CN115268045A - Microscope focus drift correction system and method based on liquid lens - Google Patents

Abstract

The invention belongs to the technical field of microscope automatic focusing, and provides a microscope focus drift correction system and method based on a liquid lens, which comprises a light source module, a light source module and a control module, wherein the light source module is configured to emit illumination light to a sample to be detected; the bias module comprises a liquid lens, and the liquid lens is configured to change the focal length thereof according to the change of the voltage value loaded on the liquid lens; a defocus measurement module configured to acquire defocus images of a plurality of samples to be measured while the focal length of the liquid lens is varied; and the central control unit is configured to perform difference calculation on the collected multiple out-of-focus images, and obtain out-of-focus relation curves of the objective lens and the liquid lens through calculation. The invention has the advantages that the microscope can realize rapid focusing and accurate focusing, and the mechanical displacement structure in the focusing system is removed, so that the focusing system has compact structure, and the focusing speed and precision are greatly improved.

Description

Microscope focus drift correction system and method based on liquid lens

Technical Field

The invention relates to the technical field of microscope automatic focusing, in particular to a microscope focus drift correction system and method based on a liquid lens.

Background

In the existing biological microscopes, the sample is often observed for a long time to research the change property of the sample. However, due to factors such as temperature variation, mechanical drift and some unexpected vibration, the focus of the microscope often drifts, which affects the accuracy of long-term observation and even leads to failure of the whole experiment. Therefore, many manufacturers have introduced autofocus techniques. The main ones include Nikang, lyca, chuiss and Orlinbas. An automatic focusing system based on a differential system is published by Nikon, the focusing precision is high, but the whole system is very large, and a plurality of image sensors are adopted to acquire differential images (the patent number is US10509199B 2); the Lycra uses a triangular reflector to separate an illumination light path from a detection light path so as to avoid interference between the light paths, has the advantages of compact structure and slightly insufficient precision and speed (the patent number is US8829402B 2); zeiss's difference processing is to obtain the two images required for difference by rotating mirrors (patent No.: US6825454B 2).

When observing biological samples, the existing automatic focusing system has two main problems: 1. the existing active focusing device mostly adjusts the focus of a detection light path based on the physical displacement of a mechanical structure to realize the observation of different thickness layers of a sample, so that the problems of inaccurate measurement of a focal plane, low focusing speed and the like can be caused by the physical displacement of the focusing device, and the automatic focusing speed and accuracy are influenced. 2. The existing differential processing method usually depends on three image sensors, one for collecting a focusing image, one for collecting a front focus image and one for collecting a rear focus image, the method makes the structure of the focusing device complex and the volume large, and a focus drift correction system based on a liquid lens only needs one image sensor, thereby simplifying the system structure and improving the automatic focusing speed.

Disclosure of Invention

The objective of the present invention is to provide a system and a method for correcting focus drift of a microscope based on a liquid lens, so as to solve the above problems.

In order to achieve the purpose, the invention adopts the technical scheme that:

the utility model provides a microscope focus drift correction system based on liquid lens, includes microscope system, and it is equipped with immersion medium including objective and microscope light source on the objective, and its effect includes for the formation of image to the sample that awaits measuring:

a light source module configured to emit illumination light to a sample to be measured;

the bias module comprises a liquid lens, and the liquid lens is configured to enable the focal length of the liquid lens to change according to the change of the voltage value loaded on the liquid lens;

a defocus measurement module configured to acquire defocus images of a plurality of samples to be measured while the focal length of the liquid lens is varied;

a central control unit configured to perform a difference calculation on the plurality of acquired out-of-focus images, obtain an out-of-focus relationship curve of the objective lens and the liquid lens through calculation, and change a voltage applied to the liquid lens.

Furthermore, a semi-transparent and semi-reflective mirror is arranged between the offset module, the defocusing measurement module and the light source module, and reflects the illumination light emitted by the system light source module to the offset module through the reflection of a sample to be measured, so that the reflected illumination light is emitted into the defocusing measurement module from the offset module.

Furthermore, the defocusing measurement module comprises a half-moon-shaped diaphragm, a second collimating mirror and an image sensor which are sequentially arranged according to the optical path transmission path; and the optical axes of the half-moon-shaped diaphragm, the second collimating mirror and the image sensor are all superposed with the optical axis of the liquid lens.

Further, the bias module further comprises a convex lens and a concave lens, and the convex lens and the concave lens are used for being combined with the liquid lens to expand the light beam, and the optical axis of the convex lens coincides with the system light source and the liquid lens.

Further, the central control unit is also configured to adjust the voltage value loaded on the liquid lens according to the defocus relation curve to compensate the defocus amount of the liquid lens.

Further, the central control unit is also configured to drive the objective lens to move by a preset height according to the defocus relation curve so as to compensate the defocus amount of the objective lens.

Further, the light source module also comprises a first collimating mirror and a half-moon-shaped diaphragm, wherein the first collimating mirror is used for collimating light emitted by the system light source into parallel light, the half-moon-shaped diaphragm is used for blocking off half of the illuminating light beam, and the first collimating mirror is superposed with the optical axis of the system light source;

and the illumination light emitted by the system light source sequentially passes through the half-moon-shaped diaphragm, the first reflector and the first collimating mirror and is emitted into the bias module.

Further, the system light source is LED light.

The invention also provides a liquid lens-based microscope focus drift correction method, which comprises the following steps:

s1, installing an objective lens with a preset multiplying power on a microscope, arranging an immersion medium on the objective lens, and placing a sample to be detected on one side of the objective lens to enable the sample to be detected to be located at the focal position of the objective lens;

s2, starting a light source module, irradiating illumination light rays emitted by the light source module onto a sample to be measured sequentially through a liquid lens module and an objective lens, reflecting the illumination light rays by the sample to be measured, allowing the reflected light rays to enter an offset measurement module sequentially through the objective lens and the liquid lens module, and imaging on an image sensor of the offset measurement module;

s3, starting the liquid lens module, and sending an instruction to the liquid lens by the central control unit to adjust the focal length of the liquid lens to a preset value;

s4, adjusting the focal length of the liquid lens to enable the light reflecting surface of the deviation measuring module to coincide with a plane which an operator wants to observe;

s5, adjusting the distance between the objective lens and the sample, and acquiring a defocused image of the sample from the image sensor;

s6, adjusting a preset voltage value loaded on the liquid lens through the central controller, and collecting a plurality of images of the sample to be detected on the image sensor under different focal lengths;

s7, carrying out difference calculation on the collected multiple images under different focal lengths to obtain a difference curve under an out-of-focus state, and obtaining the position of a zero-crossing point of the difference curve;

s8, repeating the steps S5-S7, and obtaining a defocusing relation curve according to the position of the zero-crossing point and the preset interval distance of the movement of the objective lens;

and S9, according to the obtained defocusing relation curve, adjusting a voltage value loaded on the liquid lens to compensate the defocusing amount of the liquid lens module, and driving the objective lens to move by a preset height to compensate the defocusing amount of the objective lens, so that the sample to be measured is always kept at the focal position of the microscope.

Compared with the prior art, the invention at least comprises the following beneficial effects:

(1) The method can realize the rapid focusing and the accurate focusing of the microscope, and can solve the problem of focusing plane deviation caused by different immersion media on each objective when the objective is switched by the microscope;

(2) The control of the position of the focal plane of the objective lens is realized by changing the focal length of the liquid lens, the mechanical displacement structure in the traditional offset system is reduced, and the focusing speed and precision are greatly improved;

(3) The method comprises the steps that the voltage applied to the liquid lens is rapidly changed when an image is collected to change the focal length of the liquid lens and collect the image, namely a quasi-focus curve, a front-focus curve and a back-focus curve are respectively recorded under the three conditions of current voltage, first preset voltage and second preset voltage, images formed by reflected light on an image sensor are all axial light intensity distribution curves, the front-focus curve and the back-focus curve are subjected to differential operation to obtain a differential curve, and the distance between the current position and an accurate focusing position can be obtained by analyzing the zero-crossing position of the differential curve;

(4) The liquid lens is adopted to quickly adjust the focal length to realize the collection of the front focal curve, the back focal curve and the focusing curve on the same image collector, so that the defect that the traditional differential operation needs three image sensors at different positions is avoided, the structure is simplified, and the space utilization rate of a focusing part is improved.

Drawings

FIG. 1 is a schematic view of an optical focusing structure of a microscope according to an embodiment of the present invention;

FIG. 2 is a flow chart of a method for correcting focus drift of a microscope according to an embodiment of the present invention;

FIG. 3 is a flowchart illustrating operation of a microscope for focus drift correction in accordance with an embodiment of the present invention;

FIG. 4 is an in-focus image of an image sensor linear array CCD in an embodiment of the present invention;

FIG. 5 is an out-of-focus image of a linear CCD array of an image sensor in an embodiment of the present invention;

FIG. 6 is a Gaussian fit curve of an in-focus image of an image sensor linear array CCD in an embodiment of the present invention;

FIG. 7 is a schematic diagram of an in-focus image, a front-focus image and a back-focus image of an image sensor linear array CCD according to an embodiment of the present invention;

FIG. 8 is a difference curve diagram obtained by performing difference calculation using two out-of-focus images in the embodiment of the present invention;

FIG. 9 is a graph showing the defocus relationship between the zero crossing point and the defocus amount of the fitted difference curve in the embodiment of the present invention.

In fig. 1: 1. a system light source; 2. a half-moon shaped diaphragm; 3. a first reflector; 4. a first collimating mirror; 5. a light source module; 6. a semi-transparent semi-reflective mirror; 7. a concave lens; 8. a second reflector; 9. a convex lens; 10. a liquid lens; 11. a biasing module; 12. a third reflector; 13. a fourth mirror; 14. an objective lens; 15. a sample; 16. a half-moon shaped diaphragm; 17. a second collimating mirror; 18. an image sensor; 19. a defocus measurement module; 20. a central processing unit; 21. a microscope light source; 22. and (4) a microscope.

Detailed Description

It should be noted that all directional indicators (such as up, down, left, right, front, and back \8230;) in the embodiments of the present invention are only used to explain the relative positional relationship between the components, the motion situation, etc. in a specific posture (as shown in the attached drawings), and if the specific posture is changed, the directional indicators are changed accordingly.

Moreover, descriptions of the present invention as relating to "first," "second," "a," etc. are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicit ly indicating a number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly, and for example, "secured" may be a fixed connection, a removable connection, or an integral part; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood according to specific situations by those of ordinary skill in the art.

In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of those skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination of technical solutions should not be considered to exist, and is not within the protection scope of the present invention.

The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described with reference to the drawings, but the present invention is not limited to these embodiments.

As shown in FIG. 1, according to one embodiment of the present invention, in a focus drift correction system for a liquid lens-based microscope, a light source module 5 includes a system light source 1 (e.g., L10596-03, hamamatsu, JP), a half-moon shaped diaphragm 2, a first reflector 3 (e.g., NIB1000.010, novel, CHN), and a first collimator 4 (e.g., #32-856, edmund optics, USA). Wherein the illumination light that system light source 1 sent jets into biasing module 11 through half moon type diaphragm 2 in proper order, first speculum 3, first collimating mirror 4 and semi-transparent semi-reflecting mirror 6 on, wherein half moon type diaphragm 2 can restrict the beam radius that light source 1 sent, thereby it limits the light beam size of follow-up incident to keep off half through the light beam that system light source 1 sent, prevent the interference between incident light and the reverberation, illumination light becomes the parallel light after through first speculum 3 and first collimating mirror 4, then the incidence biasing module 11, and expand in biasing module 11.

The system light source 1 can drive the LED lamp set to emit infrared illumination light by the central control unit 20.

A half-mirror 6 (for example, NIB1000.010-2g, novel, chn) is disposed between the bias module 11, the defocus measurement module 20 and the light source module 5, the half-mirror 6 reflects the illumination light emitted from the light source module 5 to the bias module 11, and the light reflected from the specified layer of the sample 15 passes through the bias module 11 and enters the defocus measurement module 20.

The bias module 11 of the present invention comprises a concave lens 7 (e.g. LC1439, thorlabs, USA), a second mirror 8 (e.g. NIB1000.010, novel, CHN), a convex lens 9 (e.g. LA1207, thorlabs, USA), and a liquid lens 10 (e.g. EL-12-30-TC, optitune, switzerland), wherein the concave lens 7 is located at a side close to the half mirror 6, the liquid lens 10 is located at a side of the concave lens 7 close to the objective lens 11 and connected to a central control unit 20, and the liquid lens 10 and the concave lens 7 in combination can expand the light beam. The second reflector 8 is used for deflecting the light path, so that the light path is more compact. The convex lens 9 is located between the liquid lens 10 and the concave lens 7, and is used for combining with the liquid lens 10 to achieve a beam expanding effect. The liquid lens 10 can change the position of the illumination light reflected on the sample layer by changing the focal length.

A third reflector 12 (for example, NIB1000.010, novel, CHN) and a fourth reflector 13 (for example, NIB1000.010, novel, CHN) are disposed between the bias module 11 and the objective lens 14, and the illumination light emitted by the system light source 1 passes through the first reflector 3, the first collimator 4, the half-mirror 6, the bias module 11, the third reflector 12, and the fourth reflector 13 in sequence, then is focused by the objective lens 14 and illuminated on the sample 15 to be measured, is reflected at the interface between the sample 15 to be measured and the air (the oil mirror is generated at the interface between the sample and the oil), and then enters the defocus measurement module 17 through the fourth reflector 13, the third reflector 12, the bias module 11, and the half-mirror 6.

The defocus measurement module 19 of the present invention comprises a half-moon shaped diaphragm 16, a second collimating mirror 17 (e.g., #32-856, edmund optics, USA), and an image sensor 18 (e.g., TCD1254GFG, TOSHIBA, JP), wherein the image sensor 18 is conjugate to the system light source 1, and the image on the image sensor 18 is an axial light intensity distribution curve, which facilitates the difference calculation. The reflected light rays are emitted into an out-of-focus measuring module 19 from the bias module 11 through the half-mirror 6, and are transmitted to an image sensor 18 in the out-of-focus measuring module through a half-moon-shaped diaphragm 16 and a second collimating mirror 17 in sequence.

The microscope 22 (e.g., ECLIPSE Ti2-E, nikon, JP) used in the present invention has an objective lens 14 and a microscope light source 21, which can image and capture a sample to be measured by an observer.

In the present embodiment, the initial voltage value is set as X, the first preset voltage value is set as Y, the second preset voltage value is set as Z, X = Y + v, X = Z-v, and v is the same change voltage amount, that is, the difference between the first preset voltage and the initial voltage is equal to the difference between the second preset voltage and the initial voltage, and the difference is only that the change voltage amount is different in positive and negative, so that the image sensor 18 acquires the front focus image and the back focus image.

The central control unit 20 can perform difference calculation on the collected front focus image and back focus image, obtain the defocus relationship curve of the objective lens and the liquid lens through calculation, and obtain the defocus amount of the current microscope system according to the generated defocus relationship curve. Finally, according to the obtained defocus amount, the central control unit 20 controls the objective lens 14 to move up and down along the z axis so that the focus of the objective lens 14 is always located on the observation layer of the sample 15 to be measured, real-time focusing of a microscope system is achieved, the problem of focusing plane offset caused by different immersion media on each objective lens when the microscope switches the objective lenses is solved, a mechanical displacement structure in the focusing system is removed, and focusing speed and quality are greatly improved.

According to the above embodiment of the present invention, the present invention further provides a method for correcting focus drift of a microscope based on a liquid lens, as shown in fig. 2, which includes the steps of:

s1, installing an objective lens with a preset multiplying power on a microscope, arranging an immersion medium on the objective lens, and placing a sample to be detected on one side of the objective lens to enable the sample to be detected to be located at the focal position of the objective lens;

the immersion medium may be air, water or oil, which may affect the focusing accuracy when focusing, and the refractive index of the immersion medium is different for different immersion mediums, so that there is a certain effect on the focusing accuracy, and the magnification of the objective lens is selected according to the actual needs of the operator.

S2, starting a light source module, irradiating illumination light rays emitted by the light source module onto a sample to be measured sequentially through a bias module and an objective lens, reflecting the illumination light rays by the sample to be measured, and enabling the reflected light rays to enter a defocusing measurement module sequentially through the objective lens and the bias module;

s3, starting the bias module, and enabling the central control unit to send an instruction to the liquid lens to adjust the focal length of the liquid lens to a preset value, namely controlling the parallel light emitted from the bias module at the moment;

s4, adjusting the focal length of the liquid lens to enable the light ray reflecting surface of the defocusing measuring module to coincide with a plane which an operator wants to observe;

according to different immersion media used by the objective lens, the position of the light reflection plane can be correspondingly changed, and the light reflection plane of the microscope defocusing measurement module needs to be manually adjusted to be superposed with the plane to be observed, so that the initial focusing position is ensured, and the objective lens is suitable for different immersion media. At this time, the reflected light of the defocus measurement module is imaged on the image sensor, and an in-focus image as shown in fig. 4 is obtained.

S5, adjusting the distance between the objective lens and the sample, and acquiring a defocusing image of the sample from the image sensor to obtain a defocusing image shown in FIG. 5;

s6, adjusting a preset voltage value loaded on the liquid lens through the central controller, and collecting two images of the sample to be detected on the image sensor under different focal lengths;

first, a preset voltage value is added to the liquid lens, and a first defocused image is obtained on the image sensor. And then changing the first preset voltage value for the liquid lens to enable the focal length of the liquid lens to be larger than the initial focal length and obtain a second out-of-focus image on the image sensor, namely a front focus image, and then changing the second preset voltage value for the liquid lens to enable the focal length of the liquid lens to be smaller than the initial focal length and obtain a third out-of-focus image on the image sensor, namely a back focus image. The comparison between the obtained front focus image and rear focus image and the in-focus image is shown in fig. 7.

S7, carrying out differential calculation on the acquired front focus image and the acquired rear focus image to obtain a differential curve in a defocused state and obtain a zero-crossing point position of the differential curve;

the three obtained out-of-focus images are not consistent in imaging conditions, gaussian fitting is firstly carried out on the three acquired out-of-focus images to obtain a Gaussian fitting curve as shown in fig. 6, then difference calculation is carried out on the Gaussian fitting curves of the front focus image and the rear focus image, out-of-focus amounts of the objective lens and the liquid lens are measured according to the zero-crossing positions of the difference curve to obtain an out-of-focus difference curve as shown in fig. 8, and out-of-focus amounts of the objective lens and the liquid lens are measured through the out-of-focus difference curve.

S8, repeating the steps S4-S6, and obtaining a defocusing relation curve according to the position of the zero-crossing point and the preset movement interval distance of the objective lens;

and S9, adjusting a voltage value loaded on the liquid lens according to the obtained defocusing relation curve to compensate the defocusing amount of the offset module, and driving the objective lens to move by a preset height to compensate the defocusing amount of the objective lens, so that the sample to be measured is always kept at the focal position of the microscope.

In the present embodiment, step S4 is repeated, and the objective lens is controlled to move in the same direction a plurality of times at intervals of 1 μm. And acquiring a defocused image of the sample to be detected under an initial voltage value by moving once, then adding a first preset voltage value to the liquid lens to acquire a defocused image of a second sample to be detected, and then adding a second preset voltage value to the liquid lens to acquire a defocused image of a third sample. A difference curve as shown in fig. 8 is obtained and the position of the zero crossing point of the difference curve (i.e. the distance of the zero crossing point of the difference curve on the x-axis) is recorded. And changing the voltage of the liquid lens to the initial voltage value and continuing the next objective lens moving operation.

In this embodiment, the central control unit performs multiple differences on the defocus image acquired by the image sensor and records the relationship between the zero-crossing point position and the movement interval of the objective lens, so as to draw a coordinate point in a rectangular coordinate system according to the defocus amount (i.e., the length of movement of the objective lens at a predetermined interval) and the zero-crossing point position, and fit the drawn coordinate point to obtain a defocus relationship curve as shown in fig. 9. In the present embodiment, the defocus relationship curve obtained by fitting should satisfy the following relationship:

y＝kx+b

wherein y is defocus and x is the zero-crossing position. The values of k and b can be solved after fitting a curve by utilizing the defocus amount and the zero-crossing point position. And obtaining a corresponding defocus value after obtaining a zero-crossing point position x according to the defocus relation curve.

In the embodiment, the sample to be measured is kept at the focal position of the objective lens according to the curve of the relationship between the zero-crossing point position and the defocusing. In this embodiment, the central control unit obtains an image of the sample to be measured through the image sensor, obtains a difference curve of the sample according to steps S4 to S6, obtains a zero-crossing point position of the processed curve, and can obtain the defocus amount of the current microscope system according to the generated defocus relationship curve. According to the acquired defocus amount, the central control unit controls the objective lens to move up and down to enable the sample to be detected to be always located at the focus of the objective lens, and real-time focusing of the microscope system is achieved.

In this embodiment, thereby can also change the initial focus of liquid lens through changing the initial voltage value of loading on liquid lens, further can realize changing the convergent point axial position of illumination light path light, make the measurement light converge at the different thickness layers of the sample that awaits measuring to the realization is focused to the precision of the different thickness regions of sample awaiting measuring.

In the present embodiment, the minimum focus drift resolution of the focus drift correction of the microscope can be calculated from parameters of the objective lens of the microscope, and the following calculation is performed according to parameters related to the objective lens of one hundred times, and 20 times, 60 times and other objective lenses which are not calculated in the present specification should also be calculated in the same calculation manner, and are included in the protection scope of the present patent:

by referring to the relevant 100 times objective parameters (e.g. CFI Apochromat TIRF 100XC, nikon, JP), a numerical aperture NA of 1.49 and a focal length of 2mm can be obtained, and by designing the microscope objective with an infinite parallel light structure, the rise of the beam can be calculated according to the magnification and the focal length as follows:

H＝f'×n·sinU＝f'×NA＝2mm×1.49＝2.98mm

thus, an incident light pupil diameter of D =2h =5.96mm can be obtained

Total depth of field d of a microscopic imaging system_dotThe device consists of two parts of wave optics and geometric optics depth of field. At high numerical apertures of the microscope, the depth of field is mainly determined by wave optics, whereas at lower numerical apertures the geometrical optical depth of field dominates.

Wherein λ represents the wavelength of the incident light; e denotes the minimum distance resolvable by the detector in the image plane and M denotes the magnification of the objective lens.

In the system, the wavelength lambda =860nm, the medium refractive index n =1.515, the CCD pixel size e =5.25 μ M, and the objective lens magnification M =100.

The total depth of field d of the microscopic imaging system can be obtained by substituting the parameters and calculating_dot＝0.64μm。

According to empirical formulas, the imaging system resolution is less than 1/3 of the total depth of field, i.e., δ ≦ 0.213 μm.

According to the Rayleigh criterion resolution formula, the calculation result of the transverse (vertical to the optical axis) resolution of the microscope optical imaging system is as follows:

the axial (parallel to the optical axis) resolution is:

in the actual calibration process, the minimum resolution of the obtained object space can be smaller than the theoretical parameter, so that the delta is taken under a 100-time microscope by combining the embodiment and referring to the design indexes of foreign related products_xy＝0.24μm，δ_z=0.44 μm as the object-side minimum resolution.

In this embodiment, a system calibration process is performed on the focus drift of the microscope by changing objective lenses with different multiples, so as to obtain a focus drift correction curve and a corresponding maximum correctable focus drift amount under each objective lens with different multiples, which are specifically shown in table 1 below:

TABLE 1 design index requirements for microscope focus drift correction

According to the above embodiment of the present invention, the present invention further provides a method for using a liquid lens based microscope focus drift correction system, as shown in fig. 3, which includes the steps of:

a1, installing an objective lens with a preset multiplying power on a microscope, arranging an immersion medium on the objective lens, and placing a sample to be detected on one side of the objective lens to enable the sample to be detected to be located at the focal position of the objective lens;

a2, collecting images through an image sensor, quickly changing the focal length of a liquid lens by a preset amount, collecting to obtain a front focus image and a rear focus image, calculating to obtain a difference curve and obtaining a corresponding zero crossing point position;

after the focus drift correction is started, if the focal length of the liquid lens is not in the preset value at the moment, the central control unit sends a signal to change the focal length of the liquid lens into the preset focal length. The preset focal length is the zero position of the focus drift correction, and the light emitted from the liquid lens is parallel light at the moment. If the sample is positioned near the focus of the objective lens (the defocusing amount does not exceed the maximum defocusing amount under the objective lens corresponding to the corresponding focus drift correction), the focus drift correction starts to work, otherwise, an error is reported, and the normal operation cannot be carried out.

A3, judging whether the current position is at a focusing zero point or not according to the zero crossing point position;

a4, if the objective lens is not located at a focusing zero point, calculating the displacement required by the objective lens according to the position of the zero crossing point, and controlling the objective lens to displace a corresponding distance along the z axis; repeating the steps A2-A3 until the zero crossing point position of the differential curve is positioned at the focusing zero point;

a5, when the zero-crossing point position of the differential curve is just at the focusing zero point, if an observer does not need to change the position of the sample observation layer, performing the step A6, and if the position of the sample observation layer needs to be changed, performing the step A7;

a6, fixing the position of the focus drift correction system, and carrying out long-time focus locking;

and A7, changing the voltage applied to the liquid lens, calculating the displacement required by the objective lens through the displacement of the zero-crossing point position of the differential curve acquired by the linear array CCD, and synchronously changing the position of the objective lens so that an observer can see the change of the observation layer of the sample. Until the desired viewing layer is determined, steps A2-A5 are resumed after the voltage across the liquid lens has stopped being changed.

After the voltage applied to the liquid lens is changed, the reflection point of the illumination light emitted by the focus drift correction is changed, the zero crossing point position of the differential curve acquired by the linear array CCD is changed, the central control unit synchronously sends a signal to the objective lens to control the same displacement of the objective lens so as to correct the zero crossing point position of the differential curve back to the zero position of the CCD, and an observer can observe that the sample layer observed by the microscope at the moment is changed.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims (9)

1. The utility model provides a microscope focus drift correction system based on liquid lens, includes microscope system, and it is equipped with immersion medium including objective and microscope light source on the objective, and its effect is for the formation of image to the sample that awaits measuring, its characterized in that includes:

a light source module configured to emit illumination light to a sample to be measured;

the bias module comprises a liquid lens, and the liquid lens is configured to enable the focal length of the liquid lens to change according to the change of the voltage value loaded on the liquid lens;

a defocus measurement module configured to acquire defocus images of a plurality of samples to be measured while the focal length of the liquid lens is varied;

a central control unit configured to perform a difference calculation on the plurality of captured out-of-focus images, obtain an out-of-focus relationship curve of the objective lens and the liquid lens through calculation, and change a voltage applied to the liquid lens.

2. The system of claim 1, wherein a half-mirror is disposed between the offset module, the defocus measurement module and the light source module, the half-mirror reflects the illumination light emitted from the system light source module to the offset module, and the reflected illumination light is incident from the offset module to the defocus measurement module by reflection of the sample to be measured.

3. The liquid lens based microscope focus drift correction system according to claim 1, wherein the defocus measurement module comprises a half-moon shaped diaphragm, a second collimating mirror and an image sensor arranged in sequence according to the optical path transmission path; and the optical axes of the half-moon-shaped diaphragm, the second collimating mirror and the image sensor are superposed with the optical axis of the liquid lens.

4. The liquid lens based microscope focus drift correction system of claim 1, wherein the bias module further comprises a convex lens and a concave lens for expanding the light beam in combination with the liquid lens, the optical axis of which coincides with the system light source and the liquid lens.

5. The liquid lens based microscope focus drift correction system of claim 1, wherein the central control unit is further configured to adjust the voltage value applied to the liquid lens according to the defocus relationship curve to compensate for the defocus of the liquid lens.

6. The liquid lens based microscope focus drift correction system of claim 1, wherein the central control unit is further configured to drive the objective lens to move by a preset height according to the defocus relationship curve to compensate for the defocus amount of the objective lens.

7. The system of claim 1, wherein the light source module further comprises a first collimating mirror for collimating light emitted from the system light source into parallel light and a half-moon type aperture for blocking off half of the illumination beam, the first collimating mirror being coincident with the optical axis of the system light source;

and the illumination light emitted by the system light source sequentially passes through the half-moon-shaped diaphragm, the first reflector and the first collimating mirror and is emitted into the bias module.

8. The liquid lens based microscope focus drift correction system of claim 6, wherein the system light source is an LED lamp.

9. A liquid lens-based microscope focus drift correction method, based on the liquid lens-based microscope focus drift correction system of any one of claims 1-8, comprising the steps of:

s1, installing an objective lens with a preset multiplying power on a microscope, arranging an immersion medium on the objective lens, and placing a sample to be detected on one side of the objective lens to enable the sample to be detected to be located at the focal position of the objective lens;

s2, starting a light source module, irradiating illumination light rays emitted by the light source module onto a sample to be measured sequentially through a liquid lens module and an objective lens, reflecting the illumination light rays by the sample to be measured, allowing the reflected light rays to enter a deviation measurement module sequentially through the objective lens and the liquid lens module, and imaging on an image sensor of the deviation measurement module;

s3, starting the liquid lens module, and sending an instruction to the liquid lens by the central control unit to adjust the focal length of the liquid lens to a preset value;

s4, adjusting the focal length of the liquid lens to enable the light reflecting surface of the deviation measuring module to coincide with a plane which an operator wants to observe;

s5, adjusting the distance between the objective lens and the sample, and acquiring a defocused image of the sample from the image sensor;

s6, adjusting a preset voltage value loaded on the liquid lens through the central controller, and collecting a plurality of images of the sample to be detected on the image sensor under different focal lengths;

s7, carrying out differential calculation on the collected multiple images under different focal lengths to obtain a differential curve under an out-of-focus state, and obtaining the position of a zero-crossing point of the differential curve;

s8, repeating the steps S5-S7, and obtaining a defocusing relation curve according to the position of the zero-crossing point and the preset movement interval distance of the objective lens;

and S9, according to the obtained defocusing relation curve, adjusting a voltage value loaded on the liquid lens to compensate the defocusing amount of the liquid lens module, and driving the objective lens to move by a preset height to compensate the defocusing amount of the objective lens, so that the sample to be measured is always kept at the focal position of the microscope.

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