WO2024045889A1

WO2024045889A1 - Focus locking method and apparatus for microscope

Info

Publication number: WO2024045889A1
Application number: PCT/CN2023/105812
Authority: WO
Inventors: 汤伯超; 王宏达; 孙佳音
Original assignee: 广东粤港澳大湾区黄埔材料研究院
Priority date: 2022-09-01
Filing date: 2023-07-05
Publication date: 2024-03-07
Also published as: CN115356840A

Abstract

Disclosed are a focus locking method and apparatus for a microscope. The method comprises: collecting displacement data of a Z-axis shifter and image data of a slide in real time (101); according to the displacement data and the image data, performing curve fitting processing, so as to obtain a linear relationship between the displacement data and the image data (102); and according to the linear relationship, adjusting the position of the Z-axis shifter, so as to realize focus locking (103). Therefore, curve fitting processing and the acquisition of a linear relationship require no additional hardware device, but can be realized on a computer by means of software, thereby reducing the cost generated by using an additional hardware device. In addition, by means of the real-time collection of displacement data of a Z-axis shifter and image data of a slide, in combination with a calculated linear relationship, the adjustment of the Z-axis shifter can be performed in real time, thereby improving the precision of focus locking.

Description

A focus locking method and device for a microscope

Technical field

The present invention relates to the technical field of microscope distance measurement, and in particular, to a focus locking method and device for a microscope.

Background technique

Microscope focus locking mainly locks the distance between the objective lens of the microscope and the sample, that is, the focal plane of the objective lens; during the focus locking process, on the one hand, the detector of the microscope is used to detect the distance between the objective lens and the sample in real time; on the other hand, due to Normally the displacement of the objective lens and sample is 20-50nm, so a sophisticated detector is used to detect values of 20nm or less. Therefore, the existing technology mainly measures the distance between the objective lens and the sample through hardware including detectors to determine whether the distance remains within the preset locking distance value. If a deviation occurs, the distance between the objective lens and the sample is adjusted to achieve the focus locking function. ; To implement this focus locking method, additional hardware equipment is required, resulting in lower accuracy.

Contents of the invention

The present invention provides a focus locking method and device for a microscope to solve the technical problems of the existing technology requiring additional hardware equipment and low precision.

In order to solve the above technical problems, embodiments of the present invention provide a focus locking method for a microscope, which includes:

Collect the displacement data of the Z-axis displacer and the image data of the glass slide in real time;

According to the displacement data and the image data, through curve fitting processing, a linear relationship between the displacement data and the image data is obtained;

According to the linear relationship, the position of the Z-axis displacer is adjusted to achieve focus locking.

The curve fitting processing and the acquisition of the linear relationship in the present invention do not need to be obtained by additional hardware equipment, and can be realized through software on the computer, thereby reducing the cost caused by the use of additional hardware equipment; in addition, by adjusting the Z-axis displacer Real-time collection of displacement data and slide image data, combined with calculation Based on the calculated linear relationship, the Z-axis shifter can be adjusted in real time, improving the focus lock accuracy.

Further, the real-time collection of the displacement data of the Z-axis displacer and the image data of the glass slide is specifically as follows:

Change the position of the Z-axis displacer, and obtain several first displacement values according to the movement distance of the Z-axis displacer; wherein the displacement data is the several first displacement values;

Image data of the slide corresponding to each of the displacement values is acquired.

Further, changing the position of the Z-axis displacer and obtaining several first displacement values according to the movement distance of the Z-axis displacer can be replaced by recording several first displacement values generated by the Z-axis displacer when the stage sinks. The first displacement value.

The present invention collects or records the Z-axis displacement value by changing the position of the Z-axis displacer, or the stage changes the position of the Z-axis displacer due to automatic sinking, and collects image data corresponding to each displacement value. Furthermore, when the stage sinks due to sinking, the Z-axis displacer is adjusted in real time based on the linear relationship and the collected displacement value and image data, thereby improving the focus locking accuracy.

Further, according to the displacement data and the image data, through curve fitting processing, a linear relationship between the displacement data and the image data is obtained, specifically:

Wherein, the displacement data are several first displacement values of the Z-axis displacer;

According to the image data corresponding to each first displacement value, after curve fitting, several first coordinates are obtained;

Set the first displacement value corresponding to each of the first coordinates as a second coordinate, and select several first coordinate pairs based on several first coordinates and several second coordinates;

According to the plurality of first coordinate pairs, a linear equation is calculated; wherein the linear relationship includes the linear equation.

The present invention obtains a linear equation through the displacement value of the Z displacer and the centroid calculation of the image data. The linear equation represents the linear relationship between the image data and the position of the Z-axis displacer. By substituting the current collection data into the linear equation, the Z adjustment can be obtained The data required by the axis displacer can be used to realize real-time adjustment of the Z-axis displacer; in addition, the calculations required to obtain linear equations can be realized on the computer, thus avoiding the use of additional hardware equipment.

Further, according to the image data corresponding to each of the first displacement values, after curve fitting, several first coordinates are obtained, specifically:

Select the centroid area in each image data;

Perform Gaussian fitting on each of the centroid regions to obtain a single-peak Gaussian curve;

Select the position corresponding to the maximum value of each unimodal Gaussian curve, and set the position as the first coordinate.

Further, several coordinate pairs are selected based on several first coordinates and several second coordinates, specifically:

Combining each first coordinate with the second coordinate corresponding to each first coordinate to generate several second coordinate pairs;

According to the consistency sampling algorithm, error points in the plurality of second coordinate pairs are eliminated to obtain the plurality of first coordinate pairs.

The present invention eliminates erroneous points in the second coordinate pair through a consistent sampling algorithm, eliminating errors generated during acquisition, thereby eliminating errors in subsequent calculations, and improving focus lock accuracy; in addition, the process of eliminating erroneous points does not require additional hardware The equipment can be implemented on a computer, thereby reducing costs.

Further, a linear equation is calculated based on the several first coordinate pairs, specifically:

Establish a fitting straight line equation according to the several first coordinate pairs;

According to the straight line equation, the error value is calculated;

According to the least square method and the error value, the slope is calculated;

Based on the slope, the linear equation is established.

The present invention calculates the slope based on the least square method and several first coordinate pairs; through the linear equation established by the slope, after substituting the centroid and offset of the current image, the required amount of movement of the Z-axis displacer can be calculated displacement value to adjust the Z-axis displacer according to the required displacement value.

Further, according to the linear relationship, the position of the Z-axis displacer is adjusted to achieve focus locking, specifically as follows:

Wherein, the linear relationship includes a linear equation;

According to the center of mass of the image to be locked and the current position of the Z-axis displacer, the offset is obtained by solving;

Substitute the center of mass and the offset into the linear equation, and solve to obtain the second displacement value of the Z-axis displacer required for focus locking;

According to the second displacement value, adjust the position of the Z-axis displacer so that the center of mass returns to the focus-locked position.

Further, the image data is one-dimensional image data generated by linear CCD collecting the reflected light spot of the glass slide.

On the other hand, the present invention also provides a focus locking device for a microscope, including: a collection module, a calculation module and a focus locking module;

Among them, the acquisition module is used to collect the displacement data of the Z-axis displacer and the image data of the glass slide in real time;

The calculation module is used to obtain a linear relationship between the displacement data and the image data through curve fitting processing according to the displacement data and the image data;

The focus lock module is used to adjust the position of the Z-axis displacer according to the linear relationship to achieve focus lock.

The curve fitting processing and the acquisition of the linear relationship in the present invention do not need to be obtained by additional hardware equipment, and can be realized through software on the computer, thereby reducing the cost caused by the use of additional hardware equipment; in addition, by adjusting the Z-axis displacer Real-time collection of displacement data and slide image data, combined with the calculated linear relationship, enables real-time adjustment of the Z-axis displacer, improving focus lock accuracy.

Description of drawings

Figure 1 is a schematic flow chart of an embodiment of a focus locking method for a microscope provided by the present invention;

Figure 2 is a schematic flow chart of another embodiment of a focus locking method for a microscope provided by the present invention;

Figure 3 is a schematic flow chart of another embodiment of the focus locking method of a microscope provided by the present invention;

Figure 4 is a schematic flow chart of another embodiment of the focus locking method of a microscope provided by the present invention;

Figure 5 is a schematic structural diagram of an embodiment of the focus locking device of a microscope provided by the present invention;

Figure 6 is a schematic structural diagram of another embodiment of the focus locking device of the microscope provided by the present invention;

Figure 7 is a schematic structural diagram of another embodiment of the focus locking device of the microscope provided by the present invention;

FIG. 8 is a schematic structural diagram of another embodiment of the focus locking device of a microscope provided by the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Embodiment 1

Please refer to Figure 1, which is a schematic flow chart of an embodiment of a focus locking method for a microscope provided by the present invention. It mainly includes steps 101 to 103, specifically as follows:

Step 101: Collect the displacement data of the Z-axis displacer and the image data of the glass slide in real time.

In this embodiment, the displacement data of the Z-axis displacer is the distance that the Z-axis displacer controls the movement of the objective lens, and the image data is the image generated by the camera by collecting the reflected light spot on the glass slide.

In this embodiment, the image data may be one-dimensional image data generated by a linear CCD collecting the reflected light spot of the glass slide.

Step 102: According to the displacement data and the image data, obtain a linear relationship between the displacement data and the image data through curve fitting processing.

In this embodiment, through the linear relationship between the moving distance of the Z-axis displacer and the image data, combined with the centroid of the current image data, the distance required to move the Z-axis displacer is calculated, and then the Z-axis displacer is Adjust so that the center of mass moves to the focus lock position.

Step 103: According to the linear relationship, adjust the position of the Z-axis displacer to achieve focus locking.

Please refer to FIG. 2 , which is a schematic flow chart of another embodiment of a focus locking method for a microscope provided by the present invention. The main difference between Figure 2 and Figure 1 is that Figure 2 also includes step 201 and step 202, as follows:

In this embodiment, step 201 is specifically step 201-step 202.

Step 201: Change the position of the Z-axis displacer, and obtain several first displacement values according to the movement distance of the Z-axis displacer; wherein the displacement data is the several first displacement values.

In this embodiment, changing the position of the Z-axis displacer and obtaining several first displacement values according to the movement distance of the Z-axis displacer can be replaced by: when the recording stage sinks, the Z-axis displacer generates several first displacement values.

The present invention changes the position of the Z-axis displacer, or the stage generates the Z-axis position due to automatic sinking. When the position of the shifter changes, the Z-axis displacement value is collected or recorded, and the image data corresponding to each displacement value is collected. Then when the stage sinks due to sinking, the linear relationship is combined with the collected displacement value and image data. Real-time adjustment of the Z-axis shifter improves focus lock accuracy.

Please refer to FIG. 3 , which is a schematic flow chart of another embodiment of a focus locking method for a microscope provided by the present invention. The main difference between Figure 3 and Figure 1 is that Figure 3 also includes steps 301 to 303, which are as follows:

In this embodiment, step 102 specifically includes steps 301 to 303.

In this embodiment, the displacement data is several first displacement values of the Z-axis displacer.

Step 301: According to the image data corresponding to each first displacement value, obtain several first coordinates after curve fitting.

In this embodiment, several first coordinates are obtained after curve fitting based on the image data corresponding to each first displacement value, specifically: selecting the centroid area in each image data; Perform Gaussian fitting on each centroid area to obtain a unimodal Gaussian curve; select the position corresponding to the maximum value of each unimodal Gaussian curve, and set the position as the first coordinate.

Step 302: Set the first displacement value corresponding to each first coordinate as a second coordinate, and select several first coordinate pairs based on several first coordinates and several second coordinates.

In this embodiment, the number of the first coordinate pairs is greater than three.

In this embodiment, several coordinate pairs are selected based on several first coordinates and several second coordinates, specifically: combining each first coordinate with the second coordinate corresponding to each first coordinate. , generate several second coordinate pairs; according to the consistency sampling algorithm, eliminate the error points in the several second coordinate pairs, and obtain the several first coordinate pairs.

Step 303: Calculate a linear equation according to the several first coordinate pairs; wherein the linear relationship includes the linear equation.

In this embodiment, calculating a linear equation based on the several first coordinate pairs specifically includes: establishing a fitting straight line equation based on the several first coordinate pairs; calculating a linear equation based on the straight line equation. The error value is calculated; the slope is calculated according to the least square method and the error value; and the linear equation is established based on the slope.

In this embodiment, the linear equation is expressed as y=kx+b, where x is the variable of the first coordinate, y is the variable of the second coordinate, k is the slope, and b is the offset.

Please refer to FIG. 4 , which is a schematic flow chart of another embodiment of a focus locking method for a microscope provided by the present invention. The main difference between Figure 4 and Figure 1 is that Figure 3 also includes steps 301 to 303, which are as follows:

In this embodiment, the linear relationship includes a linear equation.

Step 301: According to the center of mass of the image to be locked and the current position of the Z-axis displacer, solve to obtain the offset.

Step 302: Substitute the center of mass and the offset into the linear equation, and solve to obtain the second displacement value of the Z-axis displacer required for focus locking.

Step 303: According to the second displacement value, adjust the position of the Z-axis displacer so that the center of mass returns to the focus-locked position.

Please refer to FIG. 5 , which is a schematic structural diagram of an embodiment of the focus locking device of a microscope provided by the present invention. It mainly includes: an acquisition module 501 , a calculation module 502 and a focus locking module 503 .

In this embodiment, the acquisition module 501 is used to acquire the displacement data of the Z-axis displacer and the image data of the glass slide in real time.

The calculation module 502 is used to obtain a linear relationship between the displacement data and the image data through curve fitting processing according to the displacement data and the image data.

The focus lock module 503 is used to adjust the position of the Z-axis displacer according to the linear relationship to achieve focus lock.

Please refer to Figure 6 , which is a schematic structural diagram of another embodiment of the focus locking device of a microscope provided by the present invention. The main difference between Figure 6 and Figure 5 is that Figure 6 also includes: a first collection unit 601 and a second collection unit 602.

In this embodiment, the collection module 501 includes a first collection unit 601 and a second collection unit 602.

The first acquisition unit 601 is used to change the position of the Z-axis displacer, and obtain several first displacement values according to the movement distance of the Z-axis displacer; wherein the displacement data is the several first displacement values.

The second acquisition unit 602 is used to acquire image data of the slide corresponding to each displacement value.

In this embodiment, the first acquisition unit 601 can be replaced by a third acquisition unit; the third acquisition unit is used to record several first displacement values generated by the Z-axis displacer when the stage sinks.

Please refer to Figure 7 , which is a schematic structural diagram of another embodiment of the focus locking device of a microscope provided by the present invention. The main difference between Figure 7 and Figure 5 is that Figure 7 also includes: a curve fitting unit 701 and a coordinate pair generation unit. 702 and equation building unit 703.

In this embodiment, the calculation module 502 includes: a curve fitting unit 701, a coordinate pair generating unit 702, and an equation establishing unit 703.

The curve fitting unit 701 is configured to obtain several first coordinates after curve fitting based on the image data corresponding to each first displacement value.

In this embodiment, the curve fitting unit 701 also includes a centroid selection subunit, a curve fitting subunit and a first setting subunit; the centroid selection subunit is used to select the centroid area in each image data; the curve fitter The unit is used to perform Gaussian fitting on each of the centroid regions to obtain a unimodal Gaussian curve; the first setting subunit is used to select the position corresponding to the maximum value of each unimodal Gaussian curve, and set the position to The first coordinate.

The coordinate pair generating unit 702 is used to set the first displacement value corresponding to each of the first coordinates as a second coordinate, and select several first coordinate pairs according to several first coordinates and several second coordinates. .

In this embodiment, the coordinate pair generation unit 702 also includes: a coordinate pair generation subunit and an error point elimination subunit; the coordinate pair generation subunit is used to generate each first coordinate corresponding to each first coordinate. The second coordinate combination generates several second coordinate pairs; the erroneous point elimination subunit is used to eliminate erroneous points in the several second coordinate pairs according to the consistency sampling algorithm to obtain the several first coordinate pairs.

The equation establishing unit 703 is configured to calculate a linear equation according to the plurality of first coordinate pairs; wherein the linear relationship includes the linear equation.

In this embodiment, the equation establishing unit 703 includes a straight line fitting subunit, an error value calculating subunit, a slope calculating subunit and an equation establishing subunit; the straight line fitting subunit is used to establish a linear equation based on the several first coordinate pairs. Fitting the straight line equation; the error value calculation subunit is used to calculate the error value according to the straight line equation; the slope calculation subunit is used to calculate the slope according to the least square method and the error value; the equation establishment subunit is used to calculate the slope according to the least squares method and the error value. The slope, establishes the linear equation.

Please refer to Figure 8, which is a schematic structural diagram of another embodiment of the focus locking device of a microscope provided by the present invention. The main difference between Figure 8 and Figure 5 is that Figure 8 also includes: an offset calculation unit 801 and a displacement value calculation unit. Unit 802 and focus lock unit 803.

In this embodiment, the focus lock module 503 includes: an offset calculation unit 801, a displacement value calculation unit 802, and a focus lock unit 803.

The offset calculation unit 801 is used to obtain the offset based on the centroid of the image to be focused and the current position of the Z-axis displacer.

The displacement value calculation unit 802 is used to substitute the center of mass and the offset into the linear equation, and solve to obtain the second displacement value of the Z-axis displacer required for focus locking.

The focus lock unit 803 is used to adjust the position of the Z-axis displacer according to the second displacement value so that the center of mass returns to the focus lock position.

The above-mentioned specific embodiments further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above-mentioned are only specific embodiments of the present invention and are not intended to limit the scope of the present invention. . It is particularly pointed out that for those skilled in the art, anything in the present invention Within the spirit and principles of the present invention, any modifications, equivalent substitutions, improvements, etc. shall be included in the protection scope of the present invention.

Claims

A focus locking method for a microscope, which is characterized by including:

Collect the displacement data of the Z-axis displacer and the image data of the glass slide in real time;

According to the displacement data and the image data, through curve fitting processing, a linear relationship between the displacement data and the image data is obtained;

According to the linear relationship, the position of the Z-axis displacer is adjusted to achieve focus locking.
The focus locking method of a microscope according to claim 1, wherein the real-time collection of the displacement data of the Z-axis displacer and the image data of the glass slide is:

Change the position of the Z-axis displacer, and obtain several first displacement values according to the movement distance of the Z-axis displacer; wherein the displacement data is the several first displacement values;

Image data of the slide corresponding to each of the displacement values is acquired.
The focus locking method of a microscope according to claim 2, characterized in that said changing the position of the Z-axis displacer, obtaining several first displacement values according to the movement distance of the Z-axis displacer, can be replaced by, recording When the object platform sinks, several first displacement values are generated by the Z-axis displacer.
The focus locking method of a microscope according to claim 1, characterized in that, according to the displacement data and the image data, a linear relationship between the displacement data and the image data is obtained through curve fitting processing, specifically as follows: :

Wherein, the displacement data are several first displacement values of the Z-axis displacer;

According to the image data corresponding to each first displacement value, after curve fitting, several first coordinates are obtained;

Set the first displacement value corresponding to each of the first coordinates as a second coordinate, and select several first coordinate pairs based on several first coordinates and several second coordinates;

According to the several first coordinate pairs, a linear equation is calculated; wherein the linear relationship includes include the linear equation.
The focus locking method of a microscope according to claim 4, characterized in that, based on the image data corresponding to each of the first displacement values, several first coordinates are obtained after curve fitting, specifically:

Select the centroid area in each image data;

Perform Gaussian fitting on each of the centroid regions to obtain a single-peak Gaussian curve;

Select the position corresponding to the maximum value of each unimodal Gaussian curve, and set the position as the first coordinate.
The focus locking method of a microscope according to claim 4, wherein several coordinate pairs are selected based on several first coordinates and several second coordinates, specifically:

Combining each first coordinate with the second coordinate corresponding to each first coordinate to generate several second coordinate pairs;

According to the consistency sampling algorithm, error points in the plurality of second coordinate pairs are eliminated to obtain the plurality of first coordinate pairs.
The focus locking method of a microscope according to claim 4, wherein a linear equation is calculated based on the plurality of first coordinate pairs, specifically:

Establish a fitting straight line equation according to the several first coordinate pairs;

According to the straight line equation, the error value is calculated;

According to the least square method and the error value, the slope is calculated;

Based on the slope, the linear equation is established.
The focus locking method of a microscope according to claim 1, wherein the position of the Z-axis displacer is adjusted according to the linear relationship to achieve focus locking, specifically:

Wherein, the linear relationship includes a linear equation;

According to the center of mass of the image to be locked and the current position of the Z-axis displacer, the offset is obtained by solving;

Substitute the center of mass and the offset into the linear equation, and solve to obtain the second displacement value of the Z-axis displacer required for focus locking;

According to the second displacement value, adjust the position of the Z-axis displacer so that the center of mass returns to the focus-locked position.
The focus locking method of a microscope according to any one of claims 1 to 8, wherein the image data is one-dimensional image data generated by a linear CCD collecting the reflected light spot of a glass slide.
A focus locking device for a microscope, characterized by comprising: an acquisition module, a calculation module and a focus locking module;

Among them, the acquisition module is used to collect the displacement data of the Z-axis displacer and the image data of the glass slide in real time;

The calculation module is used to obtain a linear relationship between the displacement data and the image data through curve fitting processing according to the displacement data and the image data;

The focus lock module is used to adjust the position of the Z-axis displacer according to the linear relationship to achieve focus lock.