CN113655610B - Automatic focusing method and control device for photothermal reflection microscopic thermal imaging - Google Patents

Automatic focusing method and control device for photothermal reflection microscopic thermal imaging Download PDF

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CN113655610B
CN113655610B CN202110795894.8A CN202110795894A CN113655610B CN 113655610 B CN113655610 B CN 113655610B CN 202110795894 A CN202110795894 A CN 202110795894A CN 113655610 B CN113655610 B CN 113655610B
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thermal imaging
measured piece
intensity value
illumination light
total intensity
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CN113655610A (en
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刘岩
乔玉娥
邹学峰
丁立强
吴爱华
赵英伟
李锁印
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CETC 13 Research Institute
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Abstract

The invention provides an automatic focusing method and a control device for photothermal reflection microscopic thermal imaging. The method comprises the following steps: acquiring an acquisition image of a measured piece acquired by a photothermal reflection microscopic thermal imaging device with the illumination intensity monotonically changing within a preset maximum defocusing range when the measured piece is positioned at a to-be-focused position, and a reference image of the measured piece when the measured piece is positioned at the focused position; calculating a first total intensity value according to the acquired image, and calculating a second total intensity value according to the reference image; comparing the first total intensity value with the second total intensity value, and determining the defocusing direction of the measured piece based on the comparison result and the monotonous change trend of the illumination intensity in the preset maximum defocusing range; acquiring the defocus depth of a measured piece; the defocusing direction and the defocusing depth are used for focusing the measured piece. The invention can improve focusing efficiency, avoid the problem of insufficient consistency of manual focusing, ensure focusing stability of multi-frame collected images and improve accuracy of measurement results when temperature measurement is performed based on photo-thermal reflection.

Description

Automatic focusing method and control device for photothermal reflection microscopic thermal imaging
Technical Field
The invention relates to the technical field of microscopic temperature imaging, in particular to an automatic focusing method and a control device for photothermal reflection microscopic thermal imaging.
Background
The photothermal reflection temperature measurement technology is a non-contact temperature measurement technology, and is based on the photothermal reflection phenomenon, wherein the photothermal reflection phenomenon is basically characterized in that the reflectivity of an object can change along with the temperature change of the object.
When performing thermometry based on photothermal reflection, in order to realize microscopic thermal imaging with high spatial resolution, a photothermal reflection microscopic imaging device is generally constructed based on a high-performance optical microscope. The light path system of the light microscope is used for providing detection light, a high-performance camera is used for recording microscopic imaging, and the output camera reading is used as a measured value.
However, in the temperature measurement process, in order to ensure measurement accuracy, the camera reading at the reference temperature and the camera reading at the temperature to be measured generally need to take the average value of multiple frames of images. This requires a stable correspondence between the data on each pixel of the camera and the spatial position of the surface of the measured object in the whole measurement process, and if the correspondence is disturbed, the accuracy of the temperature measurement result is affected. However, there are several temperature changes in the test process, and thermal expansion of the corresponding measured piece may cause defocusing of the acquired image, so that the image is blurred, and multiple focusing is required. However, the existing manual focusing method is poor in repeatability and insufficient in focusing consistency, so that focusing stability of multiple frames of collected images is affected, and extra measurement errors are introduced, so that accuracy of measurement results is affected.
Disclosure of Invention
The embodiment of the invention provides an automatic focusing method and a control device for photothermal reflection microscopic thermal imaging, which are used for solving the problems that the focusing stability of a multi-frame acquisition image is affected and a measurement result is inaccurate due to insufficient consistency of the existing manual focusing method.
In a first aspect, an embodiment of the present invention provides an auto-focusing method for photothermal reflection microscopy thermal imaging, including:
Acquiring an acquired image of a measured piece acquired by a photothermal reflection microscopic thermal imaging device when the measured piece is positioned at a position to be focused; wherein the illumination intensity of the photothermal reflection microscopic thermal imaging device monotonically changes within a preset maximum defocus range;
calculating a first total intensity value of the acquired image according to the acquired image, and calculating a second total intensity value of the reference image according to the reference image; the reference image is an image acquired by the photothermal reflection microscopic thermal imaging device when the measured piece is positioned at a focusing position;
Comparing the first total intensity value with the second total intensity value, and determining the defocus direction of the measured piece based on the comparison result and the monotonous change trend of the illumination light intensity in the preset maximum defocus range;
Acquiring the defocus depth of the measured piece; the defocusing direction and the defocusing depth are used for focusing the measured piece.
In one possible implementation manner, the determining the defocus direction of the measured piece based on the comparison result and the tendency of the illumination intensity to monotonically change in the preset maximum defocus range includes:
When the monotonous change trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously increases along with the increase of the object distance in the preset maximum defocus range, if the first total intensity value is larger than the second total intensity value, determining that the defocus direction of the measured piece is the direction of the increase of the object distance; if the first total intensity value is smaller than the second total intensity value, determining that the defocusing direction of the measured piece is the direction of object distance reduction;
When the monotonous change trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously decreases along with the increase of the object distance in the preset maximum defocus range, if the first total intensity value is larger than the second total intensity value, determining that the defocus direction of the measured piece is the object distance decreasing direction; and if the first total intensity value is smaller than the second total intensity value, determining that the defocusing direction of the measured piece is the direction of increasing the object distance.
In one possible implementation manner, the calculating the first total intensity value of the acquired image according to the acquired image and the calculating the second total intensity value of the reference image according to the reference image includes:
According to Calculating a first total intensity value of the acquired image and according toCalculating a second total intensity value of the reference image;
Wherein I c is the first total intensity value, c (x, y) is the gray value of the (x, y) pixel in the acquired image, I r is the second total intensity value, and r (x, y) is the gray value of the (x, y) pixel in the reference image.
In one possible implementation manner, obtaining the defocus depth of the measured piece includes:
calculating a first Fourier transform of the acquired image according to the acquired image, and calculating a second Fourier transform of the reference image according to the reference image;
determining a fitting diameter of a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device according to the first Fourier transform and the second Fourier transform;
and calculating out the defocusing depth of the measured piece according to the fitting diameter and imaging parameters of an optical subsystem in the photothermal reflection microscopic thermal imaging device.
In one possible implementation, the determining the fitting diameter of the point spread function of the optical subsystem in the photothermal reflection microscopy thermal imaging device according to the first fourier transform and the second fourier transform includes:
According to OrCalculating to obtain a point spread function of an optical subsystem in the photothermal reflection microscopic thermal imaging device;
determining the fitting diameter of the point spread function according to the point spread function;
Wherein p (x, y) is a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device, R (u, v) is the first Fourier transform, C (u, v) is the second Fourier transform, Is an inverse fourier transform.
In a second aspect, an embodiment of the present invention provides a control device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, the processor implementing the steps of the method according to the first aspect or any one of the possible implementations of the first aspect when the computer program is executed.
In a third aspect, an embodiment of the present invention provides a photothermal reflection microscopic thermal imaging apparatus, including an illumination light path system, an imaging light path system, and a camera;
And the distance between the imaging position of the light source corresponding to the maximum illumination light intensity in the illumination light path system and the ideal focusing position is at least preset to the maximum defocusing range.
In one possible implementation, the illumination mode of the illumination light path system is critical illumination,
The light source imaging position corresponding to the maximum illumination light intensity in the critical illumination is above or below the focal plane of the image side, and the distance between the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination and the focal plane of the image side is larger than the preset maximum defocus range;
or the illumination mode of the illumination light path system is kohler illumination,
The illumination light in the Kohler illumination diverges or converges outside the preset maximum defocus range.
In a fourth aspect, an embodiment of the present invention provides a photothermal reflection microscopy thermal imaging system, including a control device as described in the second aspect, a photothermal reflection microscopy thermal imaging device as described in the third aspect or any one of possible implementation manners of the third aspect, and a displacement table;
the control device is respectively and electrically connected with the photothermal reflection microscopic thermal imaging device and the displacement table;
The photothermal reflection microscopic thermal imaging device is used for collecting an image collected when the measured piece is positioned at a to-be-focused position and collecting a reference image when the measured piece is positioned at the focused position;
the displacement table is used for placing a measured piece and moving the measured piece according to the defocusing direction and the defocusing depth so as to focus the measured piece.
In one possible implementation, the photothermal reflection microscopy thermal imaging system further comprises: a temperature control table; the photothermal reflection microscopic thermal imaging device comprises an optical platform and an optical subsystem; the optical subsystem comprises an illumination light path system, an imaging light path system and a camera;
The temperature control table is positioned on the displacement table and is electrically connected with the control device; the optical subsystem and the displacement table are respectively positioned on the optical platform;
the temperature control table is used for placing the tested piece, and the optical subsystem is used for collecting an collected image when the tested piece is positioned at a position to be focused and collecting a reference image when the tested piece is positioned at the focusing position; the optical stage is configured to provide support for the optical subsystem and the displacement stage.
In a fifth aspect, embodiments of the present invention provide a computer readable storage medium storing a computer program which, when executed by a processor, implements the steps of the method as described above in the first aspect or any one of the possible implementations of the first aspect.
The embodiment of the invention provides an automatic focusing method and a control device for photothermal reflection microscopic thermal imaging, which are characterized in that a light-thermal reflection microscopic thermal imaging device with illumination intensity monotonously changing in a preset maximum defocus range is used for acquiring an acquisition image when a measured piece is positioned at a to-be-focused position and a reference image when the measured piece is positioned at the focused position, so that a first total intensity value of the acquisition image can be obtained through calculation according to the acquisition image, and a second total intensity value of the reference image can be obtained through calculation according to the reference image. Comparing the first total intensity value with the second total intensity value, the defocusing direction of the measured piece relative to the focusing position when the measured piece is in the focusing position can be automatically determined based on the comparison result and the monotonous change trend of the illumination light intensity in the preset maximum defocusing range. And simultaneously acquiring the defocusing depth of the measured piece, and focusing the measured piece according to the defocusing direction and the defocusing depth. The invention can automatically determine the defocusing direction of the measured piece relative to the focusing position when the measured piece is in the focusing position according to the comparison result of the first total intensity value and the second total intensity value based on the monotonous variation trend of the illumination light intensity in the preset maximum defocusing range. On the one hand, the focusing efficiency can be improved, on the other hand, even when the temperature changes for a plurality of times to cause the need of focusing for a plurality of times, the focusing is carried out according to the reference image of the measured piece at the focusing position and the acquired image of the measured piece at the focusing position, so that the problem of insufficient consistency of manual focusing can be avoided, the focusing stability of the multi-frame acquired image is ensured, the error caused by defocusing in the acquisition process is reduced, and the accuracy of the measurement result when the temperature measurement is carried out based on the photo-thermal reflection is improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an application scenario diagram of an auto-focus method for photothermal reflection microscopy thermal imaging according to an embodiment of the present invention;
FIG. 2 is a flowchart of an implementation of an auto-focus method for photothermal reflection microscopy thermal imaging according to an embodiment of the invention;
FIG. 3 is a flowchart for obtaining the defocus depth of a measured object according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of an auto-focusing apparatus for photothermal reflection microscopy thermal imaging according to an embodiment of the invention;
fig. 5 is a schematic diagram of a control device according to an embodiment of the present invention.
Detailed Description
In the following description, for purposes of explanation and not limitation, specific details are set forth such as the particular system architecture, techniques, etc., in order to provide a thorough understanding of the embodiments of the present invention. It will be apparent, however, to one skilled in the art that the present invention may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present invention with unnecessary detail.
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the following description will be made by way of specific embodiments with reference to the accompanying drawings.
In the prior art, the change in reflectivity with temperature can be considered to be linear, and thus can be characterized by a coefficient of change, commonly referred to in the literature as the photothermal reflectance (Thermoreflectance Coefficience) or photothermal reflectance calibration coefficient (Thermoreflectance Calibration Coefficience), denoted by C TR, defined as:
Wherein R is the reference reflectivity, deltaR is the reflectivity variation, deltaT is the temperature variation.
For most metal and semiconductor materials, the range of C TR is typically (10 -2～10-5)K-1 and is related to material, incident light wavelength, angle of incidence, if the surface of the part being measured has a multi-layer structure, the material composition of each layer and the interference of light between the layers of material also directly affect the magnitude of C TR, typically by selecting the appropriate measurement wavelength for each (type or model) sample of the part being measured, and measuring C TR, typically referred to as C TR calibration (C TR calibration), and using the measured C TR for temperature measurement.
In the case of C TR, the temperature can be calculated by measuring the change in reflectivity of the test piece according to the following equation:
Wherein T x is the temperature to be measured, T 0 is the reference temperature, R x is the reflectivity at the temperature to be measured, and R 0 is the reflectivity at the reference temperature.
Since the rate of change of reflectivity is of practical interestTherefore, a beam of detection light (incident light) can be projected to the surface of the measured piece, and then the temperature measurement can be realized by measuring the change rate of the intensity of reflected light, which is also a mainstream implementation mode of the current photo-thermal reflection temperature measurement technology. Assuming that the detected light intensity is unchanged, the rate of change of reflectivity in the formula for calculating the temperature may be equivalent to the rate of change of the detector readings, i.e. the formula for calculating the temperature changes to:
Where c x is the detector reading at the temperature to be measured and c 0 is the detector reading at the reference temperature.
To achieve high spatial resolution microscopic thermal imaging, photothermal reflection microscopic thermal imaging devices are typically built based on high performance optical microscopes. The detection light is provided by an illumination light path system of the optical microscope, microscopic imaging is recorded by using a high-performance camera, and the output camera reading is taken as a measured value c.
Since the magnitude of C TR is low, in order to ensure measurement accuracy, the average value of multiple frames of images is generally required to be obtained when C 0 and C x are obtained, and the total number of measured frames is recorded as N, which is as follows:
from the above principle, the data on each pixel of the camera and the spatial position of the surface of the measured object have a stable corresponding relationship in the whole measuring process, and if the corresponding relationship is interfered, the accuracy of the temperature measuring result is affected. In the test process, the temperature changes for a plurality of times, the thermal expansion of the corresponding tested piece can cause defocusing to cause image blurring, multiple times of focusing are needed, and the focusing consistency is good enough to ensure that the corresponding relation is stable and consistent, otherwise, additional errors are introduced.
However, the existing manual focusing method is poor in repeatability and insufficient in focusing consistency, so that focusing stability of multiple frames of collected images is affected, and accuracy of a measurement result is affected.
In order to solve the above-mentioned problems, an embodiment of the present invention proposes an auto-focusing method for photothermal reflection microscopy thermal imaging, and fig. 1 is an application scenario diagram of the auto-focusing method for photothermal reflection microscopy thermal imaging provided by the embodiment of the present invention. The method can be applied to, but is not limited to, the application scenario.
By applying the automatic focusing method for the photothermal reflection microscopic thermal imaging, disclosed by the embodiment of the invention, the photothermal reflection microscopic thermal imaging system is formed by combining the photothermal reflection microscopic thermal imaging device. As shown in fig. 1, the photothermal reflection microscopic thermal imaging system comprises a photothermal reflection microscopic thermal imaging device, a displacement table 30 and the like, wherein the photothermal reflection microscopic thermal imaging device is composed of a control device 10, an optical platform 21 and an optical subsystem 22.
The method comprises the steps of collecting an image collected when a measured piece is located at a position to be focused by using a photothermal reflection microscopic thermal imaging device, and collecting a reference image when the measured piece is located at the focusing position. The photothermal reflection microscopic thermal imaging device and the displacement table 30 are electrically connected with the control device 10, the control device 10 acquires an acquisition image and a reference image acquired by the photothermal reflection microscopic thermal imaging device, and the defocus direction and the defocus depth of the measured piece are obtained after the processing procedure of the automatic focusing method for the photothermal reflection microscopic thermal imaging in the embodiment of the invention is executed. The displacement table 30 is used for placing the measured object and moving the measured object according to the defocus direction and defocus depth obtained by the control device 10 so as to focus the measured object.
Wherein displacement stage 30 and optical subsystem 22 may be located on optical stage 21, respectively, optical stage 21 being configured to provide support for optical subsystem 22 and displacement stage 30.
Alternatively, the displacement stage 30 may be a 3-axis nano displacement stage, so as to compensate for the displacement of the measured member caused by factors such as temperature variation during the temperature test, for example, the displacement of the measured member caused by defocus.
Optionally, the photothermal reflection microscopy thermal imaging system may further include a temperature control platform 40, where the temperature control platform 40 is located on the displacement platform 30, and the temperature control platform 40 may also be electrically connected to the control device 10, where the temperature control platform 30 is used for placing a measured object, so as to control a temperature environment of the measured object. Illustratively, the temperature control station 40 may be a programmable cold and hot station.
According to the photothermal reflection microscopic thermal imaging system provided by the embodiment of the invention, the photothermal reflection microscopic thermal imaging device is a photothermal reflection microscopic thermal imaging device with the illumination intensity monotonously changing in the preset maximum defocus range, and the control device is a device for processing according to the automatic focusing method for the photothermal reflection microscopic thermal imaging, so that the defocus direction of a measured piece relative to a focusing position when the measured piece is in a focusing position can be automatically determined based on the monotonous change trend of the illumination intensity in the preset maximum defocus range and the processing of the control device, and focusing is performed according to the defocus direction and the defocus depth. The focusing efficiency can be improved, when the temperature changes repeatedly to cause the requirement of focusing repeatedly, focusing is carried out according to the reference image of the measured piece at the focusing position and the acquisition image of the measured piece at the focusing position, so that the problem of insufficient consistency of manual focusing is avoided, the focusing stability of multi-frame acquisition images is ensured, the error caused by defocusing in the acquisition process is reduced, and the accuracy of the measurement result when the temperature measurement is carried out based on photo-thermal reflection is improved.
The light-heat reflection microscopic thermal imaging device for collecting the collected image when the measured piece is located at the focusing position and collecting the reference image when the measured piece is located at the focusing position can be functionally divided into an illumination light path system (device for realizing that the illumination light beam emitted by the LED in fig. 1 reaches the space where the measured piece is located), an imaging light path system (device for realizing that the reflected light beam emitted by the measured piece in fig. 1 reaches the camera) and the camera according to functions in combination with fig. 1.
In order to facilitate the automatic focusing method for photothermal reflection microscopic thermal imaging provided by the embodiment of the invention to determine the defocus direction of the measured piece within the preset maximum defocus range, the distance between the imaging position of the light source corresponding to the maximum illumination light intensity in the illumination light path system and the ideal focusing position is required to be at least the preset maximum defocus range.
Optionally, the illumination mode of the illumination light path system is critical illumination, and the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination is above or below the focal plane of the image side, and the distance between the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination and the focal plane of the image side is greater than the preset maximum defocus range. The light-heat reflection microscopic thermal imaging device is used for obtaining the distance between the imaging position of the light source corresponding to the maximum illumination light intensity in the illumination light path system and the ideal focusing position, and the distance is at least preset to the maximum defocusing range. Or adjusting the original photothermal reflection microscopic thermal imaging device with the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination at the focal plane of the image side to obtain the photothermal reflection microscopic thermal imaging device with the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination above or below the focal plane of the image side after adjustment, wherein the distance between the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination and the focal plane of the image side is larger than the preset maximum defocus range.
Optionally, the illumination mode of the illumination light path system is kohler illumination, and illumination light in the kohler illumination diverges or converges outside a preset maximum defocus range. The light-heat reflection microscopic thermal imaging device is used for obtaining the distance between the imaging position of the light source corresponding to the maximum illumination light intensity in the illumination light path system and the ideal focusing position, and the distance is at least preset to the maximum defocusing range. Or adjusting the parallel illumination light in the Kohler illumination to obtain the photothermal reflection microscopic thermal imaging device with the illumination light in the Kohler illumination after adjustment dispersed or converged outside the preset maximum defocusing range.
Referring to fig. 2, a flowchart of an implementation of an auto-focusing method for photothermal reflection microscopy thermal imaging according to an embodiment of the present invention is shown, and details are as follows:
In step 201, an acquired image of a measured object acquired by a photothermal reflection microscopy thermal imaging device is acquired when the measured object is located at a position to be focused.
The photothermal reflection microscopic thermal imaging device is provided in the embodiment, wherein the illumination light intensity is monotonically changed within a preset maximum defocus range.
In step 202, a first total intensity value of the acquired image is calculated from the acquired image, and a second total intensity value of the reference image is calculated from the reference image.
The reference image is an image of the measured piece acquired by the photothermal reflection microscopic thermal imaging device when the measured piece is positioned at the focusing position.
The measured piece can be located at the focusing position through manual focusing or an existing automatic focusing method, and then the corresponding reference image at the moment is acquired by utilizing the photothermal reflection microscopic thermal imaging device with the illumination intensity monotonically changing within the preset maximum defocusing range. And when focusing and focus following are needed in the subsequent test process, the reference image is used as a reference to process the acquired image.
Optionally, calculating the first total intensity value of the acquired image according to the acquired image and calculating the second total intensity value of the reference image according to the reference image may include:
According to Calculating a first total intensity value of the acquired image and according toA second total intensity value of the reference image is calculated.
Wherein I c is a first total intensity value, and c (x, y) is a gray value of a (x, y) pixel point in the acquired image, which is used for representing the acquired image. I r is the second total intensity value, and r (x, y) is the gray value of the (x, y) pixel point in the reference image, which is used for representing the reference image.
In step 203, the magnitudes of the first total intensity value and the second total intensity value are compared, and the defocus direction of the measured object is determined based on the comparison result and the tendency of monotonically changing illumination intensity within the preset maximum defocus range.
Optionally, determining the defocus direction of the measured object based on the comparison result and the tendency of the illumination intensity to monotonically change in the preset maximum defocus range may include:
When the monotonous change trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously increases along with the increase of the object distance in the preset maximum defocus range, if the first total intensity value I c is larger than the second total intensity value I r, determining that the defocus direction of the measured piece is the direction of the increase of the object distance; if the first total intensity value I c is smaller than the second total intensity value I r, determining the defocusing direction of the measured piece as the direction of object distance reduction.
When the monotonous change trend of the illumination intensity within the preset maximum defocus range is that the illumination intensity is monotonously reduced along with the increase of the object distance within the preset maximum defocus range, if the first total intensity value I c is larger than the second total intensity value I r, determining that the defocus direction of the measured piece is the object distance reduction direction; if the first total intensity value I c is smaller than the second total intensity value I r, determining that the defocusing direction of the measured piece is the direction of increasing the object distance.
In this embodiment, since the illumination intensity of the photothermal reflection microscopic thermal imaging device monotonically changes within the preset maximum defocus range (i.e., the illumination intensity monotonically increases or decreases with the object distance within the preset maximum defocus range), and the reference image is an image of the measured object at the focusing position, it can be determined whether the collected image is shifted in the direction of increasing the object distance or in the direction of decreasing the object distance relative to the reference image according to the magnitude of the first total intensity value of the collected image relative to the second total intensity value of the reference image, thereby determining the defocus direction of the measured object at the focusing position relative to the focusing position.
In step 204, the defocus depth of the test piece is obtained. The defocusing direction and the defocusing depth are used for focusing the measured piece.
When focusing the measured piece, besides determining the defocusing direction, the defocusing depth needs to be determined.
Optionally, referring to fig. 3, a flowchart for obtaining a defocus depth of a measured object according to an embodiment of the present invention is shown, and details are as follows:
in step 301, a first fourier transform of the acquired image is calculated from the acquired image and a second fourier transform of the reference image is calculated from the reference image.
Wherein according toCalculating a first Fourier transform C (u, v) of the acquired image according toA second fourier transform R (u, v) of the reference image is calculated. Wherein,Is a fourier transform.
In step 302, a fitted diameter of a point spread function of an optical subsystem in a photothermal reflectance microscopy thermal imaging device is determined from the first fourier transform and the second fourier transform.
Optionally, determining the fitting diameter of the point spread function of the optical subsystem in the photothermal reflection microscopy thermal imaging device according to the first fourier transform and the second fourier transform may include:
According to OrCalculating to obtain a point spread function of an optical subsystem in the photo-thermal reflection microscopic thermal imaging device; from the point spread function, a fitting diameter of the point spread function is determined.
Wherein p (x, y) is a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device, R (u, v) is a first Fourier transform, C (u, v) is a second Fourier transform,Is an inverse fourier transform.
Wherein, only the defocus effect is considered, the point spread function p (x, y) should be in an approximate airy-spot form. If required for precision, the fitting diameter of the point spread function p (x, y) in the form of an approximate airy patch can be obtained by directly counting the number of pixels (i.e. counting from the origin to two opposite directions until the value of p (x, y) in both opposite directions is 0). Or solving according to the analysis formula of the Airy spot according to discrete points on the point spread function p (x, y), determining unknown parameters in the analysis formula of the Airy spot, and further determining the fitting diameter of the point spread function p (x, y) according to the analysis formula of the Airy spot after determining the unknown parameters.
In step 303, the defocus depth of the measured object is calculated according to the fitting diameter and the imaging parameters of the optical subsystem in the photothermal reflection microscopy thermal imaging device.
Wherein imaging parameters of the optical subsystem in the photothermal reflection microscopy thermal imaging device may include one or more of: camera element size parameters, magnification parameters, and objective aperture angle parameters.
Optionally, calculating the defocus depth of the measured piece according to the fitting diameter and imaging parameters of an optical subsystem in the photothermal reflection microscopic thermal imaging device may include:
According to And calculating out the defocus depth of the measured piece.
Wherein d is a fitting diameter, a is a camera pixel size parameter, m is a magnification parameter, θ is one half of an objective aperture angle parameter, and the air has a numerical aperture of n.a. =sin θ.
After the defocusing direction and the defocusing depth are obtained, the vertical direction of a 3-axis nano displacement table in the photo-thermal reflection microscopic thermal imaging system can be operated through a proportional-integral-derivative control algorithm so as to perform closed-loop focusing.
When continuous focusing is needed for tracking, the automatic focusing method for photothermal reflection microscopic thermal imaging in the embodiment can be used for processing each acquired image to realize tracking.
According to the embodiment of the invention, the light-heat reflection microscopic thermal imaging device with the illumination light intensity monotonously changing in the preset maximum defocusing range is utilized to collect the collected image when the measured piece is positioned at the to-be-focused position and the reference image when the measured piece is positioned at the focused position, so that the first total intensity value of the collected image can be obtained through calculation according to the collected image, and the second total intensity value of the reference image can be obtained through calculation according to the reference image. Comparing the first total intensity value with the second total intensity value, the defocusing direction of the measured piece relative to the focusing position when the measured piece is in the focusing position can be automatically determined based on the comparison result and the monotonous change trend of the illumination light intensity in the preset maximum defocusing range. And simultaneously acquiring the defocusing depth of the measured piece, and focusing the measured piece according to the defocusing direction and the defocusing depth. The invention can automatically determine the defocusing direction of the measured piece relative to the focusing position when the measured piece is in the focusing position according to the comparison result of the first total intensity value and the second total intensity value based on the monotonous variation trend of the illumination light intensity in the preset maximum defocusing range. On the one hand, the focusing efficiency can be improved, on the other hand, even when the temperature changes for a plurality of times to cause the need of focusing for a plurality of times, the focusing is carried out according to the reference image of the measured piece at the focusing position and the acquired image of the measured piece at the focusing position, so that the problem of insufficient consistency of manual focusing can be avoided, the focusing stability of the multi-frame acquired image is ensured, the error caused by defocusing in the acquisition process is reduced, and the accuracy of the measurement result when the temperature measurement is carried out based on the photo-thermal reflection is improved.
It should be understood that the sequence number of each step in the foregoing embodiment does not mean that the execution sequence of each process should be determined by the function and the internal logic, and should not limit the implementation process of the embodiment of the present invention.
The following are device embodiments of the invention, for details not described in detail therein, reference may be made to the corresponding method embodiments described above.
Fig. 4 is a schematic structural diagram of an autofocus device for photothermal reflection microscopic thermal imaging according to an embodiment of the present invention, and for convenience of explanation, only a portion related to the embodiment of the present invention is shown, which is described in detail below:
As shown in fig. 4, the auto-focusing device 4 for photothermal reflection microscopic thermal imaging includes: a first acquisition module 41, a calculation module 42, a comparison module 43 and a second acquisition module 44.
A first acquiring module 41, configured to acquire an acquired image of a measured piece acquired by the photothermal reflection microscopic thermal imaging device when the measured piece is located at a position to be focused; wherein the illumination intensity of the photothermal reflection microscopic thermal imaging device monotonically changes within a preset maximum defocus range;
a calculation module 42, configured to calculate a first total intensity value of the acquired image according to the acquired image, and calculate a second total intensity value of the reference image according to the reference image; the reference image is an image acquired by the photothermal reflection microscopic thermal imaging device when the measured piece is positioned at a focusing position;
A comparing module 43, configured to compare the first total intensity value and the second total intensity value, and determine a defocus direction of the measured object based on a comparison result and a tendency of monotonically changing the illumination intensity within a preset maximum defocus range;
a second acquisition module 44, configured to acquire a defocus depth of the measured piece; the defocusing direction and the defocusing depth are used for focusing the measured piece.
According to the embodiment of the invention, the light-heat reflection microscopic thermal imaging device with the illumination light intensity monotonously changing in the preset maximum defocusing range is utilized to collect the collected image when the measured piece is positioned at the to-be-focused position and the reference image when the measured piece is positioned at the focused position, so that the first total intensity value of the collected image can be obtained through calculation according to the collected image, and the second total intensity value of the reference image can be obtained through calculation according to the reference image. Comparing the first total intensity value with the second total intensity value, the defocusing direction of the measured piece relative to the focusing position when the measured piece is in the focusing position can be automatically determined based on the comparison result and the monotonous change trend of the illumination light intensity in the preset maximum defocusing range. And simultaneously acquiring the defocusing depth of the measured piece, and focusing the measured piece according to the defocusing direction and the defocusing depth. The invention can automatically determine the defocusing direction of the measured piece relative to the focusing position when the measured piece is in the focusing position according to the comparison result of the first total intensity value and the second total intensity value based on the monotonous variation trend of the illumination light intensity in the preset maximum defocusing range. On the one hand, the focusing efficiency can be improved, on the other hand, even when the temperature changes for a plurality of times to cause the need of focusing for a plurality of times, the focusing is carried out according to the reference image of the measured piece at the focusing position and the acquired image of the measured piece at the focusing position, so that the problem of insufficient consistency of manual focusing can be avoided, the focusing stability of the multi-frame acquired image is ensured, the error caused by defocusing in the acquisition process is reduced, and the accuracy of the measurement result when the temperature measurement is carried out based on the photo-thermal reflection is improved.
In a possible implementation manner, the comparing module 43 may be configured to determine that, when the tendency of the monotonic change of the illumination intensity within the preset maximum defocus range is that the illumination intensity monotonically increases with the increase of the object distance within the preset maximum defocus range, if the first total intensity value is greater than the second total intensity value, the defocus direction of the measured object is the direction of the increase of the object distance; if the first total intensity value is smaller than the second total intensity value, determining that the defocusing direction of the measured piece is the direction of object distance reduction;
When the monotonous change trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously decreases along with the increase of the object distance in the preset maximum defocus range, if the first total intensity value is larger than the second total intensity value, determining that the defocus direction of the measured piece is the object distance decreasing direction; and if the first total intensity value is smaller than the second total intensity value, determining that the defocusing direction of the measured piece is the direction of increasing the object distance.
In one possible implementation, the computing module 42 may be configured to, based onCalculating a first total intensity value of the acquired image and according toCalculating a second total intensity value of the reference image;
Wherein I c is the first total intensity value, c (x, y) is the gray value of the (x, y) pixel in the acquired image, I r is the second total intensity value, and r (x, y) is the gray value of the (x, y) pixel in the reference image.
In one possible implementation, the second obtaining module 44 may be configured to calculate a first fourier transform of the acquired image from the acquired image, and calculate a second fourier transform of the reference image from the reference image;
determining a fitting diameter of a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device according to the first Fourier transform and the second Fourier transform;
and calculating out the defocusing depth of the measured piece according to the fitting diameter and imaging parameters of an optical subsystem in the photothermal reflection microscopic thermal imaging device.
In one possible implementation, the second obtaining module 44 may be configured to, according toOrCalculating to obtain a point spread function of an optical subsystem in the photothermal reflection microscopic thermal imaging device;
determining the fitting diameter of the point spread function according to the point spread function;
Wherein p (x, y) is a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device, R (u, v) is the first Fourier transform, C (u, v) is the second Fourier transform, Is an inverse fourier transform. /(I)
Fig. 5 is a schematic diagram of a control device according to an embodiment of the present invention. As shown in fig. 5, the control device 5 of this embodiment includes: a processor 50, a memory 51 and a computer program 52 stored in said memory 51 and executable on said processor 50. The processor 50, when executing the computer program 52, implements the steps of the various embodiments of the autofocus method for photothermal reflection microscopy thermal imaging described above, such as steps 201 to 204 shown in fig. 2, or steps 301 to 303 shown in fig. 3. Or the processor 50, when executing the computer program 52, performs the functions of the modules of the apparatus embodiments described above, such as the functions of the modules 41 to 44 shown in fig. 4.
By way of example, the computer program 52 may be partitioned into one or more modules/units that are stored in the memory 51 and executed by the processor 50 to complete the present invention. The one or more modules/units may be a series of computer program instruction segments capable of performing specific functions for describing the execution of the computer program 52 in the control means 5. For example, the computer program 52 may be partitioned into modules 41 to 44 shown in fig. 4.
The control device 5 may be a computing device such as a desktop computer, a notebook computer, a palm computer, a cloud server, etc. The control device 5 may include, but is not limited to, a processor 50, a memory 51. It will be appreciated by those skilled in the art that fig. 5 is merely an example of the control device 5 and does not constitute a limitation of the control device 5, and may include more or less components than illustrated, or may combine certain components, or different components, e.g., the control device may further include an input-output device, a network access device, a bus, etc.
The Processor 50 may be a central processing unit (Central Processing Unit, CPU), other general purpose Processor, digital signal Processor (DIGITAL SIGNAL Processor, DSP), application SPECIFIC INTEGRATED Circuit (ASIC), field-Programmable gate array (Field-Programmable GATE ARRAY, FPGA) or other Programmable logic device, discrete gate or transistor logic device, discrete hardware components, or the like. A general purpose processor may be a microprocessor or the processor may be any conventional processor or the like.
The memory 51 may be an internal storage unit of the control device 5, such as a hard disk or a memory of the control device 5. The memory 51 may be an external storage device of the control apparatus 5, such as a plug-in hard disk, a smart memory card (SMART MEDIA CARD, SMC), a Secure Digital (SD) card, a flash memory card (FLASH CARD) or the like, which are provided in the control apparatus 5. Further, the memory 51 may also include both an internal storage unit and an external storage device of the control apparatus 5. The memory 51 is used for storing the computer program and other programs and data required by the control device. The memory 51 may also be used to temporarily store data that has been output or is to be output.
The embodiment of the invention also provides a photothermal reflection microscopic thermal imaging device, which comprises an illumination light path system, an imaging light path system and a camera;
the imaging position of the light source corresponding to the maximum illumination intensity in the illumination light path system is far away from the ideal focusing position by at least a distance of a preset maximum defocusing range.
Optionally, the illumination mode of the illumination light path system is critical illumination, and the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination is above or below the focal plane of the image side, and the distance between the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination and the focal plane of the image side is greater than the preset maximum defocus range.
Optionally, the illumination mode of the illumination light path system is kohler illumination, and illumination light in the kohler illumination diverges or converges outside the preset maximum defocus range.
The photothermal reflection microscopic thermal imaging device provided by the embodiment of the invention can ensure that the imaging position of the light source corresponding to the maximum illumination light intensity in the illumination light path system is far away from the ideal focusing position by at least the distance of the preset maximum defocusing range. Furthermore, the automatic focusing method for photothermal reflection microscopic thermal imaging provided by the embodiment is convenient for determining the defocusing direction of the measured piece within the preset maximum defocusing range.
The embodiment of the invention also provides a photothermal reflection microscopic thermal imaging system, referring to fig. 1, the system comprises a control device 10, a photothermal reflection microscopic thermal imaging device and a displacement table 30;
the control device 10 is electrically connected to the photothermal reflection microscopy thermal imaging device and the displacement stage 30, respectively.
The photothermal reflection microscopic thermal imaging device is used for collecting an image when the measured piece is located at the to-be-focused position and collecting a reference image when the measured piece is located at the focused position.
The displacement table 30 is used for placing the measured piece and moving the measured piece according to the defocusing direction and the defocusing depth so as to focus the measured piece.
Optionally, the photothermal reflection microscopy thermal imaging system further comprises: a temperature control stage 40; the photothermal reflection microscopic thermal imaging device comprises an optical platform 21 and an optical subsystem 22; the optical subsystem 22 includes an illumination light path system, an imaging light path system, and a camera, among others.
The temperature control table 40 is positioned on the displacement table 30, and the temperature control table 40 is electrically connected with the control device 10; the optical subsystem 22 and the displacement stage 30 are respectively positioned on the optical stage 21; the temperature control table 40 is used for placing a measured piece, and the optical subsystem 22 is used for collecting an image collected when the measured piece is positioned at a position to be focused and collecting a reference image when the measured piece is positioned at the focusing position; the optical stage 21 is used to provide support for the optical subsystem 22 and displacement stage 30.
According to the photothermal reflection microscopic thermal imaging system provided by the embodiment of the invention, the photothermal reflection microscopic thermal imaging device is the photothermal reflection microscopic thermal imaging device with the illumination light intensity monotonously changing within the preset maximum defocus range, and the control device is a device for processing according to the automatic focusing method for the photothermal reflection microscopic thermal imaging, so that the defocus direction of a measured piece relative to a focusing position when the measured piece is in a to-be-focused position can be automatically determined based on the monotonous change trend of the illumination light intensity within the preset maximum defocus range and the processing of the control device, and focusing is performed according to the defocus direction and the defocus depth. The focusing efficiency can be improved, when the temperature changes repeatedly to cause the requirement of focusing repeatedly, focusing is carried out according to the reference image of the measured piece at the focusing position and the acquisition image of the measured piece at the focusing position, so that the problem of insufficient consistency of manual focusing is avoided, the focusing stability of multi-frame acquisition images is ensured, the error caused by defocusing in the acquisition process is reduced, and the accuracy of the measurement result when the temperature measurement is carried out based on photo-thermal reflection is improved.
It will be apparent to those skilled in the art that, for convenience and brevity of description, only the above-described division of the functional units and modules is illustrated, and in practical application, the above-described functional distribution may be performed by different functional units and modules according to needs, i.e. the internal structure of the apparatus is divided into different functional units or modules to perform all or part of the above-described functions. The functional units and modules in the embodiment may be integrated in one processing unit, or each unit may exist alone physically, or two or more units may be integrated in one unit, where the integrated units may be implemented in a form of hardware or a form of a software functional unit. In addition, the specific names of the functional units and modules are only for distinguishing from each other, and are not used for limiting the protection scope of the present application. The specific working process of the units and modules in the above system may refer to the corresponding process in the foregoing method embodiment, which is not described herein again.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
Those of ordinary skill in the art will appreciate that the various illustrative elements and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, or combinations of computer software and electronic hardware. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the solution. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.
In the embodiments provided in the present invention, it should be understood that the disclosed apparatus/control apparatus and method may be implemented in other manners. For example, the apparatus/control apparatus embodiments described above are merely illustrative, e.g., the division of the modules or units is merely a logical function division, and there may be additional divisions in actual implementation, e.g., multiple units or components may be combined or integrated into another system, or some features may be omitted or not performed. Alternatively, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection via interfaces, devices or units, which may be in electrical, mechanical or other forms.
The units described as separate units may or may not be physically separate, and units shown as units may or may not be physical units, may be located in one place, or may be distributed on a plurality of network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
In addition, each functional unit in the embodiments of the present invention may be integrated in one processing unit, or each unit may exist alone physically, or two or more units may be integrated in one unit. The integrated units may be implemented in hardware or in software functional units.
The integrated modules/units, if implemented in the form of software functional units and sold or used as stand-alone products, may be stored in a computer readable storage medium. Based on such understanding, the present invention may implement all or part of the above-described embodiment of the method, or may be implemented by a computer program for instructing related hardware, where the computer program may be stored in a computer readable storage medium, and the computer program may be executed by a processor to implement the steps of each of the above-described embodiments of the autofocus method for photothermal reflection microscopy thermal imaging. Wherein the computer program comprises computer program code which may be in source code form, object code form, executable file or some intermediate form etc. The computer readable medium may include: any entity or device capable of carrying the computer program code, a recording medium, a U disk, a removable hard disk, a magnetic disk, an optical disk, a computer Memory, a Read-Only Memory (ROM), a random access Memory (Random Access Memory, RAM), an electrical carrier signal, a telecommunications signal, a software distribution medium, and so forth. It should be noted that the computer readable medium may include content that is subject to appropriate increases and decreases as required by jurisdictions in which such content is subject to legislation and patent practice, such as in certain jurisdictions in which such content is not included as electrical carrier signals and telecommunication signals.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.

Claims (11)

1. An auto-focusing method for photothermal reflection microscopy thermal imaging, the auto-focusing method comprising:
Acquiring an acquired image of a measured piece acquired by a photothermal reflection microscopic thermal imaging device when the measured piece is positioned at a position to be focused; wherein, the illumination light intensity of the photo-thermal reflection microscopic thermal imaging device monotonously changes in a preset maximum defocus range, and the monotonously changing trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously increases or decreases along with the increase of the object distance in the preset maximum defocus range;
calculating a first total intensity value of the acquired image according to the acquired image, and calculating a second total intensity value of the reference image according to the reference image; the reference image is an image acquired by the photothermal reflection microscopic thermal imaging device when the measured piece is positioned at a focusing position;
Comparing the first total intensity value with the second total intensity value, and determining the defocus direction of the measured piece based on the comparison result and the monotonous change trend of the illumination light intensity in the preset maximum defocus range;
Acquiring the defocus depth of the measured piece; the defocusing direction and the defocusing depth are used for focusing the measured piece.
2. The auto-focusing method for photothermal reflection microscopy thermal imaging according to claim 1, wherein determining the defocus direction of the object to be measured based on the comparison result and a tendency of the illumination intensity to monotonically vary within a preset maximum defocus range comprises:
When the monotonous change trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously increases along with the increase of the object distance in the preset maximum defocus range, if the first total intensity value is larger than the second total intensity value, determining that the defocus direction of the measured piece is the direction of the increase of the object distance; if the first total intensity value is smaller than the second total intensity value, determining that the defocusing direction of the measured piece is the direction of object distance reduction;
When the monotonous change trend of the illumination light intensity in the preset maximum defocus range is that the illumination light intensity monotonously decreases along with the increase of the object distance in the preset maximum defocus range, if the first total intensity value is larger than the second total intensity value, determining that the defocus direction of the measured piece is the object distance decreasing direction; and if the first total intensity value is smaller than the second total intensity value, determining that the defocusing direction of the measured piece is the direction of increasing the object distance.
3. The auto-focusing method for photothermal reflection microscopy thermal imaging according to claim 1 or 2, wherein the calculating a first total intensity value of the acquired image from the acquired image and a second total intensity value of the reference image from the reference image comprises:
According to Calculating a first total intensity value of the acquired image and according toCalculating a second total intensity value of the reference image;
Wherein I c is the first total intensity value, c (x, y) is the gray value of the (x, y) pixel in the acquired image, I r is the second total intensity value, and r (x, y) is the gray value of the (x, y) pixel in the reference image.
4. The auto-focusing method for photothermal reflection microscopy thermal imaging according to claim 1 or 2, wherein obtaining the defocus depth of the test piece comprises:
calculating a first Fourier transform of the acquired image according to the acquired image, and calculating a second Fourier transform of the reference image according to the reference image;
determining a fitting diameter of a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device according to the first Fourier transform and the second Fourier transform;
and calculating out the defocusing depth of the measured piece according to the fitting diameter and imaging parameters of an optical subsystem in the photothermal reflection microscopic thermal imaging device.
5. The method of claim 4, wherein determining the fitted diameter of the point spread function of the optical subsystem in the photothermal reflectance microscopy thermal imaging device from the first fourier transform and the second fourier transform comprises:
According to OrCalculating to obtain a point spread function of an optical subsystem in the photothermal reflection microscopic thermal imaging device;
determining the fitting diameter of the point spread function according to the point spread function;
Wherein p (x, y) is a point spread function of an optical subsystem in the photothermal reflection microscopy thermal imaging device, R (u, v) is the first Fourier transform, C (u, v) is the second Fourier transform, Is an inverse fourier transform.
6. A control device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, characterized in that the processor implements the steps of the method according to any of the preceding claims 1 to 5 when the computer program is executed.
7. The photothermal reflection microscopic thermal imaging device is characterized by comprising an illumination light path system, an imaging light path system and a camera;
The distance of a light source imaging position corresponding to the maximum illumination light intensity in the illumination light path system away from an ideal focusing position by at least a preset maximum defocus range is set, so that the illumination light intensity of the photothermal reflection microscopic thermal imaging device monotonously changes within the preset maximum defocus range, and the monotonous change trend of the illumination light intensity within the preset maximum defocus range is that the illumination light intensity monotonously increases with the increase of the object distance or monotonously decreases with the increase of the object distance within the preset maximum defocus range, so that the steps of the method according to any one of the claims 1 to 5 are realized based on the photothermal reflection microscopic thermal imaging device.
8. The photothermal reflection micro thermal imaging apparatus according to claim 7, wherein the illumination mode of the illumination light path system is critical illumination,
The light source imaging position corresponding to the maximum illumination light intensity in the critical illumination is above or below the focal plane of the image side, and the distance between the light source imaging position corresponding to the maximum illumination light intensity in the critical illumination and the focal plane of the image side is larger than the preset maximum defocus range;
or the illumination mode of the illumination light path system is kohler illumination,
The illumination light in the Kohler illumination diverges or converges outside the preset maximum defocus range.
9. A photothermal reflection microscopy thermal imaging system comprising a control device according to claim 6, a photothermal reflection microscopy thermal imaging device according to claim 7 or 8, and a displacement stage;
the control device is respectively and electrically connected with the photothermal reflection microscopic thermal imaging device and the displacement table;
The photothermal reflection microscopic thermal imaging device is used for collecting an image collected when the measured piece is positioned at a to-be-focused position and collecting a reference image when the measured piece is positioned at the focused position;
the displacement table is used for placing a measured piece and moving the measured piece according to the defocusing direction and the defocusing depth so as to focus the measured piece.
10. The photothermal reflection microscopy thermal imaging system of claim 9, further comprising: a temperature control table; the photothermal reflection microscopic thermal imaging device comprises an optical platform and an optical subsystem; the optical subsystem comprises an illumination light path system, an imaging light path system and a camera;
The temperature control table is positioned on the displacement table and is electrically connected with the control device; the optical subsystem and the displacement table are respectively positioned on the optical platform;
the temperature control table is used for placing the tested piece, and the optical subsystem is used for collecting an collected image when the tested piece is positioned at a position to be focused and collecting a reference image when the tested piece is positioned at the focusing position; the optical stage is configured to provide support for the optical subsystem and the displacement stage.
11. A computer readable storage medium storing a computer program, characterized in that the computer program when executed by a processor implements the steps of the method according to any of the preceding claims 1 to 5.
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