CN111458835A - A kind of microscope multi-view autofocus system and using method thereof - Google Patents

Abstract

The invention provides a microscope multi-view automatic focusing system, which includes a microscope main body, a stepping motor control system, a shooting unit, and a moving object platform; the use method includes manually adjusting the microscope objective lens to a relative focusing range, and passing the step in the range. The motor changes the z-axis coordinate of the stage and acquires the video image, calculates and compares the average value of the variance of the gray value of the target object image at the end point of the moving interval, and optimizes and iterates according to the golden section method to obtain the maximum value, and realizes the automatic focusing in the z-axis direction; The stepper motor controls the stage to change according to the s-shaped advancing direction in the xy-axis plane, so as to realize the scanning of continuous positions on the plane, and finally stitch the scanned images. The automatic focusing system method provided by the invention has the advantages of low computational complexity and the like, and can capture high-resolution and large-field medical images.

Description

A kind of microscope multi-view autofocus system and using method thereof

Technical field:

The invention relates to the technical field of digital images, in particular to a microscope multi-view automatic focusing system and a method for using the same.

Background technique:

Since the invention of the microscope by Jansen and his son in the Netherlands in 1590, the microscope can help researchers observe tiny objects invisible to the naked eye and open the door to the microscopic world.

Objective lens focusing is an important part of the microscope observation process. However, with the rapid development of science and technology, the traditional manual focusing method is difficult to meet the requirements of modern science. The final result of the measurement is easily deviated by the researchers through visual observation and work fatigue, and manual focusing also has defects such as low efficiency. The use of microscope autofocus technology can solve the above problems very well, freeing researchers from boring repetitive work and starting more creative work.

At present, the exploration of microscope autofocus technology at home and abroad is still in its infancy, and the main research and application are concentrated in the medical field. The research of autofocus system is also mainly based on static single image, lack of dynamic focus of continuous position of the sample, which affects the integrity of the obtained sample.

Therefore, the present invention proposes a multi-view multi-image auto-focusing system for microscopes based on the variance value of gray values, aiming at realizing fast and accurate continuous auto-focusing of microscopes.

Invention content:

The purpose of the present invention is to solve the above-mentioned problems, and provide a microscope multi-view auto-focusing system and a method for using the same, which realizes the rapid focusing of samples with different fields of view.

A microscope multi-view automatic focusing system at least includes a microscope main body, a stepping motor control system, a shooting unit, and a moving object platform, and the using method includes the following steps:

Step 1: roughly manually adjust the focus of the microscope objective, so that the image observed at a certain position is relatively clear;

Step 2: control the moving object platform by the stepper motor, change the z-axis coordinate of the moving object platform, and use the shooting unit to collect sample video images;

Step 3: carry out sharpness evaluation calculation to the video image of each frame of the sample, find the maximum value of sharpness evaluation function by one-dimensional linear optimization method in the moving range of the z-axis of the loading platform, and record it as a focus approximate point ;

Step 4: upload the focus image and corresponding coordinates to the software visualization window, wait for subsequent image splicing;

Step 5: Control the loading platform by the stepper motor in the s-shaped advancing direction along the xy-axis plane at a fixed step length, repeat the step 2, step 3, step 4 at each measurement point, until the software The interface end presents the complete focus mosaic image of the sample.

As a further improvement of the present invention, the video image sharpness is measured by using the average value of gray value variance as an evaluation function, and the image contrast value is calculated in the processor. The loading platform first moves in the positive direction within the predetermined z-axis direction interval, and judges the size trend of the image sharpness evaluation function in the distance. If it is increasing, continue to move in this direction and find out. The sharpest image in the collection; if it is decreasing, move the stage in the opposite direction and calculate the sharpness of the captured image to find the sharpest image.

As a further improvement of the present invention, the one-dimensional linear optimization of the image sharpness evaluation function adopts the golden section method to find the maximum value of the function, and compares the gray value variance of the two positions of the end points of the moving interval in the z-axis direction of the stage. The average value of , and optimization iteration is performed until the moving interval is less than the given threshold, then the iteration is stopped, the approximate solution of the focus is obtained, the automatic focus in the z direction is realized, and the focused image and the corresponding three-axis coordinates are saved.

As a further improvement of the present invention, when the stepper motor controls the moving object platform to move along the xy-axis plane, if a displacement offset is given in the x-axis direction, the y-axis direction coordinate remains unchanged. If a displacement offset is given in the y-axis direction, the x-axis direction coordinates remain unchanged. According to the above-mentioned moving rules, the linear s-shaped advance is carried out to realize the scanning of continuous positions on the plane.

Beneficial effects:

The invention collects and calculates the average value of the variance of the gray value of the image, and uses the golden section one-dimensional linear optimization method to iterate to realize the automatic focusing of the microscope lens and obtain the focused picture. Compared with the prior art, the automatic focusing method has small calculation amount and high precision, greatly saves manpower and material resources, and improves focusing efficiency.

The invention breaks the limitation of the traditional static single-picture focusing, and uses the stepping motor to control the object platform to advance in the s-shaped direction along the xy-axis plane with a fixed step length, thereby realizing the dynamic focusing of multiple fields of view. Through the automatic stitching of the focused image set by the software, a clear and complete image of the measured object can be obtained, which improves the measurement integrity of the system, especially when the surface of the measured object is uneven, which has great advantages.

Description of drawings:

1 is a schematic diagram of a microscope autofocus control platform system of the present invention;

Fig. 2 is the microscope autofocusing and splicing flow chart of the present invention;

Fig. 3 is the focus splicing scanning process diagram of the present invention;

FIG. 4 is a software diagram of a system scanning interface constructed based on the Labview development environment of the present invention.

List of reference numbers:

1-Camera, 2-Objective lens, 3-Three-coordinate stage, 4-Motor controller, 5-Multifunctional capture card.

Detailed ways:

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

Example

Build the entire microscope multi-view multi-image focusing system platform, including the control system, the video image acquisition system and the data processing system. The overall system schematic diagram is shown in Figure 1. The position of the stage is automatically adjusted by the xyz three-coordinate mobile platform 3, and the multi-function acquisition card 5 uses the gray-scale judgment function to evaluate and output the pulse sequence to the motor control according to the image collected by the camera 1. The device 4 is used to control the asynchronous motor on the three-coordinate table 3. Place the sample to be tested on the mobile carrier platform, and prepare for testing after the system parameters are debugged.

The objective lens of the microscope is roughly manually adjusted to make the image observed at a certain position relatively clear, the video image acquisition system is turned on, and the system is turned on to automatically perform fine focusing.

The moving object platform is controlled by the stepper motor, and 10000 pulses correspond to 1mm to change the z-axis coordinate of the moving object platform. The sample video images are collected by the shooting unit and sent to the data processor for analysis. Take care to keep the position of the microscope objective fixed throughout the test.

The collected true-color image sets are first processed by the system in grayscale, and the focused images have clear outlines, rich details and high contrast. As shown in Figure 2, the size of the scanning field of view is pre-set in the system. After the loading platform moves to the initial position, it moves in the positive direction along the set z-axis direction interval to calculate the sharpness evaluation value. And judge the size trend of the image sharpness evaluation function in the moving direction. If it is increasing, continue to move along this direction and calculate the sharpness of the captured image; if it is decreasing, move the image in the opposite direction. material platform.

The grayscale evaluation function has the characteristic of unimodality, and the one-dimensional linear optimization method can be used to find the maximum value of the sharpness evaluation function. The invention adopts the golden section method to iterate, the calculation amount is small and the efficiency is high, and the high efficiency of the automatic focusing system of the microscope can be realized. When the z-axis movement interval is small enough, the iteration is stopped and an approximate solution of the focus is obtained. The coordinates of the image captured by the system at this time are the coordinates of the first focused image point, and at the same time, the image is saved as the first frame of focused image and presented in the terminal software system window.

After obtaining the current focused image, the stepper motor controls the stage to move along the xy-axis plane, and keeps the z-axis direction unchanged. During the moving process, if the object platform has a movement displacement offset in the x-axis direction, the y-axis direction coordinate remains unchanged. If the stage has a displacement offset in the y-axis direction, the x-axis direction coordinates remain unchanged. According to this moving rule, a straight s-shaped advance is performed to realize the scanning of continuous positions on the plane. The scanned images are sequentially evaluated for image sharpness, and the golden section is linearly optimized to find the maximum value.

The acquired series of in-focus images are saved in the processor terminal with the corresponding three-dimensional coordinate names, and the experimenter can observe the complete sample in-focus image set in the terminal software system window, realizing the rapid auto-focusing of the microscope with multiple fields of view and multiple images. . Figure 3 and Figure 4 are the specific focus splicing scanning process diagram and the system scanning interface software diagram based on Labview software, respectively.

The above-mentioned examples are only preferred examples of the present invention, and do not limit the scope of implementation of the present invention. Without departing from the spirit and essence of the present invention, the skilled person can make various corresponding changes and deformations according to the present invention, but these corresponding changes and deformations should all belong to the protection scope of the appended claims of the present invention.

Claims (5)

1. A microscope multi-field autofocus system, characterized in that, comprising a microscope main body, a stepping motor control system, a photographing unit and a moving object-carrying platform. 2. the using method of a kind of microscope multi-field autofocus system according to claim 1, is characterized in that, comprises the following steps: Step 1: Manually focus the objective lens of the microscope body to make the observed image clear; Step 2: control the mobile carrier platform by the stepper motor control system, change the z-axis coordinate of the mobile carrier platform, and utilize the shooting unit to collect sample video images; Step 3: carry out the definition evaluation calculation to the video image of each frame of the sample, find the maximum value of the definition evaluation function by one-dimensional linear optimization method in the moving range of the z-axis of the moving object platform, and record it as a focus approximate point; Step 4: upload the focused image and the corresponding coordinates to the software visualization window, and wait for subsequent image stitching; Step 5: Control the moving object platform through the stepper motor control system according to the fixed step length along the xy-axis plane in the s-shaped forward direction, repeat steps 2, 3 and 4 at each measurement point until the software interface shows a complete Sample in-focus stitched images. 3. the using method of a kind of microscope multi-view auto-focusing system according to claim 2, is characterized in that, described video image sharpness adopts gray value variance mean value to measure as evaluation function, calculates in processor and obtains The image contrast value; the moving object platform first moves in the positive direction within the predetermined z-axis direction interval, and judges the size trend of the image sharpness evaluation function within the distance. If it is increasing, continue along the Move in that direction and find the sharpest image in the collection; if it is decreasing, move the moving stage in the opposite direction and calculate the sharpness of the captured image to find the sharpest image. 4. the using method of a kind of microscope multi-view autofocus system according to claim 2, is characterized in that, the one-dimensional linear optimization of image sharpness evaluation function adopts golden section method to find the maximum value of this function, compares described Move the average value of the variance of the gray values of the two end points of the moving interval in the z-axis direction of the platform, and perform optimization iteration until the moving interval is smaller than the given threshold, stop the iteration, obtain the approximate solution of the focus, and realize the automatic focusing in the z-direction , and save the focused image and the corresponding three-axis coordinates. 5. The method of using a microscope multi-view autofocus system according to claim 2, wherein when the stepper motor control system controls the moving object platform to move along the xy-axis plane, if the If a displacement offset is given, the coordinates of the y-axis direction remain unchanged; if a displacement offset is given in the y-axis direction, the coordinates of the x-axis direction remain unchanged; according to the above moving rules, a straight line s-shaped forward is performed to achieve continuous on the plane. location scan.

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