CN110646933A - Automatic focusing system and method based on multi-depth plane microscope - Google Patents
Automatic focusing system and method based on multi-depth plane microscope Download PDFInfo
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Abstract
The invention discloses an automatic focusing system and method based on a multi-depth plane microscope. The system comprises: an illumination system for illuminating the sample; the motion platform is used for bearing a sample and moves under the control of the motion control system; the motion control system is used for controlling the motion platform to perform x and y direction mobile scanning and controlling the microscope objective lens or the cylindrical lens to perform z axis focusing scanning; the image acquisition equipment comprises a main camera and an auxiliary camera, and is used for imaging an illuminated sample on the main camera through an objective lens and a cylindrical lens respectively, and imaging on the auxiliary camera through the objective lens, the cylindrical lens and a light splitting system, wherein the auxiliary camera can simultaneously image a plurality of different depth planes. The optimal focusing position is calculated through the focusing definition of the depth plane images, global focusing scanning is not needed, searching scanning is not needed, automatic focusing time is greatly shortened, and meanwhile, high focusing precision is achieved. The definition of the collected picture is obviously superior to that of the similar products.
Description
Technical Field
The invention belongs to the technical field of microscope automatic focusing, and relates to an automatic focusing system and method based on a multi-depth plane microscope.
Background
The automatic focusing, that is, the manual operation is not needed, the internal automatic processing of the optical imaging system, and the effective combination of the machine and the program can automatically adjust each parameter, such as the object distance and the image distance, and clearly calculate the definition of the imaged picture and the specific imaging position, so that the quality of the finally imaged picture is optimal and the resolution is highest. In other words, the subject is focused and shot on the plane of the photosensitive chip to form a clear image, and the main image on the screen is clear.
The surface of a common detected object is a complex curved surface, when the surface of the object is scanned, if the bending degree of the detected object is larger than the depth of field of equipment, an automatic focusing system needs to be designed, and each part of each image is kept clear during detection and scanning, so that the observation and analysis of people are facilitated. When the detected object is scanned, two key indexes are provided, one is scanning quality and the other is scanning speed, and the two indexes are mutually restricted.
Auto-focusing can be divided into active ranging based on hardware and passive focusing based on digital image processing techniques. Active ranging methods are complex to implement and therefore passive focusing is often used in microscopes. Passive focusing can be further divided into an out-of-focus depth method and a focused definition scanning method. The defocusing depth method determines depth information by comparing the size of the diffuse spots after processing the defocusing image, and the error is large. The focus definition scanning method may be classified into a global focus scanning method and a scout scanning method. The global focus scanning method requires a large number of position scans of the z-axis to find the best resolution position, which takes a long time. The scout scan algorithm reduces the number of z-axis scans, but increases the algorithm time, which can also take longer.
Disclosure of Invention
In view of the above problems, the technical problem to be solved by the present invention is to provide an auto-focusing system and method based on a multi-depth planar microscope.
In order to achieve the purpose, the invention adopts the following technical scheme:
a multi-depth planar microscope based autofocus system comprising:
an illumination system for illuminating the sample;
the motion platform is used for bearing a sample and moves under the control of the motion control system;
the motion control system is used for controlling the motion platform to perform x and y direction mobile scanning and controlling the microscope objective lens or the cylindrical lens to perform z axis focusing scanning;
the image acquisition equipment comprises a main camera and an auxiliary camera, wherein an illuminated sample is imaged on the main camera through an objective lens and a cylindrical lens respectively, and is imaged on the auxiliary camera through the objective lens, the cylindrical lens and a light splitting system, and the auxiliary camera can be used for imaging a plurality of different depth planes simultaneously.
Preferably, the illumination system is a kohler illumination system, and the light source is guided through an optical fiber.
Preferably, the light splitting system is selected from one or more of a semi-transparent semi-reflective lens, a light splitting prism or a secondary grating. More preferably, the secondary grating is part of a fresnel grating.
Preferably, the light splitting system adopts a cascade mode.
The automatic focusing method based on the multi-depth plane microscope is realized by the system, and comprises the following steps:
1) before automatic focusing, the main camera and the auxiliary camera are corrected, so that the focusing definition of the main camera is the same as the definition of a middle depth plane of the auxiliary camera;
2) after the main camera and the auxiliary camera are corrected, the sample is subjected to global scanning at a single position to obtain an optimal focusing position;
3) scanning a sample in the x and y directions, acquiring an image by an auxiliary camera when the sample is scanned to a position, calculating the definition of three focus surfaces of the auxiliary camera, and performing curve fitting on three definition values to obtain an optimal focus position;
4) and according to the movement z axis corresponding to the optimal focusing position obtained by calculation, the main camera image is clearest, and the main camera image is acquired.
Preferably, step 1) comprises:
1-4) carrying out z-axis focusing scanning on the objective lens to obtain a plurality of pictures of planes with different depths;
1-5) calculating the focusing definition of the z-axis focusing scanning pictures acquired by the main camera and the z-axis focusing scanning pictures of a plurality of depth planes acquired by the auxiliary camera to obtain a focusing definition curve;
1-6) adjusting the positions of the main camera and the auxiliary camera to ensure that the focusing definition curve of the main camera is superposed with the focusing definition curve of the middle plane of the auxiliary camera.
Preferably, in step 1-1), the method for performing z-axis focus scanning includes: the moving platform, the objective lens and the lens cone are moved integrally, and one of the lens cone and the lens cone is moved, or the combination of two or more of the lens cone and the lens cone is moved.
Preferably, in step 1-2), the algorithm for calculating the focus sharpness includes: absolute central moment algorithm, image contrast algorithm (Nanda), image curvature algorithm (Helmli), DCT energy ratio algorithm (shen), gaussian derivative algorithm (geuseberroek), gray standard deviation algorithm (Krotkov), gray local standard deviation algorithm (Pech), energy gradient algorithm (subbao), threshold gradient algorithm (snaots), Brenner algorithm, squared gradient algorithm (Eskicioglu), Helmli mean algorithm, histogram entropy algorithm (Krotkov), laplacian energy algorithm (subbao), modified laplacian algorithm (Nayar), laplacian variance algorithm (Pech), diagonal algorithm (Thelen), Steerable filter algorithm (Minhas), spatial frequency algorithm (Eskicioglu), Tenebrad algorithm (Krotkov), Tenebrad variance algorithm (Pench), wavelet coefficient and variance algorithm (Yang), wavelet coefficient variance algorithm (Yang).
Preferably, in steps 3) and 4), curve fitting is performed on the plurality of focus definition values of the auxiliary camera to obtain a focus definition peak position, and the amount of movement required by the z-axis is an offset between the peak position and the central plane.
Preferably, the method further comprises the following steps: and predicting the focusing z value of the next scanning position by curve fitting or artificial intelligence learning of the focusing z value of the scanned position, wherein the predicted focusing z value and the calculated focusing z value are combined by different weights to form the required focusing z value.
The invention has the following beneficial effects:
the multi-plane focusing image is obtained through a hardware system, the optimal focusing position is calculated through a software algorithm in combination with the focusing definition of a plurality of planes, global focusing scanning is not needed, searching scanning is not needed, the time for scanning one sample by mainstream products in the market is 3-5 minutes (the same area is scanned) at the fastest speed, the scanning time can reach 30 seconds by using the scanner provided by the invention, and the automatic focusing time is greatly accelerated.
Meanwhile, the method has high focusing precision, and the definition of the acquired picture is obviously superior to that of the similar products.
Drawings
FIG. 1 is a schematic view of a scanning microscope system for splitting light using a plurality of prisms according to example 1;
FIG. 2 is a schematic view of a scanning microscope system using a secondary grating according to example 2;
FIG. 3 is a schematic view of a scanning microscope system employing two mutually orthogonal secondary gratings according to example 3;
FIG. 4 is a schematic view of the beam splitting prism;
FIG. 5 is a schematic diagram of a secondary grating;
FIG. 6 is a schematic diagram of secondary grating light splitting;
FIG. 7 is a schematic diagram of two mutually orthogonal secondary grating beam splitting;
FIG. 8 is a series of multi-depth planar pictures obtained by scanning the z-axis of the secondary camera;
FIG. 9 shows images acquired by three depth planes with different sharpness;
FIG. 10 shows the focus sharpness curves of the primary camera and the sharpness curves of the three depth planes of the secondary camera;
FIG. 11 shows three plane focus sharpness fit curves of the auxiliary camera when the z-axis is at different focus positions;
FIG. 12 shows a z-axis coordinate surface obtained without scanning with a prediction algorithm;
FIG. 13 shows a z-axis coordinate surface scanned in conjunction with a prediction algorithm;
the labels in the above figures are: 1-a light source; 2-an optical fiber; 3-a lighting system; 4-a motion platform; 5-a motion control system; 6-sample; 7-objective lens; 8, a lens barrel; 9-a cylindrical mirror; 10. 12-a spectroscopic system; 11-a main camera; 13-secondary camera.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
The technical scheme adopted by the invention mainly comprises a hardware system and a software algorithm system. And obtaining a multi-plane focusing image through a hardware system, and calculating the optimal focusing position by combining the focusing definition of a plurality of planes through a software algorithm.
The hardware system mainly comprises a light source, a lighting system, a motion platform, a motion control system, an objective lens, a cylindrical lens, a light splitting system and image acquisition equipment. Wherein:
an illumination system for illuminating the sample;
the motion platform is used for bearing a sample and moves under the control of the motion control system;
the motion control system is used for controlling the motion platform to perform x and y direction mobile scanning and controlling the microscope objective lens or the cylindrical lens to perform z axis focusing scanning;
the light splitting system can be one or more of a semi-transparent semi-reflective lens, a light splitting prism or a secondary grating. The sawtooth shape, the width, the step number and the depth of the secondary grating need to be specially designed, so that the lighting effect is maximum, and the image brightness of each depth plane is uniform. In order to scan a thicker sample, the spectroscopic system can be used in a cascaded manner to acquire images of more depth planes. For example, the beam splitting prism set shown in fig. 4 can be cascaded to obtain images of 6 depth planes. In order to not make the image of each depth plane too small, multiple cameras may be used. Two of the secondary gratings shown in fig. 5 can also be cascaded orthogonally (fig. 6) to obtain images of 9 depth planes. By adopting a cascading mode, the number of depth planes and the focusing range can be increased, so that even if the main camera is out of focus far, the focusing position can be accurately found.
The image acquisition equipment is the camera, is equipped with two at least (main camera and supplementary camera) for with the sample that will be lighted through objective and section of thick bamboo mirror formation of image on main camera respectively to and through objective, section of thick bamboo mirror and the formation of image on supplementary camera of beam splitting system, supplementary camera can form a plurality of different depth planes simultaneously.
The software algorithm determines the primary camera's best focus position by calculating the focus sharpness of the secondary camera's multiple focus planes. Before automatic focusing, the main camera and the auxiliary camera need to be corrected, so that the focusing definition of the main camera is the same as the definition of the middle depth plane of the auxiliary camera.
Specifically, the objective lens of the microscope is subjected to z-axis focusing scanning to obtain a plurality of pictures of planes with different depths. The z-axis focusing scanning can move the motion platform, the whole object lens and the lens barrel; combinations of two or more of the above various methods may also be moved. And then, calculating the focusing definition of the z-axis focusing scanning pictures acquired by the main camera and the z-axis focusing scanning pictures of the plurality of depth planes acquired by the auxiliary camera to obtain a focusing definition curve. And adjusting the positions of the main camera and the auxiliary camera to ensure that the focusing definition curve of the main camera is superposed with the focusing definition curve of the middle plane of the auxiliary camera.
The focus sharpness may be calculated using one or more of absolute central moment algorithm, image contrast algorithm (Nanda), image curvature algorithm (Helmli), DCT energy ratio algorithm (shen), gaussian derivative algorithm (geuseberroek), gray standard deviation algorithm (Krotkov), gray local standard deviation algorithm (Pech), energy gradient algorithm (subband), threshold gradient algorithm (Snatos), Brenner algorithm, squared gradient algorithm (eskiveloglu), Helmli mean algorithm, entropy algorithm (Krotkov), laplacian energy algorithm (subband), modified laplacian algorithm (nayarr), laplacian variance algorithm (Pench), diagonal algorithm (Thelen), stererable filter algorithm (Minhas), spatial frequency algorithm (esradkiveloglu), Tenengrad algorithm (Krotkov), tenneng algorithm (Pench), wavelet coefficient and variance algorithm (Yang), and variance algorithm (variance) in combination.
When automatic focusing is carried out, the sample on the motion platform is scanned in the x and y horizontal directions, and the main camera and the auxiliary camera at each scanning position take one image. Whether the main camera is in clear focus or not can be judged by carrying out focusing definition calculation on the images of the plurality of focusing surfaces of the auxiliary camera; and in which direction it should move if not in sharp focus.
And performing curve fitting on a plurality of focusing definition values of the auxiliary camera to obtain a focusing definition peak position, wherein the offset between the peak position and the central plane is the amount of movement required by the z axis.
Thus, large-range global focusing scanning (for example, scanning a plurality of points, respectively calculating focusing definition, and the point with the maximum definition is a focusing position) on the z axis is not required; and the focusing position can be obtained only by collecting and calculating once without adopting algorithms for searching the focusing position such as a hill climbing method and the like.
Example 1
The hardware system is shown in fig. 1. The light source 1 is led into a Kohler illumination system 3 through an optical fiber 2, and an illuminated sample 6 is imaged on a main camera 11 through an objective lens 7 and a tube lens 9; at the same time, the sample 6 is imaged on the secondary camera 13 by the objective lens 7, the barrel mirror 9 and the spectroscopic systems 10 and 12. For example, in the present embodiment, a Plan na0.2510x objective lens and a Plan na0.7520x objective lens are selected as the objective lenses, and the spectroscopic systems 10 and 12 are prisms (sets) and can be mounted by being held by a machining member. The splitting ratio of the splitting systems 10 and 12 is designed so that the image brightness of each depth plane is uniform and neither overexposure nor too dark is achieved; the beam splitting system 12 needs to be designed so that the imaged size of each depth plane is substantially uniform and has similar aberrations. In this embodiment, the light splitting system 12 is a light splitting prism, and taking the prism group as an example, the light splitting system can be designed as follows: the first prism reflects 33% transmission 66%, the second prism reflects 50% transmission 50%, and the third prism is totally reflective, the beam splitting diagram is shown in fig. 4. The secondary camera 13 can simultaneously image three different focal planes. The motion control system 5 controls the motion platform 4 to move and scan in the x and y directions and controls the objective lens 7 or the barrel mirror 9 to perform z-axis focusing scanning, for example, the motion control system 5 can adopt a Copley driver and a Galil DMC-2x00 digital motion controller, and the motion platform 4 can adopt a PI M406.2PD high-precision linear motion platform.
Before auto-focusing, the primary camera 11 and the secondary camera 13 are corrected to make the focusing resolution of the primary camera 11 and the mid-depth plane resolution of the secondary camera 13 the same, and in this embodiment, the FLIR BFS-U3-32S4C camera may be used. Specifically, the microscope objective lens 7 is subjected to z-axis focusing scanning to obtain a plurality of pictures of planes with different depths, as shown in fig. 8, 3 depth planes are displayed in the pictures, and the images acquired by the three depth planes have different definitions, as shown in fig. 9, the difference of the average gray values is within 10%, and three more uniform pictures can be obtained by removing the background. Then, the z-axis focusing scanning pictures acquired by the main camera 11 and the z-axis focusing scanning pictures of a plurality of depth planes acquired by the auxiliary camera 13 are subjected to focusing definition calculation to obtain a focusing definition curve. The primary and secondary camera positions are adjusted so that the primary camera 11 focus sharpness curve coincides with the secondary camera 13 mid-plane focus sharpness curve (fig. 10 circle and diamond data).
After the main camera and the auxiliary camera are corrected, the sample 6 is globally scanned at a single position to obtain the optimal focusing position. The sample 6 is then scanned in the x, y direction. When the auxiliary camera 13 acquires an image when scanning to a position, the definition of three focus surfaces of the auxiliary camera is calculated, then curve fitting is carried out on the three definition values to obtain the best focus position, and the image of the main camera is made to be the clearest according to the movement z axis (the objective lens 7 or the cylindrical lens 9) corresponding to the best focus position obtained by calculation. FIG. 11 shows three plane focus sharpness fit curves for the secondary camera with the z-axis at different focus positions, the peaks representing the best focus position, where the middle plot: the main camera is focused basically clearly; left and right panels: the z-axis needs to be moved in the corresponding direction so that the three sharpness fit curve peaks of the secondary camera lie in the middle plane (median).
Finally, a main camera image is acquired.
Example 2
The hardware system is shown in fig. 2. The light source 1 is led into a Kohler illumination system 3 through an optical fiber 2, and an illuminated sample 6 is imaged on a main camera 11 through an objective lens 7 and a tube lens 9; at the same time, the sample 6 is imaged on the secondary camera 13 by the objective lens 7, the barrel mirror 9 and the spectroscopic systems 10 and 12. The light splitting system 12 is a secondary grating, which may be part of a fresnel grating, as shown in fig. 5. Light passing through the secondary grating can be emitted from different angles through diffraction, as shown in fig. 6, 0-level and ± 1-level emergent light is emitted through the secondary grating, and images with different focusing depths can be imaged on a camera by different-level light.
The secondary camera 13 can simultaneously image three different focal planes. The motion control system 5 controls the motion platform 4 to move in the x and y directions for scanning, and controls the objective lens 7 or the tube lens 9 to perform z-axis focusing scanning.
The automatic focusing scanning flow and the algorithm are the same as the first embodiment.
Example 3
The hardware system is shown in fig. 3. The light source 1 is led into a Kohler illumination system 3 through an optical fiber 2, and an illuminated sample 6 is imaged on a main camera 11 through an objective lens 7 and a tube lens 9; at the same time, the sample 6 is imaged on the secondary camera 13 by the objective lens 7, the barrel mirror 9 and the spectroscopic systems 10 and 12. The beam splitting system 12 is two mutually orthogonal secondary gratings, as shown in fig. 7. The secondary camera 13 can simultaneously image 9 different focal planes. The motion control system 5 controls the motion platform 4 to move in the x and y directions for scanning, and controls the objective lens 7 or the tube lens 9 to perform z-axis focusing scanning.
The automatic focusing scanning flow and the algorithm are the same as the first embodiment.
In order to ensure the consistency and continuity of the focusing during the scanning of the microscope, curve fitting or artificial intelligence learning can be carried out on the focusing z value of the scanned position to predict the focusing z value of the next scanning position. The predicted focus z-value and the calculated focus z-value are combined with different weights to form the desired focus z-value. FIG. 12 shows a scanned z-axis coordinate surface obtained without prediction; FIG. 13 shows a scan z-axis coordinate surface using a combination of prediction and calculation.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. A multi-depth planar microscope based autofocus system comprising:
an illumination system for illuminating the sample;
the motion platform is used for bearing a sample and moves under the control of the motion control system;
the motion control system is used for controlling the motion platform to perform x and y direction mobile scanning and controlling the microscope objective lens or the cylindrical lens to perform z axis focusing scanning;
the image acquisition equipment comprises a main camera and an auxiliary camera, and is used for imaging an illuminated sample on the main camera through an objective lens and a cylindrical lens respectively, and imaging on the auxiliary camera through the objective lens, the cylindrical lens and a light splitting system, wherein the auxiliary camera can simultaneously image a plurality of different depth planes.
2. The multi-depth planar microscope-based autofocus system of claim 1, wherein the illumination system is a kohler illumination system, and the light source is guided through an optical fiber.
3. The multi-depth planar microscope-based automatic focusing system according to claim 1, wherein the light splitting system is selected from one or more of a semi-transparent and semi-reflective lens, a light splitting prism or a secondary grating.
4. The multi-depth planar microscope-based autofocus system of claim 1, wherein the beam splitting system is in a cascaded manner.
5. The multi-depth plane microscope-based automatic focusing method is realized by the multi-depth plane microscope-based automatic focusing system of any one of claims 1 to 4, and comprises the following steps:
1) before automatic focusing, the main camera and the auxiliary camera are corrected, so that the focusing definition of the main camera is the same as the definition of a middle depth plane of the auxiliary camera;
2) after the main camera and the auxiliary camera are corrected, the sample is subjected to global scanning at a single position to obtain an optimal focusing position;
3) scanning a sample in the x and y directions, acquiring an image by an auxiliary camera when the sample is scanned to a position, calculating the definition of three focus surfaces of the auxiliary camera, and performing curve fitting on three definition values to obtain an optimal focus position;
4) and according to the movement z axis corresponding to the optimal focusing position obtained by calculation, the main camera image is clearest, and the main camera image is acquired.
6. The multi-depth planar microscope-based autofocus method of claim 5, wherein step 1) comprises:
1-1) carrying out z-axis focusing scanning on an objective lens to obtain a plurality of pictures of planes with different depths;
1-2) carrying out focusing definition calculation on a z-axis focusing scanning picture acquired by a main camera and z-axis focusing scanning pictures of a plurality of depth planes acquired by an auxiliary camera to obtain a focusing definition curve;
1-3) adjusting the positions of the main camera and the auxiliary camera to enable the focusing definition curve of the main camera to be superposed with the focusing definition curve of the middle plane of the auxiliary camera.
7. The multi-depth planar microscope-based auto focusing method according to claim 6, wherein in the step 1-1), the method of performing the z-axis focusing scan comprises: the moving platform, the objective lens and the lens cone are moved integrally, and one of the lens cone and the lens cone is moved, or the combination of two or more of the lens cone and the lens cone is moved.
8. The multi-depth plane microscope-based auto focusing method according to claim 6, wherein in the step 1-2), the algorithm for calculating the focus sharpness comprises: absolute central moment algorithm, image contrast algorithm, image curvature algorithm, DCT energy ratio algorithm, Gaussian derivative algorithm, gray standard deviation algorithm, gray local standard deviation algorithm, energy gradient algorithm, threshold gradient algorithm, Brenner algorithm, squared gradient algorithm, Helmli mean algorithm, histogram entropy algorithm, Laplace energy algorithm, modified Laplace algorithm, Laplace variance algorithm, diagonal algorithm, Steerable filter algorithm, spatial frequency algorithm, Tenengrad variance algorithm, wavelet coefficients and algorithms, wavelet coefficient variance algorithm, or a combination of more than one.
9. The method of claim 5, wherein in steps 3) and 4), the plurality of focus resolution values of the secondary camera are curve-fitted to obtain the peak position of the focus resolution, and the amount of the z-axis movement required is the offset of the peak position from the central plane.
10. The multi-depth planar microscope-based autofocus method of claim 5, further comprising: and predicting the focusing z value of the next scanning position by curve fitting or artificial intelligence learning of the focusing z value of the scanned position, wherein the predicted focusing z value and the calculated focusing z value are combined by different weights to form the required focusing z value.
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| CN112040136A (en) * | 2020-09-23 | 2020-12-04 | 重庆邮电大学 | Automatic focusing optimization method based on clear domain and scotopic vision |
| CN112040136B (en) * | 2020-09-23 | 2021-08-10 | 重庆邮电大学 | Automatic focusing optimization method based on clear domain and scotopic vision |
| CN112432607A (en) * | 2020-11-10 | 2021-03-02 | 四川欧瑞特光电科技有限公司 | Automatic zooming three-dimensional shape measurement system and method |
| CN114166843A (en) * | 2021-11-23 | 2022-03-11 | 长园运泰利视觉科技(珠海)有限公司 | Camera system and imaging method thereof |
| CN115546114A (en) * | 2022-09-16 | 2022-12-30 | 魅杰光电科技(上海)有限公司 | Focusing method for critical dimension measurement |
| CN115546114B (en) * | 2022-09-16 | 2024-01-23 | 魅杰光电科技(上海)有限公司 | Focusing method for critical dimension measurement |
| CN116600201A (en) * | 2023-07-17 | 2023-08-15 | 北京中科科仪股份有限公司 | Automatic focusing method and device based on scanning electron microscope |
| CN116600201B (en) * | 2023-07-17 | 2023-11-03 | 北京中科科仪股份有限公司 | Automatic focusing method and device based on scanning electron microscope |
| CN117974464A (en) * | 2024-02-28 | 2024-05-03 | 上海为旌科技有限公司 | Image FV confidence determining method, device, equipment and storage medium |
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