Automatic focusing method, device and equipment for fluorescence microscope

Abstract

The invention discloses an automatic focusing method, device and equipment for a fluorescence microscope. The method comprises the following steps: S10, acquiring an image acquired by the microscope and extracting a target cell contour in the image acquired by the microscope; s20, the contour of the target cell is identified, an internal area and an external area are obtained, the internal area is a cell main body part, and the external area is a part except the cell main body part; s30, the definition value of the internal area and the definition value of the external area are calculated respectively, weighting processing is carried out respectively, and the weight corresponding to the definition value of the internal area is larger than the weight corresponding to the definition value of the external area; s40, calculating a global definition score according to a preset formula; and S50, automatically adjusting the focal length of the microscope based on the global definition score. The definition values of the internal region and the external region are respectively calculated and weighted, and the detail definition of the cell main body part is preferentially considered, so that the interference of the halo effect on the focusing result is effectively reduced, and the focusing precision and the imaging quality are improved.

Description

Technical Field

[0001] The invention relates to the technical field of optical microscopes, and in particular to an automatic focusing method, device and equipment for a fluorescence microscope. Background Art

[0002] In the field of fluorescence microscope image processing, image clarity evaluation is crucial to obtain high-quality imaging results. However, there may be large halos in the fluorescence microscope imaging process, which affects the image clarity and the accuracy of the analysis results.

[0003] Current autofocus algorithms are mostly based on image contrast, gradient or spectrum analysis, and tend to process the overall image uniformly. However, when faced with high halo imaging in fluorescence microscopy, these methods often misjudge the halo area as a clear area, resulting in reduced focus accuracy. Especially when the halo effect around cells is obvious, it is difficult for the algorithm to focus on the main part of the cell, which ultimately affects the imaging quality.

[0004] Therefore, in the out-of-focus state during fluorescence microscopy imaging, the existing automatic focusing method for fluorescence microscopy has a poor focusing effect, and a halo effect is easily generated around cells, leading to a misjudgment of image clarity. Summary of the invention

[0005] The purpose of the present invention is to provide a fluorescence microscope automatic focusing method, device and equipment, aiming to solve the technical problem that the existing fluorescence microscope automatic focusing method has poor focusing effect.

[0006] To achieve the above object, the present invention provides a method for automatic focusing of a fluorescence microscope, which comprises the following steps:

[0007] S10, acquiring a microscope image and extracting the target cell contour in the microscope image;

[0008] S20, identifying the target cell contour to obtain an inner region and an outer region, wherein the inner region is the main body of the cell, and the outer region is the portion other than the main body of the cell;

[0009] S30, respectively calculating the clarity values ​​of the inner region and the outer region, and performing weighted processing on them respectively, wherein the weight corresponding to the clarity value of the inner region is greater than the weight corresponding to the clarity value of the outer region;

[0010] S40, calculating a global clarity score according to a preset formula;

[0011] S50, automatically adjust microscope focus based on global sharpness score.

[0012] Optionally, the step of automatically adjusting the focus of the microscope based on the global clarity score in S50 comprises at least the following steps:

[0013] S51, focusing the microscope at a preset initial position, and setting an initial focal length value, which is marked as a current focal length;

[0014] S52, setting the focus range, and starting to focus from the current focal length minus half of the focus range;

[0015] S53, executing S10-S40 to obtain the current global clarity score;

[0016] S54, determining whether the current global clarity score is greater than the global clarity score calculated last time, if so, executing S55, if not, stopping the focus adjustment, automatically recording the last current focus, and locking the microscope at the focus position;

[0017] S55, adjusting the focal length by a preset increment, updating the current focal length, and executing S52-S54 until the global clarity score reaches a peak value. During the adjustment process, it is determined in real time whether the stop condition is met. If so, executing S56;

[0018] S56, automatically recording the focal length value when the focus adjustment is stopped, and locking the microscope at the focus position.

[0019] Optionally, the stop condition is specifically: whether the difference between the current global clarity score and the global clarity score calculated last time is less than a threshold value for N consecutive times, where N is a preset value;

[0020] Alternatively, whether the current global clarity score is 1 / 2 of the peak global clarity score.

[0021] Optionally, before extracting the target cell contour in the image acquired by the microscope in S10, filtering processing is further performed on the image acquired by the microscope.

[0022] Optionally, in S10, the contour of the target cell in the image captured by the microscope is extracted by an edge detection algorithm or a morphological algorithm.

[0023] Optionally, the preset formula is as follows:

[0024] Double result=a*resultCellInside+b*resultCellOutside;

[0025] Among them, Double result is the global clarity score, resultCellInside is the clarity value of the internal area, a is the weight corresponding to the clarity value of the internal area, resultCellOutside is the clarity value of the external area, and b is the weight corresponding to the clarity value of the external area.

[0026] Optional, a is 500 and b is 1.

[0027] Optionally, in S30 , the clarity values ​​of the inner area and the outer area are calculated respectively by Laplace transform or variance function.

[0028] Corresponding to the automatic focusing method for a fluorescence microscope, the present invention provides an automatic focusing device for a fluorescence microscope, comprising:

[0029] An image processing unit, used for acquiring images collected by a microscope and extracting the contours of target cells in the images collected by the microscope;

[0030] The recognition unit is used to recognize the contour of the target cell to obtain an inner region and an outer region, wherein the inner region is the main body of the cell and the outer region is the part other than the main body of the cell;

[0031] A calculation unit, used to calculate the clarity values ​​of the internal area and the external area respectively, and perform weighted processing respectively, wherein the weight corresponding to the clarity value of the internal area is greater than the weight corresponding to the clarity value of the external area; and used to calculate the global clarity score according to a preset formula;

[0032] Focus adjustment unit for automatically adjusting the microscope focus based on the global sharpness score.

[0033] In addition, to achieve the above objectives, the present invention also provides a fluorescence microscope automatic focusing device, which includes a fluorescence microscope and automatic focusing software stored in the fluorescence microscope, and the device is used to implement the fluorescence microscope automatic focusing method as described above.

[0034] The beneficial effects of the present invention are:

[0035] (1) Compared with the prior art, the present invention calculates the clarity values ​​of the inner and outer regions separately and performs weighted processing, giving priority to the detail clarity of the main part of the cell, effectively reducing the interference of the halo effect on the focusing result, and improving the focusing accuracy and imaging quality;

[0036] (2) Compared with the prior art, the present invention ensures the systematicness and accuracy of the focusing process by automatically adjusting the focal length of the microscope based on the global clarity score, and achieves precise focusing by gradually adjusting the focal length and evaluating the clarity in real time;

[0037] (3) Compared with the prior art, the present invention stops focusing in advance when the score changes are less than a threshold for multiple consecutive times or the global clarity score is reduced to 1 / 2 of the maximum value of the global clarity score, thereby optimizing the focus rate, improving the focus efficiency, reducing unnecessary focus adjustments, and saving time and computing resources;

[0038] (4) Compared with the prior art, the present invention reduces noise interference and improves image quality by filtering the microscope-collected image before extracting the target cell contour, thereby improving the accuracy and reliability of subsequent clarity evaluation;

[0039] (5) Compared with the prior art, the present invention uses edge detection algorithm or morphological algorithm to extract the target cell contour, ensuring the accuracy and robustness of contour extraction, providing a reliable basis for subsequent area marking and clarity evaluation, and improving the overall performance of the focusing algorithm;

[0040] (6) Compared with the prior art, the present invention highlights the importance of the internal details of the cell by weighted processing of the clarity values ​​of the internal area and the external area, so that the focusing algorithm is more in line with the needs of users and the focusing accuracy and imaging quality are improved;

[0041] (7) Compared with the prior art, the present invention calculates the clarity values ​​of the internal area and the external area through Laplace transform or variance function, provides an effective clarity evaluation method, ensures that the focusing algorithm can accurately evaluate the clarity of the image, and improves the accuracy and reliability of focusing;

[0042] (8) Compared with the prior art, the present invention provides an automatic focusing device for a fluorescence microscope, comprising an image processing unit, a recognition unit, a calculation unit and a focal length adjustment unit, which realizes an automated process from image acquisition to focus adjustment, improves the efficiency and accuracy of focusing, and is suitable for various fluorescence microscope imaging applications;

[0043] (9) Compared with the prior art, the present invention provides an automatic focusing device for a fluorescence microscope, which integrates a fluorescence microscope and automatic focusing software, realizes the automation and intelligence of the focusing method, improves the ease of use and imaging quality of the microscope, and is suitable for microscope imaging needs in scientific research, medical treatment, industry and other fields.

[0044] These and other aspects of the present application will be more concise and understandable in the following description of the embodiments. It should be understood that the above general description and the following detailed description are only exemplary and explanatory and cannot limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other embodiments can be obtained based on these drawings without paying creative work.

[0046] In the figure:

[0047] Figure 1The figure is a flow chart of an embodiment of the automatic focusing method for a fluorescence microscope of the present invention.

[0048] Figure 2 This is a framework diagram of an embodiment of an automatic focusing device for a fluorescence microscope of the present invention. DETAILED DESCRIPTION

[0049] Below, the present application is further described in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0050] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in combination with specific embodiments and with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0051] It should be noted that all expressions using "first" and "second" in the embodiments of the present invention are intended to distinguish two non-identical entities or non-identical parameters with the same name. It can be seen that "first" and "second" are only for the convenience of expression and should not be understood as limitations on the embodiments of the present invention. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, other steps or units inherent to a process, method, device, product or equipment that includes a series of steps or units.

[0052] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0053] The flowcharts shown in the accompanying drawings are only examples and do not necessarily include all the contents and operations / steps, nor must they be executed in the order described. For example, some operations / steps may also be decomposed, combined or partially merged, so the actual execution order may change according to actual conditions.

[0054] In conjunction with the accompanying drawings, some embodiments of the present application are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0055] See also Figure 1 As shown, an automatic focusing method for a fluorescence microscope provided by an embodiment of the present invention comprises the following steps: S10, acquiring a microscope-collected image and extracting the target cell contour in the microscope-collected image;

[0056] S20, identifying the target cell contour to obtain an inner region and an outer region, wherein the inner region is the main body of the cell, and the outer region is the portion other than the main body of the cell;

[0057] S30, respectively calculating the clarity values ​​of the inner region and the outer region, and performing weighted processing on them respectively, wherein the weight corresponding to the clarity value of the inner region is greater than the weight corresponding to the clarity value of the outer region;

[0058] S40, calculating a global clarity score according to a preset formula;

[0059] S50, automatically adjust microscope focus based on global sharpness score.

[0060] Preferably, S20 includes at least the following steps:

[0061] S21, performing adaptive threshold segmentation on the microscope collected image by using a histogram double threshold method (including a triangle method and an Otsu method) to extract bright spots and target contour areas;

[0062] S22, after removing the larger and smaller contours, the remaining contour area is identified as the external area;

[0063] S23, structurally processing the outer contour using operators such as dilation and erosion to obtain an inner area and an outer area.

[0064] The outer region is mainly used to calculate the clarity of the cell boundary contour, and the inner region is mainly used to calculate the clarity of the detail features within the cell interior.

[0065] It should be noted that in specific application scenarios, the larger and smaller standards can be set according to the observed cell diameter. For example, if the observed cell diameter is 10um, then the larger is larger than 15um and the smaller is smaller than 5um. In this application scenario, contours larger than 15um and smaller than 5um are removed, and contours with a diameter of 5um to 15um are retained.

[0066] That is, in this embodiment, the specific standards of "larger" and "smaller" are set according to the diameter of the observed cells. Based on the cell diameter, the larger standard is set to 1.5 times the cell diameter, and the smaller standard is set to 1 / 2 of the cell diameter.

[0067] The present invention calculates the clarity values ​​of the inner area and the outer area respectively and performs weighted processing, giving priority to the detail clarity of the main part of the cell, effectively reducing the interference of the halo effect on the focusing result, and improving the focusing accuracy and imaging quality.

[0068] In this embodiment, automatically adjusting the focus of the microscope based on the global clarity score in S50 at least includes the following steps:

[0069] S51, focusing the microscope at a preset initial position, and setting an initial focal length value, which is marked as a current focal length;

[0070] S52, setting the focus range, and starting to focus from the current focal length minus half of the focus range;

[0071] S53, executing S10-S40 to obtain the current global clarity score;

[0072] S54, determining whether the current global clarity score is greater than the global clarity score calculated last time, if so, executing S55, if not, stopping the focus adjustment, automatically recording the last current focus, and locking the microscope at the focus position;

[0073] S55, adjusting the focal length by a preset increment, updating the current focal length, and executing S52-S54 until the global clarity score reaches a peak value. During the adjustment process, it is determined in real time whether the stop condition is met. If so, executing S56;

[0074] S56, automatically recording the focal length value when the focus adjustment is stopped, and locking the microscope at the focus position.

[0075] In this embodiment, in S52, focusing starts from the current focal length - focus range / 2. The focus range is mainly set according to the sample. If the sample is relatively flat, a smaller range can be set. If the sample is not flat, a larger range needs to be set. "The sample is relatively flat" means that when the focus adjustment is performed, the processed image surface is relatively flat, without obvious ups and downs or complex textures. In this case, the focus range can be set smaller, because the smaller focus range is sufficient to cover the entire image or object to ensure its clarity. On the contrary, if the sample surface is not flat, with obvious ups and downs or complex textures, then the focus range needs to be set larger to ensure that areas of different depths can be correctly focused, thereby obtaining better overall clarity.

[0076] Preferably, the preset increment in S55 can be preset according to actual needs. The preset increment is specifically related to the depth of field of the objective lens, the exposure time during focusing, and the Z-axis movement speed. The smaller the preset increment, the higher the focusing accuracy and the longer the focusing time.

[0077] The present invention ensures the systematicness and accuracy of the focusing process by automatically adjusting the focal length of the microscope based on the global clarity score, and achieves precise focusing by gradually adjusting the focal length and evaluating the clarity in real time.

[0078] In this embodiment, the stop condition is specifically: whether the difference between the current global clarity score and the last calculated global clarity score is less than a threshold value for N consecutive times, where N is a preset value;

[0079] Alternatively, whether the current global clarity score is 1 / 2 of the peak global clarity score.

[0080] Preferably, the threshold is 1 / 20 of the current global clarity score.

[0081] It should be noted that the global clarity score peak is specifically the maximum value of the global clarity score.

[0082] The present invention stops focusing in advance when the score changes are less than a threshold for multiple consecutive times or the global clarity score is reduced to 1 / 2 of the maximum value of the global clarity score, thereby optimizing the focus rate, improving the focus efficiency, reducing unnecessary focus adjustments, and saving time and computing resources.

[0083] In this embodiment, before extracting the target cell contour in the image collected by the microscope in S10, the image collected by the microscope is filtered.

[0084] The present invention reduces noise interference and improves image quality by filtering the image collected by the microscope before extracting the target cell contour, thereby improving the accuracy and reliability of subsequent clarity evaluation.

[0085] In this embodiment, in S10, the contour of the target cell in the image collected by the microscope is extracted by an edge detection algorithm or a morphological algorithm.

[0086] The present invention uses an edge detection algorithm or a morphological algorithm to extract the target cell contour, ensures the accuracy and robustness of the contour extraction, provides a reliable basis for subsequent area marking and clarity evaluation, and improves the overall performance of the focusing algorithm.

[0087] In this embodiment, the preset formula is as follows:

[0088] Double result=a*resultCellInside+b*resultCellOutside;

[0089] Among them, Double result is the global clarity score, resultCellInside is the clarity value of the internal area, a is the weight corresponding to the clarity value of the internal area, resultCellOutside is the clarity value of the external area, and b is the weight corresponding to the clarity value of the external area.

[0090] In this embodiment, a is 500 and b is 1.

[0091] It should be noted that the weight a corresponding to the internal area clarity value and the weight b corresponding to the external area clarity value are not fixed at 500 and 1. If the samples are very different (determined according to actual needs), they can be adjusted according to actual needs. The value range of a and b is [1, 10000]. The principle for adjustment according to actual needs is: set a higher weight for the detailed texture inside the cell (i.e. a>b). By adjusting the weight a corresponding to the internal area clarity value and the weight b corresponding to the external area clarity value, the focus evaluation can be dynamically optimized according to different sample types, with higher adaptability.

[0092] The present invention highlights the importance of internal details of cells by weighted processing of the clarity values ​​of internal and external areas, so that the focusing algorithm better meets the needs of users and improves the focusing accuracy and imaging quality.

[0093] In this embodiment, in S30 , the clarity values ​​of the inner area and the outer area are calculated respectively by Laplace transform or variance function.

[0094] The present invention calculates the clarity values ​​of the internal area and the external area through Laplace transform or variance function, provides an effective clarity evaluation method, ensures that the focusing algorithm can accurately evaluate the clarity of the image, and improves the accuracy and reliability of focusing.

[0095] like Figure 2 As shown, the present invention also provides a corresponding automatic focusing device for a fluorescence microscope, which comprises:

[0096] An image processing unit 10 is used to obtain a microscope-collected image and extract the target cell contour in the microscope-collected image;

[0097] The recognition unit 20 is used to recognize the target cell contour to obtain an inner region and an outer region, wherein the inner region is the main body of the cell and the outer region is the part other than the main body of the cell;

[0098] A calculation unit 30, for calculating the clarity values ​​of the inner region and the outer region respectively, and performing weighted processing on them respectively, wherein the weight corresponding to the clarity value of the inner region is greater than the weight corresponding to the clarity value of the outer region; and for calculating the global clarity score according to a preset formula;

[0099] The focus adjustment unit 40 is used to automatically adjust the focus of the microscope based on the global clarity score.

[0100] The present invention provides an automatic focusing device for a fluorescence microscope, comprising an image processing unit 10, a recognition unit 20, a calculation unit 30 and a focal length adjustment unit 40, which realizes an automated process from image acquisition to focus adjustment, improves the efficiency and accuracy of focusing, and is suitable for various fluorescence microscope imaging applications.

[0101] An embodiment of the present invention further provides an automatic focusing device for a fluorescence microscope, which includes a fluorescence microscope and automatic focusing software stored in the fluorescence microscope, and the device is used to implement the automatic focusing method for a fluorescence microscope as described above.

[0102] The present invention provides an automatic focusing device for a fluorescence microscope, which integrates a fluorescence microscope and automatic focusing software, realizes automation and intelligence of a focusing method, improves the convenience of use and imaging quality of the microscope, and is suitable for microscope imaging needs in the fields of scientific research, medical treatment, and industry.

[0103] It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments can be referred to each other. For the device embodiments and equipment embodiments, since they are basically similar to the method embodiments, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiments.

[0104] The above description shows and describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention, through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not depart from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for automatic focusing of a fluorescence microscope, characterized in that: The following steps are involved: S10, acquiring a microscope image and extracting the target cell contour in the microscope image; S20, identifying the target cell contour to obtain an inner region and an outer region, wherein the inner region is the main body of the cell, and the outer region is the portion other than the main body of the cell; S30, respectively calculating the clarity values ​​of the inner region and the outer region, and performing weighted processing on them respectively, wherein the weight corresponding to the clarity value of the inner region is greater than the weight corresponding to the clarity value of the outer region; S40, calculating a global clarity score according to a preset formula; S50, automatically adjust microscope focus based on global sharpness score.

2. The automatic focusing method for a fluorescence microscope according to claim 1, characterized in that: Automatically adjusting the microscope focus based on the global sharpness score in S50 includes at least the following steps: S51, focusing the microscope at a preset initial position, and setting an initial focal length value, which is marked as a current focal length; S52, setting the focus range, and starting to focus from the current focal length minus half of the focus range; S53, executing S10-S40 to obtain the current global clarity score; S54, determining whether the current global clarity score is greater than the global clarity score calculated last time, if so, executing S55, if not, stopping the focus adjustment, automatically recording the last current focus, and locking the microscope at the focus position; S55, adjusting the focal length by a preset increment, updating the current focal length, and executing S52-S54 until the global clarity score reaches a peak value. During the adjustment process, it is determined in real time whether the stop condition is met. If so, executing S56; S56, stop focus adjustment, automatically record the current focus, and lock the microscope at the focus position.

3. The automatic focusing method for a fluorescence microscope according to claim 2, characterized in that: The stop condition is specifically: whether the difference between the current global clarity score and the last calculated global clarity score is less than a threshold value for N consecutive times, where N is a preset value; Alternatively, whether the current global clarity score is 1 / 2 of the peak global clarity score.

4. The automatic focusing method for a fluorescence microscope according to claim 1, characterized in that: In S10, before extracting the target cell contour in the image collected by the microscope, the image collected by the microscope is filtered.

5. The automatic focusing method for a fluorescence microscope according to claim 1, characterized in that: In S10, the contour of the target cell in the image collected by the microscope is extracted by an edge detection algorithm or a morphological algorithm.

6. The automatic focusing method for a fluorescence microscope according to claim 1, characterized in that: The preset formula is as follows: Double result=a*resultCellInside+b*resultCellOutside; Among them, Double result is the global clarity score, resultCellInside is the clarity value of the internal area, a is the weight corresponding to the clarity value of the internal area, resultCellOutside is the clarity value of the external area, and b is the weight corresponding to the clarity value of the external area.

7. The automatic focusing method for a fluorescence microscope according to claim 6, characterized in that: a is 500 and b is 1.

8. The automatic focusing method for a fluorescence microscope according to claim 1, characterized in that: In S30 , the clarity values ​​of the inner region and the outer region are calculated respectively by Laplace transform or variance function.

9. An automatic focusing device for a fluorescence microscope, characterized in that: include: An image processing unit, used for acquiring images collected by a microscope and extracting the contours of target cells in the images collected by the microscope; The recognition unit is used to recognize the contour of the target cell to obtain an internal area and an external area, wherein the internal area is the main part of the cell and the external area is the part outside the main part of the cell; A calculation unit, used to calculate the clarity values ​​of the internal area and the external area respectively, and perform weighted processing respectively, wherein the weight corresponding to the clarity value of the internal area is greater than the weight corresponding to the clarity value of the external area; and used to calculate the global clarity score according to a preset formula; Focus adjustment unit for automatically adjusting the microscope focus based on the global sharpness score.

10. An automatic focusing device for a fluorescence microscope, characterized in that: The fluorescence microscope automatic focusing device comprises a fluorescence microscope and automatic focusing software stored in the fluorescence microscope, and the device is used to implement the fluorescence microscope automatic focusing method according to any one of claims 1 to 8.

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