CN116699821A - Microscopic imaging defocusing amount automatic compensation system, microscopic imaging defocusing amount automatic compensation method and microscope - Google Patents
Microscopic imaging defocusing amount automatic compensation system, microscopic imaging defocusing amount automatic compensation method and microscope Download PDFInfo
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- 239000007788 liquid Substances 0.000 claims description 3
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/008—Details of detection or image processing, including general computer control
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- G—PHYSICS
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Abstract
The invention provides a microscopic imaging defocusing amount automatic compensation system, a microscopic imaging defocusing amount automatic compensation method and a microscope, and belongs to the technical field of automatic focusing of microscopes; the system comprises a cover glass, an objective lens, a tube lens, a camera, an electronic zoom lens and a defocusing measurement module; the defocusing measurement module comprises a non-polarized beam splitter, a dichroic mirror, a laser and a position detector, wherein the non-polarized beam splitter, the dichroic mirror and the sample imaging light path are arranged in a non-parallel mode, light emitted by the laser is processed by the non-polarized beam splitter, the dichroic mirror, the electronic zoom lens, the tube lens, the objective lens and the cover glass to form light spots, the axial defocusing amount is obtained through the position detector, the focal length strain amount is obtained according to the axial defocusing amount, and the electronic zoom lens changes the focal length according to the focal length strain amount to realize focal plane locking of a sample object plane. By the method, imaging of different depths of the sample under the condition of no mechanical displacement is realized, and the defocusing amount change caused by the stability of the microscope and external interference in long-time microscopic imaging is automatically compensated.
Description
Technical Field
The invention belongs to the technical field of automatic focusing of microscopes, and particularly relates to an automatic compensation system and method for microscopic imaging defocus amount and a microscope.
Background
In general, when a conventional microscope needs to image samples at different depths, the distance between the objective lens 30 and the sample 10 is usually adjusted by adjusting the displacement device 31, and the adjustment principle is as shown in fig. 1, and by adjusting the displacement device 31 of the mechanical structure of the objective lens 30, the distance between the objective lens 30 and the sample 10 is changed from d1 to d2, and the corresponding object plane is changed from P1 to P2. Of course, some microscopes do this by moving the sample, but the principle is the same, all mechanically moving, with vibration and slow speed in the process. Furthermore, microscopes for biological and medical applications often require long-term microscopic imaging of the sample to explore the varying properties of the sample; due to the influences of vibration, temperature, operation and the like in the environment, when microscopic imaging is carried out, the sample inevitably deviates axially, so that the defocus problem is caused, the defocus of the sample surface is caused, and the long-time observation precision is influenced; further, the larger the magnification, the larger the influence of the focal plane deviation is. Therefore, many manufacturers have introduced an automatic focusing technique, which can compensate for focal drift of a microscope system caused by environmental temperature change, mechanical vibration, motion vibration and the like in the process of acquiring long-time image data, so as to ensure that a clear microscopic image is acquired.
At present, most of automatic focusing technologies adopt a mode of real-time detection and mechanical compensation to compensate defocus amount so as to realize focal plane locking. The common displacement device adopted in the technology realizes mechanical compensation, and the defects of poor stability, external interference, poor operation precision and the like of the microscope lead to defocusing of a sample surface; even if mechanical compensation is achieved with a high-precision displacement device (such as a displacement device employing the piezoelectric actuation principle), the influence of external vibrations cannot be avoided, and an increase in the cost of the apparatus results.
Therefore, how to improve the existing method and system for locking the focal plane of microscopic imaging, so as to realize imaging of different depths of a sample without mechanical displacement, and simultaneously automatically compensate for the variation of defocus caused by the stability of a microscope and external interference in long-time microscopic imaging, is a problem to be solved by those skilled in the art.
Disclosure of Invention
In order to solve the technical problems, the invention provides a microscopic imaging defocus amount automatic compensation system, a microscopic imaging defocus amount automatic compensation method and a microscope, which can realize imaging of different depths of a sample without mechanical displacement and simultaneously automatically compensate defocus amount changes caused by self stability and external interference of the microscope in long-time microscopic imaging.
In a first aspect, the invention provides an automatic compensation system for microscopic imaging defocus, which comprises a cover glass for carrying a sample, and an objective lens, a tube lens and a camera which are positioned on one side of the cover glass far away from the sample, wherein the objective lens, the tube lens and the camera are sequentially arranged along an imaging optical path of the sample and are arranged along the same optical axis, and the automatic compensation system further comprises:
the electronic zoom lens is positioned between the tube mirror and the camera and is arranged with the same optical axis as the tube mirror and is used for changing the focal length of the electronic zoom lens to adjust the effective focal length of the tube mirror;
the defocusing measurement module is positioned between the electronic zoom lens and the camera and is used for realizing focal plane locking of the sample object plane by means of the zooming function of the electronic zoom lens;
the defocusing measurement module comprises a non-polarized beam splitter, a dichroic mirror, a laser and a position detector which are arranged in parallel, wherein the dichroic mirror is positioned on a sample imaging optical path, the laser is positioned between the non-polarized beam splitter and the dichroic mirror, and the position detector is positioned on one side of the non-polarized beam splitter far away from the dichroic mirror; the unpolarized beam splitter and the bicolor lens are arranged in a non-parallel manner with a sample imaging light path, light emitted by the laser is processed by the unpolarized beam splitter, the bicolor lens, the electronic zoom lens, the tube lens, the objective lens and the cover glass to form light spots, the position detector detects the position change of the light spots to obtain axial defocusing quantity, the focal length strain quantity of the electronic zoom lens is obtained according to the axial defocusing quantity, and the focal length of the electronic zoom lens is changed according to the focal length strain quantity to realize focal plane locking of a sample object plane;
the position detector is used for detecting the position change of the light spot, and calculating the axial defocus amount based on a prefabricated relation curve of the position change of the light spot and the axial defocus amount, and the prefabricated relation curve is drawn through a calibration method.
Compared with the prior art, the invention has the beneficial effects that: the electronic zoom lens is additionally arranged between the tube lens and the camera in the sample imaging light path, the effective focal length of the tube lens is changed by adjusting the focal length of the electronic zoom lens, the purpose of changing the position of an imaging surface is achieved, the imaging of different depths of a sample can be realized without any mechanical movement, and the process has no vibration and high focusing speed. Furthermore, an out-of-focus measuring module is additionally arranged between the electronic zoom lens and the camera, the axial relative displacement of the objective lens and the sample is measured, and the electronic zoom lens is utilized to realize the adjustment of the effective focal length of the tube lens, so that the axial out-of-focus amount caused by the relative axial movement of the objective lens and the cover glass is compensated, the focal length dependent variable of the electronic zoom lens is obtained according to the axial out-of-focus amount, the focal length of the electronic zoom lens is changed according to the focal length dependent variable to realize the focal plane locking of the sample object plane, and the out-of-focus amount change caused by the self stability of the microscope and external interference in long-time microscopic imaging is automatically compensated.
In some preferred embodiments, the axial defocus amount is combined with parameters of the objective lens and tube lens to derive the focal length strain amount.
In some preferred embodiments, the position detector is one of a CCD, CMOS, four-quadrant detector, or beam quality analyzer.
In some preferred embodiments, the laser is a near infrared laser.
In some preferred embodiments, the microscopic imaging defocus amount automatic compensation system further comprises an excitation filter disposed between the laser and the unpolarized beam splitter, and an emission filter disposed between the dichroic mirror and the camera.
In some preferred embodiments, the electronic zoom lens is one of a liquid crystal lens, a liquid lens, or a solid state anamorphic lens.
In a second aspect, the present invention provides a microscopic imaging defocus amount automatic compensation method, based on the microscopic imaging defocus amount automatic compensation system of the first aspect, the method comprising:
s01, installing an objective lens with preset multiplying power on a microscope, placing a sample on a cover glass, and enabling the sample to be positioned at the focal position of the objective lens;
s02, starting a laser to emit light beams, and forming first reflected light by reflection of a non-polarized beam splitter and a bicolor mirror in sequence;
s03, the first reflected light sequentially transmits the electronic zoom lens, the tube lens, the objective lens and the cover glass, and total reflection occurs on the surface of the cover glass bearing the sample to form second reflected light;
s04, the second reflected light sequentially transmits the cover glass, the objective lens, the tube lens and the electronic zoom lens, and is reflected on the dichroic mirror to form third reflected light;
s05, transmitting the unpolarized beam splitter to a position detector by the third reflected light, and detecting the position of the light spot on the position detector;
s06, if the distance between the objective lens and the cover glass changes to generate a defocusing phenomenon, acquiring an axial defocusing amount;
s07, obtaining focal length dependent variable according to the axial defocus amount and combining parameters of the objective lens and the tube lens, and controlling the electronic zoom lens to change the focal length based on the focal length dependent variable so as to realize focal plane locking of a sample object plane.
Compared with the prior art, the invention has the beneficial effects that: by adopting the automatic defocus compensation method based on the microscopic imaging defocus automatic compensation system, imaging of different depths of a sample can be realized without any mechanical movement, and the effect of locking an imaging focal plane can be achieved by automatically compensating the defocus variation caused by the stability of a microscope and external interference in long-time microscopic imaging.
In a third aspect, a microscope employs the microscopic imaging defocus amount automatic compensation system of the first aspect.
Compared with the prior art, the invention has the beneficial effects that: the automatic defocus compensation system based on the microscopic imaging defocus compensation system is adopted to carry out the defocus automatic compensation method, so that the microscope has the functions of imaging different depths of a sample without any mechanical movement, and automatically compensating for the defocus variation caused by the stability of the microscope and external interference in long-time microscopic imaging to achieve the imaging focal plane locking effect.
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 a diagram showing the mechanism by which a prior art microscope mechanically images a sample at different depths;
FIG. 2 is a schematic diagram of the auto-compensation system for defocus amount of microscopic imaging according to embodiment 1 of the present invention;
FIG. 3 is a schematic structural diagram of an automatic compensation system for microscopic imaging defocus amount according to embodiment 2 of the present invention;
FIG. 4 is a flowchart of the auto-compensation method for microscopic imaging defocus amount according to embodiment 2 of the present invention;
FIG. 5 is a schematic structural diagram of an automatic compensation system for microscopic imaging defocus amount according to embodiment 3 of the present invention;
fig. 6 is a working mechanism of the microscopic imaging defocus amount automatic compensation system provided in embodiment 4 of the present invention.
Reference numerals illustrate:
10-sample;
20-cover slips;
30-objective lens, 31-displacement device;
40-tube mirror;
a 50-camera;
60-electronic zoom lens;
a 70-defocusing measuring module, a 71-laser, a 72-unpolarized beam splitter, a 73-dichroic mirror, a 74-position detector, a 75-excitation filter and a 76-emission filter;
80-a central controller.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and intended to illustrate embodiments of the invention and should not be construed as limiting the invention.
In the description of the embodiments of the present invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like indicate orientations or positional relationships based on the orientation or positional relationships shown in the drawings, merely to facilitate description of the embodiments of the present invention and simplify description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless explicitly defined otherwise.
In the embodiments of the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," "secured" and the like are to be construed broadly and include, for example, either permanently connected, removably connected, or integrally formed; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communicated with the inside of two elements or the interaction relationship of the two elements. The specific meaning of the above terms in the embodiments of the present invention will be understood by those of ordinary skill in the art according to specific circumstances.
Example 1
As shown in fig. 2, the present embodiment provides an automatic compensation system for microscopic imaging defocus amount, which is different from the conventional microscope system shown in fig. 1 in that:an electronic zoom lens 60 is added. Specifically, the automatic compensation system of the present embodiment includes a cover glass 20, an objective lens 30, a tube lens 40, a camera 50, an electronic zoom lens 60; the sample 10 is placed on the cover glass 20, the objective lens 30, the tube lens 40, the camera 50 and the electronic zoom lens 60 are all positioned on one side of the cover glass 20 far away from the sample 10, and the objective lens 30, the tube lens 40, the electronic zoom lens 60 and the camera 50 are sequentially arranged along the sample imaging light path; preferably, the electronic zoom lens 60 is specifically a liquid crystal lens. In specific practice, an objective lens 30 with preset magnification is installed on a microscope, a sample 10 is placed on a cover glass 20, and the sample 10 is located at the focal position of the objective lens 30; the objective lens 30, the tube lens 40, the camera 50, and the electronic zoom lens 60 are disposed on the same optical axis. The present embodiment changes the focal length of the electronic zoom lens 60 from F1 to F2 by controlling the central controller 80, so that the effective focal length of the tube lens 40 can be changed, and in the case that the image plane (i.e., the plane in which the CCD or CMOS is located) is unchanged, the object-image relationship formula is as follows: 1/d+1/d´=1/f(whereindIn order to be the object distance,d´in order to be the image distance,ffocal length), the object plane conjugated with the imaging device is changed from P1 to P2, so that imaging of different depths of a sample is realized; that is, by changing the focal length of the electronic zoom lens by the control of the central controller in the present embodiment, imaging of different depths of the sample can be achieved, no mechanical movement is generated during the whole adjustment process, meaning no vibration, and the focusing speed of the mode is fast. When the conventional microscope as shown in fig. 1 is required to image different depths of a sample, the distance between the objective lens 30 and the sample 10 is adjusted by adopting a displacement device 31 matched with the objective lens 30 in a mechanical adjustment manner, so that the distance between the objective lens 30 and the sample 10 is changed from d1 to d2, the corresponding object plane is changed from P1 to P2, vibration is needed due to mechanical movement, and the focusing speed is slow in the manner.
Example 2
As shown in fig. 3, the present embodiment provides an automatic compensation system for microscopic imaging defocus amount, which is different from the conventional microscope system shown in fig. 1 in that: an electronic zoom lens 60 and a defocus measurement module 70 are added. Specifically, the automatic compensation system of the present embodiment includes a cover glass 20, an objective lens 30, a tube lens 40, a camera 50, an electronic zoom lens 60, and an defocus measurement module 70; the sample 10 is placed on the cover glass 20, the objective lens 30, the tube lens 40, the camera 50, the electronic zoom lens 60 and the defocus measurement module 70 are all located at one side of the cover glass 20 far away from the sample 10, and the objective lens 30, the tube lens 40, the electronic zoom lens 60, the defocus measurement module 70 and the camera 50 are sequentially arranged along a sample imaging light path; preferably, the electronic zoom lens 60 is embodied as a liquid lens.
Further, the defocus measurement module 70 includes a laser 71, a non-polarizing beam splitter 72, a dichroic mirror 73, and a position detector 74. Wherein the unpolarized beam splitter 72 is arranged parallel to the dichroic mirror 73 and is arranged non-parallel to the sample imaging optical path, preferably, the unpolarized beam splitter 72 forms an angle of 45 ° with the sample imaging optical path; the dichroic mirror 73 is located on the sample imaging optical path, the laser 71 is located between the unpolarized beam splitter 72 and the dichroic mirror 73, and the position detector 74 is located on the side of the unpolarized beam splitter 72 away from the dichroic mirror 73. In this embodiment, in order to facilitate separation from the visible light of the sample imaging light path, the laser is a near infrared laser, and near infrared light with emission wavelength greater than 780nm is generally selected, for example, a laser with emission wavelength of 960nm is selected, and the beam divergence angle is less than 1mrad. The dichroic mirror is exemplified by a high reflection low transmission dichroic mirror around 780 nm. The position detector may be a high frame rate CCD, a high frame rate CMOS, a four-quadrant detector, or a beam quality analyzer, such as a four-quadrant detector. In specific practice, an objective lens 30 with preset magnification is installed on a microscope, a sample 10 is placed on a cover glass 20, and the sample 10 is located at the focal position of the objective lens 30; the objective lens 30, the tube lens 40, the electronic zoom lens 60, and the camera 50 are arranged on the same optical axis, and the sample imaging optical path projects the dichroic mirror 73.
Further, in this embodiment, light emitted by the laser 71 is processed by the unpolarized beam splitter 72, the dichroic mirror 73, the electronic zoom lens 60, the tube lens 40, the objective lens 30 and the cover glass 20 to form a light spot, the position detector 74 detects the position change of the light spot to obtain an axial defocus amount, and the focal length strain amount of the electronic zoom lens 60 is obtained according to the axial defocus amount, and the electronic zoom lens 60 controls to change its focal length according to the focal length strain amount by the central controller 80 to realize focal plane locking of the sample object plane. The flow chart of the specific microscopic imaging defocus amount automatic compensation method is shown in fig. 4, and comprises the following steps:
and S01, installing an objective lens with preset multiplying power on the microscope, placing the sample on the cover glass, and enabling the sample to be positioned at the focal position of the objective lens.
Specifically, the multiplying power of the objective lens is selected according to the actual demands of an operator, the objective lens with the selected multiplying power is arranged on a microscope, and then a sample is placed on a cover glass, so that the sample is ensured to be positioned at the focal position of the objective lens.
S02, starting the laser to emit light beams, and forming first reflected light through reflection of the unpolarized beam splitter and the bicolor mirror.
Specifically, the laser emits a light beam having a wavelength of 960nm and a divergence angle of less than 1mrad, which is incident on 1 at an incident angle of 45 °:1, a half of the energy beam reaches a high-reflection low-transmission double-color mirror near 780nm on the surface of a non-polarized beam splitter, and is reflected to form first reflected light.
S03, the first reflected light sequentially transmits the electronic zoom lens, the tube lens, the objective lens and the cover glass, and total reflection occurs on the surface of the cover glass bearing the sample to form second reflected light.
Specifically, as the objective lens, the tube lens and the electronic zoom lens are arranged on the same optical axis, the first reflected light reflected by the bicolor mirror sequentially penetrates through the electronic zoom lens, the tube lens, the objective lens and the cover glass to reach the surface of the cover glass for bearing the sample to generate total reflection, so that the second reflected light is formed.
And S04, the second reflected light sequentially transmits the cover glass, the objective lens, the tube lens and the electronic zoom lens, and is reflected on the dichroic mirror to form third reflected light.
Specifically, the optical path propagation in this step is the same as the mechanism in step S03, and will not be described in detail here.
S05, transmitting the third reflected light to a position detector through a non-polarized beam splitter, and detecting the position of the light spot on the position detector.
Specifically, the position detector is electrically connected with the central controller, when the third reflected light penetrates through the beam splitter and enters the position detector, the position of the light spot is detected, the position information is uploaded to the central controller in real time, the objective lens is not required to be manually moved in the process, no vibration is generated in the process, and the focusing speed is high.
S06, if the distance between the objective lens and the cover glass changes to generate a defocusing phenomenon, acquiring an axial defocusing amount.
Specifically, when the distance between the objective lens and the cover glass changes to generate a defocus phenomenon, namely, the distance between the objective lens and the cover glass changes from d1 to d2, and correspondingly, the corresponding object plane changes from P1 to P2, the spot position detected by the position detector changes from s1 to s2, and at this time, the difference between d1 and d2, namely, the axial defocus dz, can be calculated according to the distance difference ds between s1 and s 2. Here, the prefabricated relationship curve of the spot position s and the distance d between the objective lens and the cover glass may be obtained by performing a calibration method in advance.
S07, obtaining focal length dependent variable according to the axial defocus amount and combining parameters of the objective lens and the tube lens, and controlling the electronic zoom lens to change the focal length based on the focal length dependent variable so as to realize focal plane locking of a sample object plane.
Specifically, the focal length F2 can be obtained by theoretically knowing dz and parameters of the objective lens and the tube lens, and the focal length of the electronic zoom lens can be changed from F1 to F2 by controlling the central controller so that the object plane conjugated with the image plane is changed back to P1, thereby achieving the effect of focal plane locking.
Through the steps, the axial relative displacement of the objective lens and the sample is measured, the effective focal length of the tube lens is adjusted by utilizing the electronic zoom lens, so that the axial defocusing amount caused by the relative movement of the objective lens and the cover glass in the axial direction is compensated, the focal length dependent variable of the electronic zoom lens is obtained according to the axial defocusing amount, the electronic zoom lens changes the focal length of the electronic zoom lens through the central controller according to the focal length dependent variable to realize focal plane locking of the sample object plane, and the defocusing amount change caused by the stability of the microscope and external interference in long-time microscopic imaging is automatically compensated.
Example 3
As shown in fig. 5, the present embodiment provides an automatic compensation system for microscopic imaging defocus amount, which is different from the microscope system shown in fig. 2 in that: an excitation filter 75 and an emission filter 76 are added; the excitation filter 75 is disposed between the laser 71 and the unpolarized beam splitter 72, and the emission filter 76 is disposed between the dichroic mirror 73 and the camera 50, and preferably the electronic zoom lens 60 is a solid-state anamorphic lens. In this embodiment, for imaging weak signals such as weak illumination light or fluorescence and raman, an excitation filter 75 (such as 960nm band-pass filter) is added to the optical measurement path based on the above optical path, specifically to the position in front of the laser 71; and an emission filter 76 (appropriate filters are selected according to fluorescence bandwidth) is added to the sample imaging light path, specifically to a position in front of the camera 50.
Example 4
The present embodiment provides an automatic compensation system for microscopic imaging defocus amount, which is different from the microscope system shown in fig. 2 in that: an appropriate axial defocus amount is selected as a threshold as a starting criterion for starting zooming of the electronic zoom lens. The reason for this is because: the detection and transmission of the spot position information and the zooming of the electronic zoom lens all need a certain completion time, and the test of the axial defocus amount also has a certain error, so that a proper axial defocus amount is generally selected as a threshold dz0, and when the detected axial defocus amount is greater than the threshold dz0, the zoom lens is started to perform zooming compensation, and the specific working mechanism is shown in fig. 6. The threshold dz0 is larger than the detection accuracy of the axial defocus amount, and may be set according to actual requirements.
Example 5
The embodiment provides a microscope, and the microscopic imaging defocus amount automatic compensation system related to the embodiments is applied. The microscope adopts the defocus amount automatic compensation method based on the microscopic imaging defocus amount automatic compensation system, so that the microscope has the functions of imaging different depths of a sample without any mechanical movement, and automatically compensating for the defocus amount change caused by the stability of the microscope and external interference in long-time microscopic imaging to achieve the imaging focal plane locking effect.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (8)
1. The utility model provides a microscopic imaging defocus amount automatic compensation system, includes the coverslip that bears the sample and is located objective, tube mirror and the camera that the sample one side was kept away from to the coverslip, just objective tube mirror reaches the camera is laid and is set up with the optical axis along sample imaging light path in proper order, its characterized in that still includes:
the electronic zoom lens is positioned between the tube mirror and the camera and is arranged with the same optical axis as the tube mirror and is used for changing the focal length of the electronic zoom lens to adjust the effective focal length of the tube mirror;
the defocusing measurement module is positioned between the electronic zoom lens and the camera and is used for realizing focal plane locking of the sample object plane by means of the zooming function of the electronic zoom lens;
the defocusing measurement module comprises a non-polarized beam splitter, a dichroic mirror, a laser and a position detector which are arranged in parallel, wherein the dichroic mirror is positioned on a sample imaging optical path, the laser is positioned between the non-polarized beam splitter and the dichroic mirror, and the position detector is positioned on one side of the non-polarized beam splitter far away from the dichroic mirror; the unpolarized beam splitter and the bicolor lens are arranged in a non-parallel manner with a sample imaging light path, light emitted by the laser is processed by the unpolarized beam splitter, the bicolor lens, the electronic zoom lens, the tube lens, the objective lens and the cover glass to form light spots, the position detector detects the position change of the light spots to obtain axial defocusing quantity, the focal length strain quantity of the electronic zoom lens is obtained according to the axial defocusing quantity, and the focal length of the electronic zoom lens is changed according to the focal length strain quantity to realize focal plane locking of a sample object plane;
the position detector is used for detecting the position change of the light spot, and calculating the axial defocus amount based on a prefabricated relation curve of the position change of the light spot and the axial defocus amount, and the prefabricated relation curve is drawn through a calibration method.
2. The microscopic imaging defocus amount automatic compensation system of claim 1, wherein the axial defocus amount is combined with parameters of the objective lens and the tube lens to derive the focal length strain amount.
3. The microscopic imaging defocus amount automatic compensation system of claim 1, wherein the position detector is one of a CCD, a CMOS, a four-quadrant detector, or a beam quality analyzer.
4. The microscopic imaging defocus amount automatic compensation system of claim 1, wherein the laser is a near infrared laser.
5. The auto-compensation system for microscopic imaging defocus of claim 1, further comprising an excitation filter disposed between the laser and the unpolarized beam splitter and an emission filter disposed between the dichroic mirror and the camera.
6. The auto-compensation system for microscopic imaging defocus amount according to any one of claims 1 to 5, wherein the electronic zoom lens is one of a liquid crystal lens, a liquid lens, or a solid state anamorphic lens.
7. A microscopic imaging defocus amount automatic compensation method, characterized in that based on the microscopic imaging defocus amount automatic compensation system according to any one of claims 1 to 6, the method comprises:
s01, installing an objective lens with preset multiplying power on a microscope, placing a sample on a cover glass, and enabling the sample to be positioned at the focal position of the objective lens;
s02, starting a laser to emit light beams, and forming first reflected light by reflection of a non-polarized beam splitter and a bicolor mirror in sequence;
s03, the first reflected light sequentially transmits the electronic zoom lens, the tube lens, the objective lens and the cover glass, and total reflection occurs on the surface of the cover glass bearing the sample to form second reflected light;
s04, the second reflected light sequentially transmits the cover glass, the objective lens, the tube lens and the electronic zoom lens, and is reflected on the dichroic mirror to form third reflected light;
s05, transmitting the unpolarized beam splitter to a position detector by the third reflected light, and detecting the position of the light spot on the position detector;
s06, if the distance between the objective lens and the cover glass changes to generate a defocusing phenomenon, acquiring an axial defocusing amount;
s07, obtaining focal length dependent variable according to the axial defocus amount and combining parameters of the objective lens and the tube lens, and controlling the electronic zoom lens to change the focal length based on the focal length dependent variable so as to realize focal plane locking of a sample object plane.
8. A microscope, characterized in that the microscopic imaging defocus amount automatic compensation system of any one of claims 1 to 6 is applied.
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