitting, and if not, entering the next step;
s2: moving the focusing device;
s3: taking a picture and calculating definition;
s4: updating the left endpoint and the right endpoint with the current position and the definition;
s5: judging whether the current definition is greater than the peak value, if so, entering the next step, otherwise, turning to S9;
s6: updating the peak value and the corresponding position by using the current definition and the position;
s7: updating the rising edge or the falling edge, if the current moving direction is positive (namely increasing), searching a position with the difference between the definition and the peak value larger than a given threshold value and the position smaller than and closest to the peak value in the searched position, if the current moving direction is negative (namely decreasing), searching a position with the difference between the definition and the peak value larger than the given threshold value and the position larger than and closest to the peak value in the searched position as the rising edge of the current position, if the current moving direction is negative (namely decreasing), and if the current moving direction is negative, searching the position with the difference between the definition and the peak value larger than the given threshold value and the position larger than and closest to the peak value as the falling edge;
s8: judging whether the rising edge and the falling edge are found, if so, entering a fitting step, otherwise, turning to S1;
s9: judging whether the difference between the current definition and the peak value is larger than a given threshold value, if so, entering the next step, otherwise, turning to S13;
s10: updating the rising edge or the falling edge, if the current moving direction is positive (namely increased), taking the position as the rising edge of the current position, otherwise, taking the position as the falling edge of the current position;
s11: judging whether the rising edge and the falling edge are found, if so, entering a fitting step, otherwise, entering the next step;
s12: reversing the moving direction, moving the focusing device to the left end point or the right end point, and then turning to S1;
s13: updating the number of positions successively smaller than the sharpness peak in the forward direction, and if the number is larger than a given threshold, turning to S12, otherwise, turning to S1;
fitting: fitting the peak value, the rising edge and the falling edge, finding out the optimal focal plane position, and moving a focusing device on the automatic microscope to the position to complete the focusing process;
the method comprises the following specific steps:
s1: judging whether the difference between the definition values corresponding to the rising edge and the falling edge is smaller than a given threshold value, if so, moving the focusing device to the middle position of the two, and exiting the device after the focusing is considered to be successful, otherwise, entering the next step;
s2: carrying out parabolic fitting by using the rising edge, the peak value and the falling edge to obtain the extreme value position of a parabola;
s3: moving the focusing device to the maximum position, then taking the image and calculating the definition;
s4: judging whether the difference between the definition and the peak value at the maximum value is smaller than a given threshold value, if so, moving the focusing device to the middle position of the two, and exiting the focusing device after the focusing is considered to be successful, otherwise, entering the next step;
s5: fitting a unimodal symmetric curve by using four points of a rising edge, a peak value, a maximum value and a falling edge to obtain the maximum value position of the fitted curve, wherein the unimodal symmetric curve can be set to be a parabola, a Lorentz curve, a Gaussian curve and the like;
s6: and moving the focusing device to the maximum position to exit successfully.
The working principle is as follows: the invention provides an automatic focusing method, which belongs to passive focusing, in particular to focusing of a main camera, and aims to improve the speed and the accuracy of automatic focusing in digital slice scanning, in particular to improve the accuracy of focusing of an extremely low contrast ratio or an extremely low foreground target, and in order to adapt to the situations of low contrast ratio and low foreground vision, the invention firstly improves a calculation area in a definition calculation method: adding a foreground extraction step to extract the region occupied by the foreground in the image, and taking the region as a calculation region; in terms of sharpness operator selection, an autocorrelation operator is employed in a fluorescence scanning system, and a normalized variance operator is employed in a brightfield scanning system; in order to accelerate the speed, the invention only adopts a thicker step length to search, the purpose is to obtain the peak value meeting the requirement and the rising edge and the falling edge thereof, in order to avoid falling into a local extreme value, the present value is adopted to compare with the peak value so far in the turning back judgment of the search, but the present value is not compared with the definition values of the previous times continuously; in order to improve focusing accuracy, parabolic fitting and four-point single-peak fitting are further carried out on the found peak value and the rising edge and the falling edge of the peak value in sequence after the search is finished, and the final fitting result is used as the final focal plane position.
In conclusion, the method solves the problems of insufficient focusing speed and accuracy of the traditional automatic focusing method and lower accuracy of the focusing of the extremely low-contrast or extremely low-foreground target through foreground region extraction, peak value, rising edge and falling edge measurement, parabolic fitting and four-point single-peak fitting.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. An automatic focusing method of an automatic microscope is characterized in that: the method comprises the following steps:
a pretreatment step: moving a focusing device on the automatic microscope onto a slice to obtain an image of the current position, calculating the definition of the image, setting the definition and the position of the current position as the initial definition and the position, and setting a preset step length and an initial direction;
a searching step: searching a peak value according to a preset step length, and obtaining a rising edge and a falling edge of the peak value;
fitting: and fitting the peak value, the rising edge and the falling edge to find out the optimal focal plane position, and moving a focusing device on the automatic microscope to the position to finish the focusing process.
2. The automatic microscope auto-focusing method according to claim 1, wherein: the method for calculating the definition of the image in the preprocessing step comprises the following steps:
a. extracting a foreground area of the image;
b. and taking the foreground area as a calculation area to perform definition calculation.
3. The automatic microscope auto-focusing method according to claim 1, wherein: in the preprocessing step, the definition and the position of the current position are set as the initial definition and the initial position, and the method specifically comprises the following steps:
a. initializing definition peak values and position settings thereof in a current position area;
b. initializing the left endpoint position and definition setting searched in the current position area;
c. and initializing the position and definition setting of the right end point searched in the current position area.
4. The automatic microscope auto-focusing method according to claim 1, wherein: the specific steps of searching for the peak value, the rising edge and the falling edge by preset step length in the searching step comprise;
s1: judging whether the whole stroke of the focusing device is searched, if so, determining that the focusing is failed and quitting, and if not, entering the next step;
s2: moving the focusing device;
s3: taking a picture and calculating definition;
s4: updating the left endpoint and the right endpoint with the current position and the definition;
s5: judging whether the current definition is greater than the peak value, if so, entering the next step, otherwise, turning to S9;
s6: updating the peak value and the corresponding position by using the current definition and the position;
s7: updating the rising edge or the falling edge, if the current moving direction is positive, searching a position where the difference between the definition and the peak value is greater than a given threshold value and the position is less than and closest to the peak value in the searched position, if the current moving direction is negative, using the position as the rising edge of the current position, if the current moving direction is negative, searching a position where the difference between the definition and the peak value is greater than the given threshold value and the position is greater than and closest to the peak value in the searched position, and if the current moving direction is negative, using the position as the falling edge of the current position;
s8: judging whether the rising edge and the falling edge are found, if so, entering a fitting step, otherwise, turning to S1;
s9: judging whether the difference between the current definition and the peak value is larger than a given threshold value, if so, entering the next step, otherwise, turning to S13;
s10: updating the rising edge or the falling edge, if the current moving direction is positive, taking the position as the rising edge of the current position, otherwise, taking the position as the falling edge of the current position;
s11: judging whether the rising edge and the falling edge are found, if so, entering a fitting step, otherwise, entering the next step;
s12: the moving direction is reversed, the focusing device is moved to the left end point or the right end point, and then the operation is switched to Step2.1;
s13: the number of positions successively smaller than the sharpness peak in the forward direction is updated, and if the number is larger than a given threshold, the routine goes to S12, otherwise, the routine goes to S1.
5. The automatic microscope auto-focusing method according to claim 1, wherein: the specific step of fitting the peak value and the rising edge and the falling edge in the fitting step includes:
s1: judging whether the difference between the definition values corresponding to the rising edge and the falling edge is smaller than a given threshold value, if so, moving the focusing device to the middle position of the two, and exiting the device after the focusing is considered to be successful, otherwise, entering the next step;
s2: carrying out parabolic fitting by using the rising edge, the peak value and the falling edge to obtain the extreme value position of a parabola;
s3: moving the focusing device to the maximum position, then taking the image and calculating the definition;
s4: judging whether the difference between the definition and the peak value at the maximum value is smaller than a given threshold value, if so, moving the focusing device to the middle position of the two, and exiting the focusing device after the focusing is considered to be successful, otherwise, entering the next step;
s5: fitting a unimodal symmetric curve by using four points of a rising edge, a peak value, a maximum value and a falling edge to obtain the maximum value position of the fitted curve;
s6: and moving the focusing device to the maximum position to exit successfully.
6. The automatic microscope auto-focus method of claim 5, wherein: the fitting step S5 sets the symmetric curve of the single peak to be one of a parabolic curve, a lorentz curve, or a gaussian curve.
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