CN110824685A - Focal plane determination method and device in microscopic imaging - Google Patents
Focal plane determination method and device in microscopic imaging Download PDFInfo
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- CN110824685A CN110824685A CN201911239271.1A CN201911239271A CN110824685A CN 110824685 A CN110824685 A CN 110824685A CN 201911239271 A CN201911239271 A CN 201911239271A CN 110824685 A CN110824685 A CN 110824685A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/241—Devices for focusing
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0036—Scanning details, e.g. scanning stages
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
Abstract
The application relates to a focal plane determination method and a focal plane determination device in microscopic imaging, which belong to the technical field of computers, and the method comprises the following steps: acquiring n actual focal positions corresponding to n sample positions in a designated area of an observation sample; determining a local focal plane corresponding to the designated area based on the n actual focal positions; forming a whole focal plane corresponding to an observation sample based on the local focal planes corresponding to the designated areas, wherein the observation sample is a union of the designated areas; the problem that when the focal plane is determined by using the focal positions corresponding to at least 3 positions on the observation sample, the focal plane obtained if the surface of the observation sample is uneven is not accurate can be solved; the whole focal plane is formed based on the local focal planes corresponding to the plurality of designated areas in the observation sample instead of directly determining the whole focal plane, so that the accuracy of observing the focal plane corresponding to the uneven surface of the sample can be improved.
Description
Technical Field
The application relates to a focal plane determination method and device in microscopic imaging, and belongs to the technical field of computers.
Background
When a microscope is used to acquire a microscopic image of an observation sample (such as a glass section), the observation sample needs to be automatically focused through an objective lens. The auto-focusing process includes: collecting a series of microscopic images of an objective lens at different heights; evaluating the definition of the series of microscopic images; the position where the sharpness value is maximum is determined as the in-focus position.
If the above-described auto-focusing process is performed for each position of the observation sample using the objective lens, a large amount of processing resources are consumed. Therefore, a focal plane determination method is proposed which predicts the focal position of the observation sample at each position by determining the focal plane.
The existing focal plane determination methods include: the focal positions corresponding to at least 3 positions (such as 5, 9 or 13) on the observation sample are determined, and the focal positions corresponding to the 3 positions are used for establishing a focal plane.
However, the above method is only suitable for the case of observing the surface of the sample to be flat, and the focal plane obtained for the case of observing the surface of the sample to be flat is not accurate, and accordingly, the predicted focal position is not accurate.
Disclosure of Invention
The application provides a focal plane determining method and device in microscopic imaging, which can solve the problem that when a climbing method is used for determining a focusing position, a position corresponding to a local definition maximum value is easily determined as the focusing position, so that the accuracy of determining the focusing position is not high. The application provides the following technical scheme:
in a first aspect, a method for determining a focal plane in microscopic imaging is provided, the method comprising:
acquiring n actual focal positions corresponding to n sample positions in a designated area of an observation sample, wherein n is an integer greater than 2;
determining a local focal plane corresponding to the designated area based on the n actual focal positions;
and forming a whole focal plane corresponding to the observation sample based on the local focal planes corresponding to the plurality of specified areas, wherein the observation sample is a union of the plurality of specified areas.
Optionally, the acquiring n actual focal positions corresponding to n sample positions in a specified region of an observation sample includes:
acquiring a running track when a microscopic image of the observation sample is acquired;
and when the microscopic image is acquired according to the running track, acquiring n actual focus positions corresponding to n sample positions according to a preset rule, wherein the n sample positions are not collinear.
Optionally, the moving trajectory includes a first scanning trajectory and a second scanning trajectory which are connected end to end, and a direction of the first scanning trajectory is perpendicular to a direction of the second scanning trajectory;
the preset rule comprises the steps of collecting n sample positions on at least two non-adjacent first scanning tracks; and/or acquiring n sample positions on at least two non-adjacent second scanning tracks.
Optionally, the method further comprises:
after the current local focal plane is determined, predicting a predicted focal position corresponding to the next running position on the running track by using the local focal plane;
acquiring a microscopic image of the observation sample at a next run position using the predicted focus position.
Optionally, the designated area is a rectangle including the n actual focus positions.
In a second aspect, there is provided a focal plane determination apparatus in microscopic imaging, the apparatus comprising:
the focus acquisition module is used for acquiring n actual focus positions corresponding to n sample positions in a specified region of an observation sample, wherein n is an integer greater than 2;
a first establishing module, configured to determine, based on the n actual focal positions, a local focal plane corresponding to the designated area;
and the second establishing module is used for forming an integral focal plane corresponding to the observation sample based on the local focal planes corresponding to the designated areas, and the observation sample is a union of the designated areas.
Optionally, the focus obtaining module is configured to:
acquiring a running track when a microscopic image of the observation sample is acquired;
and when the microscopic image is acquired according to the running track, acquiring n actual focus positions corresponding to n sample positions according to a preset rule, wherein the n sample positions are not collinear.
Optionally, the moving trajectory includes a first scanning trajectory and a second scanning trajectory which are connected end to end, and a direction of the first scanning trajectory is perpendicular to a direction of the second scanning trajectory;
the preset rule comprises the steps of collecting n sample positions on at least two non-adjacent first scanning tracks; and/or acquiring n sample positions on at least two non-adjacent second scanning tracks.
Optionally, the apparatus further comprises:
the focal point prediction module is used for predicting a predicted focal point position corresponding to the next running position on the running track by using the local focal plane after the current local focal plane is determined;
an image acquisition module to acquire a microscopic image of the observation sample using the predicted focus position at a next run position.
Optionally, the designated area is a rectangle including the n actual focus positions.
The beneficial effect of this application lies in: acquiring n actual focal positions corresponding to n sample positions in a designated area of an observation sample; determining a local focal plane corresponding to the designated area based on the n actual focal positions; forming a whole focal plane corresponding to the observation sample based on the local focal planes corresponding to the designated areas, wherein the observation sample is a union of the designated areas; the problem that when the focal plane is determined by using the focal positions corresponding to at least 3 positions on the observation sample, the focal plane obtained if the surface of the observation sample is uneven is not accurate can be solved; the whole focal plane is formed based on the local focal planes corresponding to the plurality of designated areas in the observation sample instead of directly determining the whole focal plane, so that the accuracy of observing the focal plane corresponding to the uneven surface of the sample can be improved.
The foregoing description is only an overview of the technical solutions of the present application, and in order to make the technical solutions of the present application more clear and clear, and to implement the technical solutions according to the content of the description, the following detailed description is made with reference to the preferred embodiments of the present application and the accompanying drawings.
Drawings
FIG. 1 is a flow chart of a method of focal plane determination in microscopic imaging as provided by one embodiment of the present application;
FIG. 2 is a schematic illustration of a trajectory provided by one embodiment of the present application;
FIG. 3 is a schematic diagram of a focal plane determination process provided by another embodiment of the present application;
fig. 4 is a block diagram of a focal plane determining apparatus in microscopic imaging provided by an embodiment of the present application.
Detailed Description
The following detailed description of embodiments of the present application will be described in conjunction with the accompanying drawings and examples. The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.
Optionally, the present application takes the implementation subject of each embodiment as an example of a microscope.
Fig. 1 is a flowchart of a focal plane determination method in microscopic imaging according to an embodiment of the present application. The method at least comprises the following steps:
Optionally, the designated area is a rectangle including n actual focus positions. Of course, in other embodiments, the designated area may also be implemented as a circle, an ellipse, etc., and the shape of the designated area is not limited in this embodiment.
In one example, acquiring n actual focus positions corresponding to n sample positions respectively in a specified region of an observation sample includes: acquiring a running track when a microscopic image of an observation sample is acquired; when microscopic images are collected according to the running track, n actual focus positions corresponding to n sample positions are collected according to a preset rule, and the n sample positions are not collinear.
In one example, the running track comprises a first scanning track and a second scanning track which are connected end to end, and the direction of the first scanning track is perpendicular to the direction of the second scanning track; at this time, the preset rule comprises collecting n sample positions on at least two non-adjacent first scanning tracks; and/or acquiring n sample positions on at least two non-adjacent second scanning tracks.
Such as: the moving track is as shown in fig. 2, the value of n is 9, and the preset rule is to collect actual focal positions corresponding to 9 sample positions on the non-adjacent first scanning track 21; actual focus positions corresponding to the 9 sample positions are acquired on the non-adjacent second scanning trajectory 22. The process of obtaining the partial focal plane according to the trajectory shown in fig. 2 is shown in fig. 3.
In another example, acquiring n actual focus positions corresponding to n sample positions respectively in a specified region of an observation sample includes: dividing an observation sample into a plurality of designated areas; determining n preset sample positions in each designated area; acquiring n actual focus positions corresponding to each sample position.
And 102, determining a local focal plane corresponding to the designated area based on the n actual focal positions.
Optionally, after the current local focal plane is determined, the microscope uses the local focal plane to predict a predicted focal position corresponding to a next operation position on the operation track; microscopic images of the observation sample are acquired at the next run position using the predicted focus position.
And 103, forming an overall focal plane corresponding to the observation sample based on the local focal planes corresponding to the plurality of designated areas, wherein the observation sample is a union of the plurality of designated areas.
Optionally, the microscope splices the partial focal planes corresponding to the multiple designated areas to obtain an overall focal plane.
In summary, in the focal plane determining method in the microscopic imaging provided by this embodiment, n actual focal positions corresponding to n sample positions in the designated area of the observation sample are obtained; determining a local focal plane corresponding to the designated area based on the n actual focal positions; forming a whole focal plane corresponding to the observation sample based on the local focal planes corresponding to the designated areas, wherein the observation sample is a union of the designated areas; the problem that when the focal plane is determined by using the focal positions corresponding to at least 3 positions on the observation sample, the focal plane obtained if the surface of the observation sample is uneven is not accurate can be solved; the whole focal plane is formed based on the local focal planes corresponding to the plurality of designated areas in the observation sample instead of directly determining the whole focal plane, so that the accuracy of observing the focal plane corresponding to the uneven surface of the sample can be improved.
Fig. 4 is a block diagram of a focal plane determining apparatus in microscopic imaging provided by an embodiment of the present application. The device at least comprises the following modules: a focus acquisition module 410, a first setup module 420, and a second setup module 430.
A focus obtaining module 410, configured to obtain n actual focus positions corresponding to n sample positions in a specified region of an observation sample, where n is an integer greater than 2;
a first establishing module 420, configured to determine, based on the n actual focal positions, a local focal plane corresponding to the designated area;
a second establishing module 430, configured to form an overall focal plane corresponding to the observation sample based on the local focal planes corresponding to the multiple specified regions, where the observation sample is a union of the multiple specified regions.
Optionally, the focus obtaining module 410 is configured to:
acquiring a running track when a microscopic image of the observation sample is acquired;
and when the microscopic image is acquired according to the running track, acquiring n actual focus positions corresponding to n sample positions according to a preset rule, wherein the n sample positions are not collinear.
Optionally, the moving trajectory includes a first scanning trajectory and a second scanning trajectory which are connected end to end, and a direction of the first scanning trajectory is perpendicular to a direction of the second scanning trajectory;
the preset rule comprises the steps of collecting n sample positions on at least two non-adjacent first scanning tracks; and/or acquiring n sample positions on at least two non-adjacent second scanning tracks.
Optionally, the apparatus further comprises: a focus prediction module and an image acquisition module.
The focal point prediction module is used for predicting a predicted focal point position corresponding to the next running position on the running track by using the local focal plane after the current local focal plane is determined;
an image acquisition module to acquire a microscopic image of the observation sample using the predicted focus position at a next run position.
Optionally, the designated area is a rectangle including the n actual focus positions.
For relevant details reference is made to the above-described method embodiments.
It should be noted that: in the above embodiment, when the focal plane determining apparatus in the microscopic imaging determines the focal plane in the microscopic imaging, only the division of the functional modules is taken as an example, and in practical applications, the functions may be distributed by different functional modules as needed, that is, the internal structure of the focal plane determining apparatus in the microscopic imaging is divided into different functional modules to complete all or part of the functions described above. In addition, the focal plane determining apparatus in the microscopic imaging and the focal plane determining method in the microscopic imaging provided in the above embodiments belong to the same concept, and specific implementation processes thereof are detailed in the method embodiments and are not described herein again.
Optionally, the present application further provides a computer-readable storage medium, in which a program is stored, the program being loaded and executed by a processor to implement the focal plane determination method in microscopic imaging of the above-mentioned method embodiments.
Optionally, the present application further provides a computer product, which includes a computer-readable storage medium, in which a program is stored, and the program is loaded and executed by a processor to implement the focal plane determination method in microscopic imaging of the above-mentioned method embodiment.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method of focal plane determination in microscopic imaging, the method comprising:
acquiring n actual focal positions corresponding to n sample positions in a designated area of an observation sample, wherein n is an integer greater than 2;
determining a local focal plane corresponding to the designated area based on the n actual focal positions;
and forming a whole focal plane corresponding to the observation sample based on the local focal planes corresponding to the plurality of specified areas, wherein the observation sample is a union of the plurality of specified areas.
2. The method of claim 1, wherein said obtaining n actual focus positions corresponding to n sample positions in a specified region of an observed sample comprises:
acquiring a running track when a microscopic image of the observation sample is acquired;
and when the microscopic image is acquired according to the running track, acquiring n actual focus positions corresponding to n sample positions according to a preset rule, wherein the n sample positions are not collinear.
3. The method of claim 2, wherein the travel path comprises a first scanning path and a second scanning path connected end to end, the direction of the first scanning path being perpendicular to the direction of the second scanning path;
the preset rule comprises the steps of collecting n sample positions on at least two non-adjacent first scanning tracks; and/or acquiring n sample positions on at least two non-adjacent second scanning tracks.
4. The method of claim 2, further comprising:
after the current local focal plane is determined, predicting a predicted focal position corresponding to the next running position on the running track by using the local focal plane;
acquiring a microscopic image of the observation sample at a next run position using the predicted focus position.
5. The method of any of claims 1 to 4, wherein the specified area is a rectangle that includes the n actual focus positions.
6. A focal plane determining apparatus in microscopic imaging, the apparatus comprising:
the focus acquisition module is used for acquiring n actual focus positions corresponding to n sample positions in a specified region of an observation sample, wherein n is an integer greater than 2;
a first establishing module, configured to determine, based on the n actual focal positions, a local focal plane corresponding to the designated area;
and the second establishing module is used for forming an integral focal plane corresponding to the observation sample based on the local focal planes corresponding to the designated areas, and the observation sample is a union of the designated areas.
7. The apparatus of claim 6, wherein the focus acquisition module is configured to:
acquiring a running track when a microscopic image of the observation sample is acquired;
and when the microscopic image is acquired according to the running track, acquiring n actual focus positions corresponding to n sample positions according to a preset rule, wherein the n sample positions are not collinear.
8. The apparatus of claim 7, wherein the travel path comprises a first scanning path and a second scanning path connected end to end, the direction of the first scanning path being perpendicular to the direction of the second scanning path;
the preset rule comprises the steps of collecting n sample positions on at least two non-adjacent first scanning tracks; and/or acquiring n sample positions on at least two non-adjacent second scanning tracks.
9. The apparatus of claim 7, further comprising:
the focal point prediction module is used for predicting a predicted focal point position corresponding to the next running position on the running track by using the local focal plane after the current local focal plane is determined;
an image acquisition module to acquire a microscopic image of the observation sample using the predicted focus position at a next run position.
10. The apparatus according to any one of claims 6 to 9, wherein the designated area is a rectangle including the n actual focus positions.
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| CN201911239271.1A CN110824685A (en) | 2019-12-06 | 2019-12-06 | Focal plane determination method and device in microscopic imaging |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106662434A (en) * | 2014-07-03 | 2017-05-10 | 阿莱恩技术有限公司 | Confocal surface topography measurement with a focal plane inclined with respect to the direction of the relative movement of confocal apparatus and sample |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106662434A (en) * | 2014-07-03 | 2017-05-10 | 阿莱恩技术有限公司 | Confocal surface topography measurement with a focal plane inclined with respect to the direction of the relative movement of confocal apparatus and sample |
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Application publication date: 20200221 |