A liquid lens-based microscope focus drift correction system and method

Abstract

The present application belongs to the technical field of microscope automatic focusing, and provides a microscope focus shift correction system and method based on a liquid lens, which comprises a light source module configured to emit illumination light to a sample to be measured; a bias module comprising a liquid lens configured to change its focal length according to the voltage value loaded thereon; a defocus measurement module configured to collect multiple defocus images of the sample to be measured when the focal length of the liquid lens changes; and a central control unit configured to perform differential calculation on the collected multiple defocus images to obtain a defocus relationship curve of the objective lens and the liquid lens. The present application has the advantages of realizing fast focusing and accurate focusing of the microscope, removing the mechanical displacement structure in the focusing system, making the focusing system compact, and greatly improving the focusing speed and accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of microscope autofocus, and in particular to a microscope focus drift correction system and method based on a liquid lens. Background Art

[0002] Existing biological microscopes often require prolonged observation of samples to investigate their changing properties. However, due to factors such as temperature fluctuations, mechanical drift, and unpredictable vibrations, the microscope's focus often drifts, compromising the accuracy of long-term observations and even causing the failure of the entire experiment. Consequently, many manufacturers have introduced autofocus technologies, with Nikon, Leica, Zeiss, and Olympus being the main players. Nikon has released an autofocus system based on a differential system, which offers high focus accuracy but is very large, employing multiple image sensors to capture differential images (Patent No.: US10509199B2). Leica uses triangular mirrors to separate the illumination and detection light paths to prevent interference between the two paths. This offers the advantage of a compact design but suffers from slightly lower accuracy and speed (Patent No.: US8829402B2). Zeiss's differential processing uses rotating mirrors to generate the two images required for differential analysis (Patent No.: US6825454B2).

[0003] When observing biological samples, existing autofocus systems have two major problems: 1. Existing active focusing devices mostly rely on the physical displacement of a mechanical structure to adjust the focus of the detection light path to enable observation of different sample thickness layers. This physical displacement of the focusing device itself can also introduce inaccurate focal plane measurement and slow focusing speeds, affecting the speed and accuracy of autofocus. 2. Existing differential processing methods typically rely on three image sensors: one to capture the focused image, one to capture the front-focus image, and one to capture the back-focus image. This method makes the focusing device itself complex and bulky. The focus drift correction system based on a liquid lens only requires a single image sensor, simplifying the system structure and improving the speed of autofocus. Summary of the Invention

[0004] The object of the present invention is to provide a liquid lens-based microscope focus drift correction system and method to solve the above-mentioned problems.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A liquid lens-based microscope focus drift correction system includes a microscope system, which includes an objective lens and a microscope light source. An immersion medium is provided on the objective lens, which functions to image a sample to be measured, including:

[0007] a light source module, configured to emit illumination light toward the sample to be tested;

[0008] The bias module includes a liquid lens, wherein the liquid lens is configured to change its focal length according to a change in a voltage value applied thereto;

[0009] a defocus measurement module, configured to collect a plurality of defocus images of the sample to be measured when the focal length of the liquid lens changes;

[0010] The central control unit is configured to perform differential calculation on the collected multiple defocused images, obtain the defocus relationship curve between the objective lens and the liquid lens through calculation, and change the voltage applied to the liquid lens.

[0011] Furthermore, a semi-transparent and semi-reflective mirror is arranged between the bias module, the defocus measurement module and the light source module. The semi-transparent and semi-reflective mirror reflects the illumination light emitted by the system light source module to the bias module, and through reflection by the sample to be tested, the reflected illumination light is emitted from the bias module into the defocus measurement module.

[0012] Furthermore, the defocus measurement module includes a half-moon diaphragm, a second collimating mirror and an image sensor which are arranged in sequence according to the optical transmission path; the optical axes of the half-moon diaphragm, the second collimating mirror and the image sensor coincide with the optical axis of the liquid lens.

[0013] Furthermore, the bias module further comprises a convex lens and a concave lens, which are used to expand the light beam in combination with the liquid lens, and the optical axis thereof coincides with the system light source and the liquid lens.

[0014] Furthermore, 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 amount of the liquid lens.

[0015] Furthermore, the central control unit is also configured to drive the objective lens to move a preset height according to the defocus relationship curve to compensate for the defocus amount of the objective lens.

[0016] Furthermore, the light source module further includes a first collimator and a half-moon diaphragm, wherein the first collimator is used to collimate the light emitted by the system light source into parallel light, and the half-moon diaphragm is used to block half of the illumination beam, and the first collimator coincides with the optical axis of the system light source;

[0017] The illumination light emitted by the system light source passes through the half-moon aperture, the first reflector, and the first collimator in sequence and enters the bias module.

[0018] Furthermore, the system light source is LED light.

[0019] Another aspect of the present invention further provides a method for correcting focus drift of a microscope based on a liquid lens, comprising the steps of:

[0020] S1. Install an objective lens with a preset magnification on a microscope, with an immersion medium on the objective lens, and place the sample to be measured on one side of the objective lens so that the sample to be measured is at the focal position of the objective lens;

[0021] S2. Start the light source module. The illumination light emitted by the light source module passes through the liquid lens module and the objective lens in sequence to illuminate the sample to be measured. After being reflected by the sample to be measured, the reflected light passes through the objective lens and the liquid lens module in sequence to enter the offset measurement module and forms an image on the image sensor of the offset measurement module.

[0022] S3, starting the liquid lens module, and the central control unit sends a command to the liquid lens to adjust the focal length of the liquid lens to a preset value;

[0023] S4. Adjust the focal length of the liquid lens so that the light reflection surface of the offset measurement module coincides with the plane the operator wants to observe;

[0024] S5, adjusting the distance between the objective lens and the sample, and acquiring a defocused image of the sample from the image sensor;

[0025] S6. Adjusting a preset voltage value applied to the liquid lens by the central controller to collect multiple images of the sample to be tested formed on the image sensor at different focal lengths;

[0026] S7, performing differential calculation on the multiple images acquired at different focal lengths, obtaining a differential curve in a defocused state, and determining a zero-crossing position of the differential curve;

[0027] S8, repeating steps S5-S7, obtaining a defocus relationship curve according to the zero-crossing position and the predetermined interval distance of the objective lens movement;

[0028] S9. According to the obtained defocus relationship curve, adjust the voltage value loaded on the liquid lens to compensate for the defocus of the liquid lens module, and drive the objective lens to move a preset height to compensate for the defocus of the objective lens, so that the sample to be measured always remains at the focal position of the microscope.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects:

[0030] (1) It can achieve fast and precise focusing of the microscope, and can solve the problem of focal plane offset caused by different immersion media on each objective lens when the microscope switches the objective lens;

[0031] (2) The focal plane position of the objective lens is controlled by changing the focal length of the liquid lens, which reduces the mechanical displacement structure in the traditional bias system and greatly improves the focusing speed and accuracy;

[0032] (3) Since the voltage applied to the liquid lens is rapidly changed during image acquisition to change the focal length of the liquid lens and acquire an image, the quasi-focus curve, the front focus curve, and the back focus curve are recorded respectively under the current voltage, the first preset voltage, and the second preset voltage. The images formed by the reflected light on the image sensor are all axial light intensity distribution curves. The front focus curve and the back focus curve are differentially calculated to obtain a differential curve. By analyzing the zero-crossing position of the differential curve, the distance between the current position and the accurate focus position can be obtained.

[0033] (4) A liquid lens is used to quickly adjust the focal length to realize the acquisition of the front focus curve, back focus curve and quasi-focus curve on the same image collector, avoiding the disadvantage that the traditional differential operation requires three image sensors in different positions, simplifying the structure and improving the space utilization of the focusing components. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the optical focusing structure of the microscope in an embodiment of the present invention;

[0035] Figure 2 is a flow chart of a microscope focus drift correction method according to an embodiment of the present invention;

[0036] Figure 3 This is a workflow diagram for operating a microscope to perform focus drift correction in an embodiment of the present invention;

[0037] Figure 4 is the in-focus image of the image sensor linear array CCD in an embodiment of the present invention;

[0038] Figure 5 is a defocused image of the image sensor linear array CCD in an embodiment of the present invention;

[0039] Figure 6 is a Gaussian fitting curve of an in-focus image of the image sensor linear array CCD in an embodiment of the present invention;

[0040] Figure 7 Schematic diagram of an in-focus image, a front-focus image, and a back-focus image of an image sensor linear array CCD in an embodiment of the present invention;

[0041] Figure 8 is a differential curve graph obtained by performing differential calculation using two defocused images in an embodiment of the present invention;

[0042] Figure 9 3 is a defocus relationship curve diagram of the zero-crossing point of the fitted differential curve and the defocus amount in an embodiment of the present invention.

[0043] Figure 1Middle: 1. System light source; 2. Half-menus diaphragm; 3. First reflector; 4. First collimator; 5. Light source module; 6. Semi-transparent and semi-reflective mirror; 7. Concave lens; 8. Second reflector; 9. Convex lens; 10. Liquid lens; 11. Bias module; 12. Third reflector; 13. Fourth reflector; 14. Objective lens; 15. Sample; 16. Half-menus diaphragm; 17. Second collimator; 18. Image sensor; 19. Defocus measurement module; 20. Central processing unit; 21. Microscope light source; 22. Microscope. DETAILED DESCRIPTION

[0044] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the various components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0045] In addition, in the present invention, descriptions such as "first," "second," and "one" are for descriptive purposes only and should not be understood to indicate or imply their relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0046] In the present invention, unless otherwise specified or limited, the terms "connection" and "fixation" should be understood in a broad sense. For example, "fixation" can mean fixed connection, detachable connection, or integration; mechanical connection or electrical connection; direct connection or indirect connection through an intermediate medium; internal communication between two elements or interaction between two elements, unless otherwise specified. Those skilled in the art will be able to understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0047] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0048] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the accompanying drawings, but the present invention is not limited to these embodiments.

[0049] like Figure 1As shown, according to one embodiment of the present invention, in a liquid lens-based microscope focus drift correction system, the light source module 5 includes a system light source 1 (e.g., L10596-03, Hamamatsu, JP), a meniscus diaphragm 2, a first reflector 3 (e.g., NIB1000.010, Novel, CHN), and a first collimator 4 (e.g., #32-856, Edmund Optics, USA). The illumination light emitted by the system light source 1 passes through the meniscus diaphragm 2, the first reflector 3, the first collimator 4, and the semi-transparent and semi-reflective mirror 6 in sequence and is incident on the bias module 11. The meniscus diaphragm 2 can limit the radius of the beam emitted by the light source 1 by blocking half of the beam emitted by the system light source 1, thereby limiting the size of the subsequent incident beam and preventing interference between the incident light and the reflected light. The illumination light becomes parallel light after passing through the first reflector 3 and the first collimator 4, and then enters the bias module 11 and is expanded in the bias module 11.

[0050] The system light source 1 can be driven by the central control unit 20 to drive the LED light group to emit infrared lighting light.

[0051] A semi-transparent, semi-reflective mirror 6 (for example: NIB1000.010-2G, Novel, CHN) is arranged between the bias module 11, the defocus measurement module 20 and the light source module 5. The semi-transparent, semi-reflective mirror 6 reflects the illumination light emitted by the light source module 5 to the bias module 11, and the light reflected back from the specified layer of the sample 15 is emitted into the defocus measurement module 20 through the bias module 11.

[0052] The bias module 11 of the present invention includes a concave lens 7 (e.g., LC1439, Thorlabs, USA), a second reflector 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, Optotune, Switzerland). The concave lens 7 is located on a side close to the semi-transparent and semi-reflective mirror 6. The liquid lens 10 is located on a side of the concave lens 7 close to the objective lens 11 and is connected to a central control unit 20. The combination of the liquid lens 10 and the concave lens 7 can expand the light beam. The second reflector 8 is used to fold the light path to make the light path more compact. The convex lens 9 is located between the liquid lens 10 and the concave lens 7 and is used to combine with the liquid lens 10 to achieve a beam expansion effect. The liquid lens 10 can change the position where the illumination light is reflected in the sample layer by changing the focal length.

[0053] A third reflector 12 (e.g., NIB1000.010, Novel, CHN) and a fourth reflector 13 (e.g., NIB1000.010, Novel, CHN) are provided between the bias module 11 and the objective lens 14. The illumination light emitted by the system light source 1 passes through the first reflector 3, the first collimating mirror 4, the semi-transparent and semi-reflecting mirror 6, the bias module 11, the third reflector 12, and the fourth reflector 13 in sequence, and then is converged by the objective lens 14 to illuminate the sample to be measured 15. The light is reflected at the interface between the sample to be measured 15 and the air (for oil mirror, it occurs at the interface between the sample and the oil), and then passes through the fourth reflector 13, the third reflector 12, the bias module 11, and the semi-transparent and semi-reflecting mirror 6 to enter the defocus measurement module 17.

[0054] The defocus measurement module 19 of the present invention includes a meniscus diaphragm 16, a second collimator 17 (e.g., #32-856, Edmund Optics, USA), and an image sensor 18 (e.g., TCD1254GFG, TOSHIBA, JP). Image sensor 18 is conjugate with the system light source 1. The image formed on image sensor 18 is an axial light intensity distribution curve, facilitating differential calculations. Reflected light from bias module 11 enters defocus measurement module 19 through semi-transparent and semi-reflective mirror 6. Within the defocus measurement module, it passes sequentially through meniscus diaphragm 16 and second collimator 17 before being transferred to image sensor 18.

[0055] The microscope 22 (eg, ECLIPSE Ti2-E, Nikon, JP) used in the present invention has an objective lens 14 and a microscope light source 21 . The microscope imaging device can image the sample to be tested and capture it by an observer.

[0056] The voltage applied to the liquid lens 10 when the reflected light moves from the sample to be tested 15 to the image sensor 18 is the initial voltage value. In this embodiment, the initial voltage value is set to X, the first preset voltage value is set to Y, and the second preset voltage value is set to Z, X=Y+v, X=Zv, and v is the same variable voltage. 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. The only difference is that the changed voltage value is different in sign, so that the image sensor 18 can obtain the front focus image and the back focus image.

[0057] The central control unit 20 is capable of performing differential calculations on the captured front-focus image and back-focus image, and through this calculation, a defocus relationship curve is obtained between the objective lens and the liquid lens. Based on the generated defocus relationship curve, the defocus value of the current microscope system can be obtained. Finally, based on the obtained defocus value, 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 to be measured 15. This achieves real-time focusing of the microscope system, solves the problem of focal plane offset caused by different immersion media on each objective lens when the microscope switches objective lenses, and eliminates the mechanical displacement structure within the focusing system, greatly improving the focusing speed and quality.

[0058] According to the above embodiment of the present invention, the present invention also provides a method for correcting focus drift of a microscope based on a liquid lens, such as Figure 2 As shown, it includes the steps of:

[0059] S1. Install an objective lens with a preset magnification on a microscope, with an immersion medium on the objective lens, and place the sample to be measured on one side of the objective lens so that the sample to be measured is at the focal position of the objective lens;

[0060] The immersion medium may be air, water or oil, which may affect the focusing accuracy during focusing. Different immersion media have different refractive indices, so there is a certain impact on the focusing accuracy. The magnification of the objective lens is selected according to the actual needs of the operator.

[0061] S2. Start the light source module. The illumination light emitted by the light source module passes through the bias module and the objective lens in sequence to illuminate the sample to be tested. After being reflected by the sample to be tested, the reflected light passes through the objective lens and the bias module in sequence to enter the defocus measurement module.

[0062] S3, starting the bias module, and the central control unit sends a command to the liquid lens to adjust the focal length of the liquid lens to a preset value, that is, controlling the light emitted from the bias module to be parallel light;

[0063] S4. Adjusting the focal length of the liquid lens so that the light reflection surface of the defocus measurement module coincides with the plane the operator wants to observe;

[0064] Depending on the immersion medium used by the objective lens, the position of the light reflection plane will change accordingly. It is necessary to manually adjust the light reflection surface of the microscope defocus measurement module to coincide with the plane to be observed to ensure the initial focus position and adapt to the objective lens with different immersion media. At this time, the reflected light of the defocus measurement module is imaged on the image sensor, and the image is obtained as follows: Figure 4 In-focus image shown.

[0065] S5. Adjust the distance between the objective lens and the sample, and obtain the defocused image of the sample from the image sensor. Figure 5 Defocused image shown;

[0066] S6. Adjusting a preset voltage value applied to the liquid lens by the central controller to capture two images of the sample to be tested formed on the image sensor at different focal lengths;

[0067] First, a preset voltage value is applied to the liquid lens to obtain a first defocused image on the image sensor. Then, the first preset voltage value is changed to make the focal length of the liquid lens larger than the initial focal length and a second defocused image, i.e., a front-focus image, is obtained on the image sensor. Then, the second preset voltage value is changed to make the focal length of the liquid lens smaller than the initial focal length and a third defocused image, i.e., a back-focus image, is obtained on the image sensor. The comparison of the obtained front-focus image, back-focus image, and the in-focus image is as follows: Figure 7 shown.

[0068] S7, performing a differential calculation on the collected front focus image and the back focus image, obtaining a differential curve in a defocused state, and determining a zero-crossing position of the differential curve;

[0069] The imaging conditions of the three defocused images are inconsistent. The three defocused images collected are firstly subjected to Gaussian fitting, and the following is obtained: Figure 6 The Gaussian fitting curve shown in FIG is then used to perform differential calculation on the Gaussian fitting curves of the front focus image and the back focus image. The defocus amount of the objective lens and the liquid lens is measured according to the zero-crossing position of the differential curve, and the final result is as follows: Figure 8 The defocus difference curve is shown, and the defocus amount of the objective lens and the liquid lens is measured by the defocus difference curve.

[0070] S8, repeating steps S4-S6, obtaining a defocus relationship curve according to the zero-crossing point position and the predetermined interval distance of the objective lens movement;

[0071] S9. According to the obtained defocus relationship curve, adjust the voltage value loaded on the liquid lens to compensate for the defocus amount of the bias module, and drive the objective lens to move a preset height to compensate for the defocus amount of the objective lens, so that the sample to be measured always remains at the focal position of the microscope.

[0072] In this embodiment, step S4 is repeated to control the objective lens to move multiple times in the same direction at intervals of 1 micron. Each movement acquires a defocused image of the sample to be tested at the initial voltage value, then applies a first preset voltage value to the liquid lens to acquire a second defocused image of the sample to be tested, and then applies a second preset voltage value to the liquid lens to acquire a third defocused image of the sample to be tested. Figure 8 The differential curve shown is recorded, and the zero crossing position of the differential curve is recorded (i.e., the distance of the zero crossing point of the differential curve on the x-axis). After the voltage of the liquid lens is changed to the initial voltage value, the next step of moving the objective lens is continued.

[0073] In this embodiment, the central control unit performs multiple differences on the defocused image collected by the image sensor and records the relationship between the zero-crossing position and the objective lens movement interval. Based on this, coordinate points are plotted in a rectangular coordinate system according to the defocus amount (i.e., the length of the objective lens moving at a predetermined interval) and the zero-crossing position. The coordinate points obtained by the plotting are fitted to obtain the following: Figure 9 In this embodiment, the defocus relationship curve obtained by fitting should satisfy the following relationship:

[0074] y=kx+b

[0075] Where y is the defocus value and x is the zero-crossing position. By fitting a curve between the defocus value and the zero-crossing position, the values ​​of k and b can be solved. Once the zero-crossing position x is determined based on the defocus relationship curve, the corresponding defocus value can be obtained.

[0076] In this embodiment, the sample to be tested is maintained at the focal position of the objective lens based on the zero-crossing position and defocus relationship curve. In this embodiment, the central control unit acquires an image of the sample to be tested via an image sensor and generates a differential curve of the sample according to steps S4-S6. The zero-crossing position of the processed curve is obtained, and the defocus value of the current microscope system is obtained based on the generated defocus relationship curve. Based on the obtained defocus value, the central control unit controls the vertical movement of the objective lens to ensure that the sample to be tested is always in the focal position of the objective lens, thus achieving real-time focusing of the microscope system.

[0077] In this embodiment, the initial focal length of the liquid lens can be changed by changing the initial voltage value loaded on the liquid lens, and the axial position of the convergence point of the illumination light path can be further changed, so that the measurement light converges on different thickness layers of the sample to be tested, thereby achieving precise focusing on different thickness areas of the sample to be tested.

[0078] In this embodiment, the minimum focus drift resolution of the focus drift correction of the microscope can be calculated based on the parameters of the microscope objective lens. The following calculation is based on the relevant parameters of the 100x objective lens. The 20x, 60x, and other objective lenses not calculated in this article should also be calculated using the same calculation method and are included in the scope of protection of this patent:

[0079] By referring to the relevant 100x objective lens parameters (for example: CFI Apochromat TIRF 100XC, Nikon, JP), we can get its numerical aperture NA is 1.49 and focal length is 2mm. The microscope objective lens adopts an infinite parallel light structure design. The sag height of the light beam can be calculated based on the magnification and focal length:

[0080] H=f'×n·sinU=f′×NA=2mm×1.49=2.98mm

[0081] Therefore, the pupil diameter of the incident light can be obtained as D = 2H = 5.96 mm

[0082] The total depth of field d of the microscopic imaging system dot It consists of two parts: wave optics and geometric optics depth of field. At high numerical apertures, the depth of field is mainly determined by wave optics, while at lower numerical apertures, the geometric optics depth of field dominates.

[0083]

[0084] Where λ is the wavelength of the incident light; e is the minimum distance that the detector can resolve on the image plane; and M is the magnification of the objective lens.

[0085] In this system, the wavelength λ = 860 nm, the medium refractive index n = 1.515, the CCD pixel size e = 5.25 μm, and the objective lens magnification M = 100.

[0086] Substituting the above parameters into the calculation, the total depth of field d of the microscopic imaging system can be obtained. dot =0.64μm.

[0087] According to the empirical formula, the resolution of the imaging system must be less than 1 / 3 of the total depth of field, that is, δ≤0.213μm.

[0088] According to the Rayleigh criterion resolution formula, the calculation result of the lateral (perpendicular to the optical axis) resolution of the microscope optical imaging system is:

[0089]

[0090] The axial (parallel to the optical axis) resolution is:

[0091]

[0092] In the actual calibration process, it can be obtained that the minimum object resolution can be smaller than the theoretical parameter. Therefore, in combination with this embodiment and referring to the design indicators of relevant foreign products, δ is taken under a 100x microscope objective lens. xy =0.24μm, δ z =0.44μm as the minimum object space resolution.

[0093] In this embodiment, the focus drift of the microscope is systematically calibrated by changing the objective lens of different magnifications, and the focus drift correction curves under each objective lens of different magnifications and the corresponding maximum correctable focus drift are obtained, as shown in Table 1 below:

[0094] Table 1 Design index requirements for microscope focus drift correction

[0095]

[0096] According to the above embodiment of the present invention, the present invention also provides a method for using a liquid lens-based microscope focus drift correction system, such as Figure 3 As shown, it includes the steps of:

[0097] A1. Install an objective lens with a preset magnification on the microscope. Place an immersion medium on the objective lens and place the sample to be tested on one side of the objective lens so that the sample is at the focal position of the objective lens.

[0098] A2. Capture images through an image sensor. Rapidly change the focal length of the liquid lens by a preset amount to acquire front-focus and back-focus images. Calculate a differential curve and obtain the corresponding zero-crossing point position.

[0099] After focus drift correction is activated, if the liquid lens focal length is not at the preset value, the central control unit sends a signal to change the focal length of the liquid lens to the preset value. The preset focal length is the zero position for focus drift correction, and the light emitted from the liquid lens is parallel light. If the sample is near the focal point of the objective lens (the defocus amount must not exceed the maximum defocus amount under the corresponding focus drift correction objective lens), focus drift correction will begin. Otherwise, an error will be reported and normal operation will not occur.

[0100] A3. Determine whether the focus is at zero point based on the zero-crossing point position.

[0101] A4. If the objective lens is not at the zero focus point, the required displacement of the objective lens is calculated based on the position of the zero crossing point, and the objective lens is controlled to move the corresponding distance along the z-axis. Repeat steps A2-A3 until the zero crossing point of the differential curve is at the zero focus point.

[0102] A5. When the zero-crossing point of the differential curve is exactly at the focus zero point, if the observer does not need to change the position of the sample observation layer, proceed to step A6; if the observer needs to change the position of the sample observation layer, proceed to step A7;

[0103] A6, fix the position of the focus drift correction system to lock the focus for a long time;

[0104] A7. Change the voltage applied to the liquid lens. Calculate the required objective lens displacement based on the zero-crossing position of the differential curve captured by the linear CCD. Simultaneously change the objective lens position so that the observer can see the changes in the sample's observation layer. Once the desired observation layer is determined, stop changing the voltage applied to the liquid lens and restart steps A2-A5.

[0105] After changing the voltage applied to the liquid lens, the reflection point of the illumination light emitted by the focus drift correction changes, and the zero-crossing position of the differential curve collected by the linear array CCD changes. The central control unit synchronously sends a signal to the objective lens to control the displacement of the objective lens by the same amount to correct the zero-crossing position of the differential curve back to the CCD zero position. The observer can observe that the sample layer observed by the microscope has changed at this time.

[0106] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A liquid lens-based microscope focus drift correction system, comprising a microscope system including an objective lens and a microscope light source, wherein an immersion medium is provided on the objective lens and serves to image a sample to be measured, characterized in that: include: a light source module, configured to emit illumination light toward the sample to be tested; The bias module includes a liquid lens, wherein the liquid lens is configured to change its focal length according to a change in a voltage value applied thereto; a defocus measurement module, configured to collect a plurality of defocus images of the sample to be measured when the focal length of the liquid lens changes; a central control unit configured to perform differential calculation on the collected multiple defocused images, obtain a defocus relationship curve between the objective lens and the liquid lens through the calculation, and change the voltage applied to the liquid lens; The plurality of defocused images are subjected to Gaussian fitting and then differential calculation is performed to obtain a differential curve in a defocused state, and the defocus amounts of the objective lens and the liquid lens are measured according to the zero-crossing position of the differential curve to obtain the defocus relationship curve; The central control unit is further configured to adjust a voltage value applied to the liquid lens according to the defocus relationship curve to compensate for a defocus amount of the liquid lens.

2. A liquid lens-based microscope focus drift correction system according to claim 1, characterized in that: A semi-transparent and semi-reflective mirror is arranged between the bias module, the defocus measurement module and the light source module. The semi-transparent and semi-reflective mirror reflects the illumination light emitted by the light source module to the bias module, and through reflection by the sample to be measured, the reflected illumination light is emitted from the bias module into the defocus measurement module.

3. The liquid lens-based microscope focus drift correction system according to claim 1, characterized in that: The defocus measurement module includes a half-moon diaphragm, a second collimating mirror, and an image sensor, which are sequentially arranged according to the optical transmission path; the optical axes of the half-moon diaphragm, the second collimating mirror, and the image sensor coincide with the optical axis of the liquid lens.

4. The liquid lens-based microscope focus drift correction system according to claim 1, characterized in that: The bias module further comprises a convex lens and a concave lens, which are used to expand the light beam in combination with the liquid lens, and the optical axis thereof coincides with the system light source and the liquid lens.

5. The liquid lens-based microscope focus drift correction system according to claim 1, characterized in that: The central control unit is further configured to drive the objective lens to move a preset height according to the defocus relationship curve to compensate for the defocus amount of the objective lens.

6. The liquid lens-based microscope focus drift correction system according to claim 1, characterized in that: The light source module further comprises a first collimator and a half-moon diaphragm, wherein the first collimator is used to collimate the light emitted by the system light source into parallel light, and the half-moon diaphragm is used to block half of the illumination beam, and the first collimator coincides with the optical axis of the system light source; The illumination light emitted by the system light source passes through the half-moon aperture, the first reflector, and the first collimator in sequence and enters the bias module.

7. The liquid lens-based microscope focus drift correction system according to claim 6, characterized in that: The system light source is LED light.

8. A liquid lens-based microscope focus drift correction method, based on the liquid lens-based microscope focus drift correction system according to any one of claims 1 to 7, characterized in that: Including steps: S1. Install an objective lens with a preset magnification on a microscope, with an immersion medium on the objective lens, and place the sample to be measured on one side of the objective lens so that the sample to be measured is at the focal position of the objective lens; S2. Start the light source module. The illumination light emitted by the light source module passes through the liquid lens and the objective lens in sequence to illuminate the sample to be tested. After being reflected by the sample to be tested, the reflected light passes through the objective lens and the liquid lens in sequence to enter the defocus measurement module and forms an image on the image sensor of the defocus measurement module. S3, starting the liquid lens, and the central control unit sends a command to the liquid lens to adjust the focal length of the liquid lens to a preset value; S4. Adjusting the focal length of the liquid lens so that the light reflection surface of the defocus measurement module coincides with the plane the 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 applied to the liquid lens by the central controller to collect multiple images of the sample to be tested formed on the image sensor at different focal lengths; S7, performing differential calculation on the multiple images acquired at different focal lengths, obtaining a differential curve in a defocused state, and determining a zero-crossing position of the differential curve; S8, repeating steps S5-S7, obtaining a defocus relationship curve according to the zero-crossing position and the predetermined interval distance of the objective lens movement; S9. According to the obtained defocus relationship curve, adjust the voltage value loaded on the liquid lens to compensate for the defocus of the liquid lens, and drive the objective lens to move a preset height to compensate for the defocus of the objective lens, so that the sample to be measured always remains at the focal position of the microscope.

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