Automatic focusing device

Abstract

The utility model discloses an automatic focusing device, and belongs to the field of microscopic optical imaging equipment. The automatic focusing device sequentially comprises a microscope objective focusing light path, a first image plane light path and a 4f relay and liquid lens focusing imaging light path. The microscope objective lens focusing light path further comprises a microscope objective lens and an objective lens focusing assembly for driving the microscope objective lens to move up and down; the first image plane light path is used for realizing infinite microscopic imaging; the 4f relay and liquid lens focusing imaging light path sequentially comprises a collimating lens assembly, a second reflector, a liquid lens focusing assembly, a third reflector, a focusing lens assembly and a camera. The automatic focusing device is applied to imaging focusing of complex biological samples containing a large number of impurities, solves the technical problem that an existing automatic focusing device cannot achieve full-automatic and high-speed focusing of the complex biological samples in a micro-fluidic chip, and can achieve full-automatic and rapid focusing of the high-complexity biological samples.

Description

Technical Field

[0001] This utility model belongs to the field of microscopic optical imaging equipment, and in particular relates to an automatic focusing device. Background Technology

[0002] In microscopic optical imaging systems, high-quality images with clear focus not only visually display the morphology of samples, but also form the basis and guarantee for many analyses such as sample identification and classification based on morphological features. However, high-magnification microscopic optical systems have a small depth of field (about 1-10μm), and are prone to image defocusing due to mechanical micro-vibrations, environmental conditions, and other reasons. As the degree of automation and the demand for clear imaging efficiency continue to increase, higher requirements are placed on the automatic focusing of the system.

[0003] There are two types of autofocusing methods in existing autofocusing devices. One method involves controlling the sample stage to move axially within a certain range, acquiring an image corresponding to the current axial position at each step, and using an algorithm to build a sharpness model to predict the optimal focal plane. Ultimately, the platform moves to the position with the best sharpness as the focal plane to complete focusing. The other method involves keeping the relative positions of the sample and the objective lens constant, inserting a liquid lens between the objective lens and the camera focusing lens. The curvature of the liquid lens changes with the driving current. A predictive model of the relationship between image sharpness and driving current is established through certain means. Based on the current sharpness, the driving current is adjusted to complete focusing.

[0004] However, current autofocus devices are unable to achieve fully automatic, high-speed focusing on complex biological samples within microfluidic chips. The reasons are as follows: First, the samples are highly complex. In addition to the sample, the suspension contains a large number of impurities of the same size as the sample that cannot be filtered out. Due to their different physical properties, these impurities and the sample are located on different focal planes. When using the above method for image sharpness analysis, the impurities often dominate the image, making the image sharpness of the impurity focal plane greater than that of the sample focal plane. Ultimately, the focal plane is calculated as the impurity focal plane, and the automatic focusing of the sample fails. Second, due to the diversity of the samples to be tested, the preset image prediction model may have low accuracy and recall or even fail due to sample differences. Therefore, it is necessary to replace the sample and remodel, which is time-consuming, labor-intensive, and requires too much manual intervention, making it impossible to achieve full automation. Finally, because excessively high chip thickness uniformity will lead to a sharp increase in chip cost, when applying the existing scheme one, the total step range needs to be set to be large (>50μm) to cover different chip thickness errors, but this greatly affects efficiency. When applying the existing scheme two, the maximum adjustable focusing range of the 50x microscopic imaging liquid lens is only about 20μm, which cannot cover chip thickness errors. Therefore, neither of the above two schemes can be applied. Utility Model Content

[0005] Details of one or more embodiments of the present invention are set forth in the following drawings and description to make other features, objects and advantages of the present application more readily apparent.

[0006] This invention proposes an automatic focusing device that solves the technical problem that existing automatic focusing devices cannot achieve fully automatic and high-speed focusing on complex biological samples within microfluidic chips, enabling fully automatic and rapid focusing for highly complex biological samples.

[0007] This utility model discloses an automatic focusing device, which sequentially includes a microscope objective focusing optical path, a first image plane optical path, and a 4f relay and liquid lens focusing imaging optical path; the microscope objective focusing optical path further includes a microscope objective and an objective focusing assembly that drives the microscope objective to move up and down; the first image plane optical path is used to realize infinity microscopic imaging; the 4f relay and liquid lens focusing imaging optical path sequentially includes a collimating lens assembly, a second reflecting mirror, a liquid lens focusing assembly, a third reflecting mirror, a focusing mirror assembly, and a camera; the collimating lens assembly controls the first image plane optical path. The imaging signal is collimated by a first mirror; the second mirror deflects the parallel beam; the liquid lens focusing assembly changes its curvature by varying its driving current, thereby changing the image distance and obtaining a series of out-of-focus blurred images and in-focus sharp images; the third mirror deflects the parallel beam, and the third mirror and the second mirror cooperate to horizontally fix the liquid lens focusing assembly; the focusing lens assembly focuses the imaging signal onto the camera's target surface and forms a 4f relay optical path with the collimating lens assembly; the camera receives the imaging signal.

[0008] In some embodiments, the system further includes a sample area to be tested, which includes a microfluidic chip and a two-dimensional motorized scanning platform. The microfluidic chip is provided with the sample to be tested. The microfluidic chip is fixed on the two-dimensional motorized scanning platform, which drives the microfluidic chip to move in the XY direction. The two-dimensional motorized scanning platform cooperates with the objective focusing assembly to realize the relative movement of the microscope objective and the microfluidic chip in three dimensions.

[0009] In some embodiments, the microfluidic chip consists of upper and lower glass layers and a PSA layer located between the upper and lower glass layers. Several cutouts in the PSA layer define several sample cell channels, and a marker point is provided next to each sample cell channel. The marker point is located on the upper surface of the lower glass layer.

[0010] In some embodiments, an illumination path is further included to provide bright-field illumination for microscopic imaging, the illumination path including a white light source and a Köhler illumination assembly; the white light source provides the light source for bright-field imaging; the Köhler illumination assembly is located between the white light source and the two-dimensional motorized scanning platform to provide a highly uniform light field.

[0011] In some embodiments, a computer control system is also included, which is electrically connected to the white light source, the two-dimensional motorized scanning platform, the objective lens focusing assembly, the liquid lens focusing assembly, and the camera.

[0012] In some embodiments, the collimating lens assembly, the focusing lens assembly, and the lens barrel have equal focal lengths, and the distance between the two assemblies is twice the focal length of the collimating lens assembly.

[0013] In some embodiments, the objective focusing assembly moves the microscope objective up and down by ±5 mm, with a minimum step value of 0.1 μm.

[0014] In some embodiments, the first image plane optical path includes a barrel lens, a first reflecting mirror, and a field lens; the barrel lens focuses the sample image onto the first image plane; the first reflecting mirror deflects the optical path; and the field lens is located at the first image plane.

[0015] In some embodiments, the first reflector, the second reflector, and the third reflector are selected from any one of a flat plate reflector and a reflecting prism; the field lens is selected from a plano-convex lens, a biconvex lens, or a cemented lens; and the collimating lens assembly and the focusing lens assembly are selected from a plano-convex lens, a cemented lens, or a combination lens group.

[0016] In some embodiments, the first, second, and third reflectors bend the light path by 90°.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention provides an automatic focusing device. By defining the composition of the focusing optical path of the microscope objective, and coordinating with a 4f relay and a liquid lens focusing imaging optical path, the focusing range is expanded through continuous movement of the objective. Simultaneously, the sample is located more quickly by using a camera for rapid shooting, achieving coarse focusing. Then, the driving current of the liquid lens focusing component is adjusted according to the relationship between the image clarity of the chip marker and the driving current of the liquid lens to achieve fine focusing of the sample. This automatic focusing device enables rapid and high-precision focusing of complex biological samples containing a large number of impurities. Compared with the traditional fixed step scanning method, the entire process time is shortened from 5-10 seconds to less than 1 second, greatly improving the efficiency of clear image acquisition and focusing speed. Attached Figure Description

[0019] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 This is a schematic diagram of the automatic focusing device provided in an embodiment of the present utility model;

[0021] Figure 2 This is a schematic diagram of the microfluidic chip provided in an embodiment of the present invention;

[0022] Figure 3 This is a schematic diagram illustrating the relationship between driving current and optical power provided in an embodiment of the present invention;

[0023] Figure descriptions: 101, White light source; 102, Köhler illumination assembly; 201, Microfluidic chip; 2011, Sample cell; 2012, Marker point; 202, Two-dimensional motorized scanning platform; 301, Microscope objective; 302, Objective focusing assembly; 401, Lens tube lens; 402, First reflecting mirror; 403, Field lens; 501, Collimating lens assembly; 502, Second reflecting mirror; 503, Liquid lens focusing assembly; 504, Third reflecting mirror; 505, Focusing lens assembly; 506, Camera; 6, Computer control system. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this utility model clearer, the present utility model will be described and explained below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present utility model and are not intended to limit the present utility model. All other embodiments obtained by those skilled in the art based on the embodiments provided by this utility model without inventive effort are within the scope of protection of this utility model.

[0025] Obviously, the accompanying drawings described below are merely some examples or embodiments of this utility model. Those skilled in the art can apply this utility model to other similar scenarios without any creative effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this utility model, any changes to the design, manufacturing, or production methods based on the disclosed technical content are merely conventional technical means and should not be construed as insufficient disclosure of this utility model.

[0026] In this utility model, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this utility model. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this utility model may be combined with other embodiments without conflict.

[0027] Unless otherwise defined, the technical or scientific terms used in this utility model shall have the ordinary meaning understood by one of ordinary skill in the art to which this utility model pertains. The terms "a," "an," "a kind," "the," and similar words used in this utility model do not indicate quantity limitation and may indicate singular or plural. The terms "comprising," "including," "having," and any variations thereof used in this utility model are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or device that includes a series of steps or modules (units) is not limited to the listed steps or units, but may also include steps or units not listed, or may include other steps or units inherent to these processes, methods, products, or devices. The terms "connected," "linked," "coupled," and similar words used in this utility model are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "A plurality" in this utility model refers to two or more. The "and / or" operator describes the relationship between related objects, indicating that three relationships can exist. For example, "A and / or B" can represent three cases: A alone, A and B simultaneously, and B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship. The terms "first," "second," and "third" used in this utility model are merely to distinguish similar objects and do not represent a specific ordering of the objects.

[0028] This utility model provides an automatic focusing device. Figure 1 This is a schematic diagram of the automatic focusing device according to an embodiment of the present invention. (Reference) Figure 1As shown, the autofocusing device sequentially includes a focusing optical path for a microscope objective 301, a first image plane optical path, and a 4f relay and liquid lens focusing and imaging optical path; the focusing optical path for the microscope objective 301 further includes a microscope objective 301 and an objective focusing assembly 302 that moves the microscope objective 301 up and down; the first image plane optical path is used to achieve infinity microscopic imaging; the 4f relay and liquid lens focusing and imaging optical path sequentially includes a collimating lens assembly 501, a second reflecting mirror 502, a liquid lens focusing assembly 503, a third reflecting mirror 504, a focusing lens assembly 505, and a camera 506; the collimating lens assembly 501... The first image plane imaging signal is collimated by mirror 501; the second reflecting mirror 502 deflects the parallel beam; the liquid lens focusing assembly 503 changes its curvature by varying its driving current, thereby changing the image distance and obtaining a series of out-of-focus blurred images and in-focus sharp images; the third reflecting mirror 504 deflects the parallel beam, and the third reflecting mirror 504 and the second reflecting mirror 502 cooperate to horizontally fix the liquid lens focusing assembly 503; the focusing mirror assembly 505 focuses the imaging signal onto the target surface of the camera 506, and forms a 4f relay optical path with the collimating mirror assembly 501; the camera 506 receives the imaging signal. Furthermore, the focal lengths of the collimating mirror assembly 501, the focusing mirror assembly 505, and the barrel lens 401 are equal, and the distance between the two assemblies is twice the focal length of the collimating mirror assembly 501. The objective lens focusing assembly 302 moves the microscope objective 301 up and down by ±5mm, with a minimum step value of 0.1μm.

[0029] The aforementioned autofocus device, by defining the composition of the focusing optical path of the microscope objective 301, and in conjunction with the 4f relay and liquid lens focusing imaging optical path, expands the focusing range through continuous movement of the objective. Simultaneously, the camera 506 captures images quickly to locate the sample, achieving coarse focusing. Then, based on the relationship between the image sharpness of the chip marker 2012 and the driving current of the liquid lens, the driving current of the liquid lens is adjusted to achieve fine focusing of the sample. This autofocus device enables rapid and high-precision focusing of complex biological samples containing numerous impurities. Compared to traditional fixed-step scanning methods, the entire process time is reduced from 5-10 seconds to less than 1 second, significantly improving the efficiency of acquiring clear images and increasing focusing speed. Notably, the liquid lens focusing component 503 is positioned between the collimating lens component 501 and the focusing lens component 505, ensuring that the liquid lens focusing component 503 performs its function while effectively avoiding any impact on the optical path caused by its addition.

[0030] Regarding the focusing optical path of the aforementioned microscope objective 301, specifically, the focusing optical path of the microscope objective 301 achieves coarse focusing by adjusting the objective position to change the object distance. This includes the microscope objective 301, which performs microscopic magnification imaging, characterized by high magnification (≥50X), high NA (≥0.8), super-flat field, and apochromatic aberration; the objective focusing assembly 302, which can be electrically controlled to move the objective up and down with a stroke of ±5mm, a minimum step value of 0.1μm, and a repeatability accuracy of 1μm during movement.

[0031] Regarding the aforementioned 4f relay and liquid lens focusing imaging optical path, specifically, the functions of the 4f relay and liquid lens focusing imaging optical path are: forming a telecentric optical path to avoid magnification changes caused by focusing; adjusting the image distance through the liquid lens focusing assembly 503 for fine focusing; and receiving the imaging signal using the camera 506. Specifically, it includes: a collimating lens assembly 501 to collimate the imaging signal of the first image plane; a second reflecting mirror 502 to bend the parallel beam by 90° to compress the optical path volume; the liquid lens focusing assembly 503, which changes its curvature through changes in its driving current, thereby changing the image distance and obtaining a series of out-of-focus blurred images and in-focus sharp images; and a third reflecting mirror 504 to bend the parallel beam by 90° to compress the optical path volume, while simultaneously cooperating with the second reflecting mirror 502 to ensure the liquid lens focusing assembly 503 is horizontally fixed, avoiding the issues that occur when vertically fixed. The liquid gravity inside the component affects the imaging quality of the lens; the focusing lens component 505 focuses the imaging signal onto the target surface of the camera 506, and at the same time forms a 4f relay optical path with the collimating lens component 501, that is, the focal lengths of the collimating lens component 501, the focusing lens component 505 and the lens barrel 401 are equal, and the distance between the two components is twice the focal length of the collimating lens component 501; the camera 506 is used to receive the imaging signal, and its characteristics are for sample imaging, and its features are large surface area (≥1 inch), high resolution (>12 million pixels), and high frame rate (>100fps).

[0032] In some embodiments, a sample area to be tested is also included, which further includes a microfluidic chip 201 and a two-dimensional motorized scanning platform 202. The sample to be tested is disposed on the microfluidic chip 201. The microfluidic chip 201 is fixed on the two-dimensional motorized scanning platform 202, which drives the microfluidic chip 201 to move in the XY direction. The two-dimensional motorized scanning platform 202 cooperates with the objective lens focusing assembly 302 to realize the relative movement of the microscope objective lens 301 and the microfluidic chip 201 in three dimensions. Figure 2 As shown, the microfluidic chip 201 consists of two glass layers and a PSA layer located between the two glass layers. Several cutouts in the PSA layer define several sample cell channels 2011. Each sample cell channel 2011 has a marker point 2012 next to it, and the marker point 2012 is located on the upper surface of the lower glass layer.

[0033] Regarding the aforementioned sample area to be tested, the sample area includes a microfluidic chip 201 containing the sample and a two-dimensional motorized scanning platform 202. The microfluidic chip 201 is fixed to the two-dimensional motorized scanning platform 202 via an adapter plate or directly, ensuring that there is no relative displacement between the chip and the scanning platform during movement. By controlling the two-dimensional motorized scanning platform 202, the microfluidic chip 201 can move in the XY directions to identify samples in different sample cells 2011. The microfluidic chip 201 in the device of this invention is as follows... Figure 2 As shown, the chip contains eight sample cells 2011 channels. It is formed by bonding three layers: the top and bottom layers are glass of different thicknesses, and the middle layer is a PSA layer. The sample cells 2011 channels are created by hollowing out the PSA layer. Each sample cell 2011 has a marker point 2012 next to it. The marker point 2012 can be a basic, easily identifiable image shape such as a circle, triangle, or crosshair, and it is etched onto the upper surface of the lower glass layer using a laser. During the pretreatment stage, the sample is deposited on the upper surface of the lower glass layer of the chip through steps such as centrifugation. Therefore, by identifying the position of the marker point 2012 and focusing, the depth information of the marker point 2012 can be compensated to obtain the focal plane position of the sample. The depth information of the marker point 2012 is a fixed bias. The etching depth of the marker point 2012 can be precisely controlled by controlling the laser exposure power. To avoid accidental errors in compensation, the depth can be measured multiple times and the average value taken. Furthermore, to avoid large or small deviations in the measured values, the maximum and minimum values ​​can be removed before averaging. The measurement method can be to measure directly under a microscope after actual sample injection, or to measure by projection magnification. The detection of this depth offset is also an important part of chip process maturation and quality inspection.

[0034] In some embodiments, an illumination path is also included to provide bright field illumination for microscopic imaging. The illumination path includes a white light source 101 and a Köhler illumination assembly 102. The white light source 101 provides the light source for bright field imaging. The Köhler illumination assembly 102 is located between the white light source 101 and the two-dimensional motorized scanning platform 202 to provide a highly uniform light field.

[0035] In some embodiments, a computer control system 6 is also included, which is electrically connected to the white light source 101, the two-dimensional motorized scanning platform 202, the objective lens focusing assembly 302, the liquid lens focusing assembly 503, and the camera 506. The computer control system 6 serves as the control and display center of the entire device, issuing control commands and processes via host computer software. Specifically, it controls the switching and brightness of the white light source 101; controls the speed, direction, step value, and distance parameters of the two-dimensional motorized scanning platform 202; controls the speed, direction, step value, and distance parameters of the objective lens focusing assembly 302; controls the driving current of the liquid lens; and controls the exposure time and white balance parameters of the camera 506. Additionally, this system is also responsible for displaying the image plane.

[0036] In some embodiments, the first image plane optical path includes a barrel lens 401, a first reflecting mirror 402, and a field lens 403. The barrel lens 401 focuses the sample image onto the first image plane; the first reflecting mirror 402 deflects the optical path; and the field lens 403 is located at the first image plane. Specifically, the first image plane optical path achieves infinity-corrected microscopic imaging. It includes the barrel lens 401, which focuses the sample image onto the first image plane, characterized by wide-field imaging, apochromaticity, and diffraction limit across the entire field of view; the first reflecting mirror 402, which deflects the optical path by 90° to compress the optical path volume; and the field lens 403, located at the first image plane, which does not contribute to the optical power of the entire optical system and therefore does not affect the performance of the optical system. Its function is to compress the field of view of the off-axis imaging beam to ensure that the aperture of optical devices in subsequent optical paths is not too large.

[0037] In some embodiments, the first reflecting mirror 402, the second reflecting mirror 502, and the third reflecting mirror 504 are selected from any one of a planar reflecting mirror and a reflecting prism; the field mirror 403 is selected from a plano-convex lens, a biconvex lens, or a cemented lens; the collimating lens assembly 501 and the focusing lens assembly 505 are selected from a plano-convex lens, a cemented lens, or a combination lens group. In some embodiments, the first reflecting mirror 402, the second reflecting mirror 502, and the third reflecting mirror 504 bend the optical path by 90°.

[0038] The process of using the aforementioned autofocus device to perform microscopic imaging and focusing on complex biological samples containing a large number of impurities includes:

[0039] Before using autofocus, the following preparations need to be completed.

[0040] 1. Establish a recognition model for chip marker 2012. Using the aforementioned device, acquire a training image set containing chip marker 2012. Specifically, place the chip parallel to or at a certain angle relative to the two-dimensional electric platform, and use the objective lens focusing assembly 302 to move the objective lens in micro-stepping motion to obtain a series of clear, blurred, and tilted images of marker 2012. Train and learn using a neural network model to ultimately obtain a prediction model for marker 2012. The neural network model can be YOLO, AlexNet, ResNet, etc., or can be freely constructed without limitation. Since marker 2012 is an easily recognizable basic image shape and is fixed on the chip glass, it is not affected by sample diversity. The model can be established after the device is assembled and adjusted, and is directly built into the control software on the computer in the device, without needing to be updated with different samples.

[0041] 2. The relationship between the image clarity of the 2012 calibrated chip marker and the driving current of the liquid lens. Figure 3To establish the relationship between driving current and optical power, the optical power of the liquid lens is initially set to 0 using this curve, and this is recorded as the initial reset position of the liquid lens. The objective lens is moved once via a tiny step (<0.2μm) using the objective lens focusing assembly 302, and the objective lens position is recorded to obtain a defocused image of the chip marker 2012. This image is then segmented to obtain sub-images, with the segmentation principle being that the chip marker 2012 is located at the center of the sub-image, and the area of ​​the sub-image is twice that of the chip marker 2012. The sharpness of the sub-images is calculated using a referenceless image quality assessment algorithm based on an optimized BRISQUE algorithm, yielding a sharpness score (0-). 1) Keeping the objective lens position unchanged, change the liquid lens drive current. Subjectively judge the defocused image to become clear, ensuring the drive current A at the moment of image clarity corresponds to the sub-image sharpness score Q of the original defocused image. Cut the image using the aforementioned cutting principle and calculate the sub-image sharpness using the same method. To reduce subjective judgment and image sharpness algorithm calculation errors, this process requires multiple operations, averaging the results, or averaging after removing candidate maximum and minimum values. Reset the liquid lens and continue moving the objective lens in steps, repeating the above steps to complete the calibration model for the image sharpness of chip marker 2012 and the liquid lens drive current. To further reduce errors, the above process needs to be executed twice, sequentially from the initial objective lens position to when the objective lens moves upward and downward. The calibration curve is built into the control software on the computer in the device and does not need to be updated with different samples. The specific method for sharpening a defocused image is as follows: First, the defocused image is made sharp through subjective judgment. Then, the subjectively sharp image is segmented using the aforementioned segmentation principle. The sharpness of the sub-images is calculated using the above method. The driving current is fine-tuned, and the above process is repeated to obtain the driving current A1 corresponding to the highest image sharpness score. To reduce subjective judgment errors and image sharpness algorithm calculation errors, this process needs to be performed multiple times, resulting in a series of A1, A2, ... An. The average value is taken to obtain A, or the maximum and minimum values ​​are removed from A1, A2, ... An, and the average value is taken to obtain A. Finally, A corresponding to Q is obtained. Then, the liquid lens is reset, and the objective lens is moved step by step, repeating the above steps. A series of driving currents Ai corresponding one-to-one with the image sharpness score Qi are obtained, thereby determining the relationship between the image sharpness of the calibration chip marker and the driving current of the liquid lens, thus completing the calibration model of the image sharpness of the chip marker and the driving current of the liquid lens.

[0042] After completing the preparations, we began fully automated, rapid focusing for highly complex biological samples. The specific method is as follows:

[0043] 1. Connect the microfluidic chip 201 to the continuous sample feeding device and place it on the two-dimensional electric scanning platform 202. The platform automatically resets and aligns with the first sample cell 2011, automatically turns on the white light source 101, and adaptively adjusts the appropriate brightness. The liquid lens focusing component 503 is reset.

[0044] 2. Coarse focusing is achieved by adjusting the objective lens position using the objective lens focusing assembly 302. Starting from the current lens position, the objective lens focusing assembly 302 is moved upward by a step size L1, denoted as position A1. Using A1 as the starting position, the speed of the objective lens focusing assembly 302 is set to v, and the objective lens is continuously moved downward by a total step size L2, where L2 = 2L1. Simultaneously, the camera 506 is continuously exposed to perform axial scanning. In the obtained scanning video, n frames are taken at average intervals. The correspondence between the i-th image and the objective lens position Ai in these n frames is:

[0045] Ai = (L2 × i) / n

[0046] 3. Based on the identification model of chip marker 2012, determine whether the n frames of images obtained contain chip marker 2012, and obtain the set n1 containing marker 2012;

[0047] 4. Cut each image in n1 to obtain a sub-image set n1'. The cutting principle is: the chip marker 2012 is located at the center of the sub-image, and the area of ​​the sub-image is twice that of the chip marker 2012. Calculate the sharpness of the sub-image. The calculation method is a no-reference image quality assessment algorithm based on the optimized BRISQUE algorithm to obtain a sharpness score (0-1). Take the image with the highest score and map it to image i in set n through timestamp index.

[0048] 5. Move the objective lens to the position Ai corresponding to i according to the formula. Since the acceleration and deceleration process of the objective lens movement is ignored in the flying scanning process, and there are problems such as uneven transfer after the camera 506 exposure when taking n frames of images, the obtained position Ai is not the clearest position of the marker point 2012. Therefore, i is recorded as the collimated image.

[0049] 6. Fine focusing is achieved by adjusting the image distance using the liquid lens focusing assembly 503. Keeping the objective lens position constant at Ai, the driving current of the liquid lens focusing assembly 503 is adjusted based on the sharpness score of i, the image sharpness of the chip marker 2012, and the calibration model of the liquid lens driving current, to obtain a final sharp image of the marker 2012.

[0050] 7. Based on this, the objective lens is moved by the objective lens focusing assembly 302 to perform offset compensation, thereby obtaining the sample focal plane position corresponding to the first sample cell 2011 and completing the automatic focusing of the sample cell 2011.

[0051] 8. Repeat the above steps to complete the autofocusing of other required sample cells 2011.

[0052] The above-mentioned automatic focusing device has the following advantages:

[0053] 1. High applicability: No model adjustment is required for different biological samples, enabling full automation of the entire process. The device's built-in recognition and calibration models are based on the physical static recognition of the microfluidic chip 201 marker point 2012, rather than the dynamic recognition of the sample image. This makes it universally applicable to different biological samples and does not require updates for different samples, enabling unattended operation of the entire focusing process.

[0054] 2. High-precision focusing can be achieved for complex biological samples containing a large number of impurities. The technical solution of this invention proposes a focusing method that combines coarse focusing by adjusting the object distance using the objective lens focusing assembly 302 and fine focusing by adjusting the image distance using the liquid lens focusing assembly 503. Compared with traditional focusing methods, this method has higher precision for layered samples with impurity interference.

[0055] 3. Fast focusing speed. The technical solution of this invention utilizes the above-mentioned autofocus device to perform axial scanning recognition based on objective lens movement. Compared with the traditional fixed step scanning method, the entire process time is shortened from 5-10 seconds to less than 1 second, which greatly improves the efficiency of acquiring clear images and increases the focusing speed.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The above embodiments only illustrate several implementation methods of this utility model, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the utility model patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this utility model, and these all fall within the protection scope of this utility model. Therefore, the protection scope of this utility model patent should be determined by the appended claims.

Claims

1. An automatic focusing device, characterized in that, It includes, in sequence, the focusing optical path of the microscope objective, the optical path of the first image plane, and the focusing and imaging optical path of the 4f relay and liquid lens; The microscope objective focusing optical path further includes a microscope objective and an objective focusing assembly that drives the microscope objective to move up and down. The first image plane optical path is used to achieve infinity microscopic imaging; The 4f relay and liquid lens focusing imaging optical path includes, in sequence: A collimating lens assembly, wherein the collimating lens assembly collimates the imaging signal of the first image plane; The second reflecting mirror deflects the parallel beam of light. A liquid lens focusing assembly, wherein the curvature of the liquid lens focusing assembly is changed by the change of its driving current, thereby changing the image distance and obtaining a series of out-of-focus blurred images and in-focus clear images; The third reflecting mirror deflects the parallel light beam, and the third reflecting mirror and the second reflecting mirror cooperate to horizontally fix the liquid lens focusing assembly; A focusing lens assembly that focuses the imaging signal onto the target surface of the camera and forms a 4f relay optical path with the collimating lens assembly; A camera that receives imaging signals.

2. The automatic focusing device according to claim 1, characterized in that, It also includes a sample area to be tested, which further includes: A microfluidic chip, wherein the microfluidic chip is provided with a sample to be tested; A two-dimensional motorized scanning platform is provided, wherein the microfluidic chip is fixed on the two-dimensional motorized scanning platform, and the two-dimensional motorized scanning platform drives the microfluidic chip to move in the XY direction. The two-dimensional motorized scanning platform cooperates with the objective lens focusing assembly to realize the relative movement between the microscope objective lens and the microfluidic chip in three dimensions.

3. The automatic focusing device according to claim 2, characterized in that, The microfluidic chip consists of two glass layers and a PSA layer located between the two glass layers. Several cutouts in the PSA layer define several sample cell channels. Each sample cell channel is marked with a marker point located on the upper surface of the lower glass layer.

4. The automatic focusing device according to claim 2, characterized in that, It also includes an illumination optical path that provides bright-field illumination for microscopic imaging, the illumination optical path comprising: A white light source, which provides a light source for bright-field imaging; A Köhler lighting assembly, located between the white light source and the two-dimensional motorized scanning platform, provides a highly uniform light field.

5. The automatic focusing device according to claim 4, characterized in that, It also includes a computer control system, which is electrically connected to the white light source, the two-dimensional motorized scanning platform, the objective lens focusing assembly, the liquid lens focusing assembly, and the camera.

6. The automatic focusing device according to claim 1, characterized in that, The collimating lens assembly, the focusing lens assembly, and the lens barrel have the same focal length, and the distance between the two assemblies is twice the focal length of the collimating lens assembly.

7. The automatic focusing device according to claim 1, characterized in that, The objective focusing assembly moves the microscope objective up and down by ±5mm, with a minimum step value of 0.1μm.

8. The automatic focusing device according to claim 1, characterized in that, The first image plane optical path includes: A telescope lens that focuses a sample image onto a first image plane; The first reflecting mirror deflects the light path. A field lens, which is located at the first image plane.

9. The automatic focusing device according to claim 8, characterized in that, The first reflector, the second reflector, and the third reflector are selected from any one of a flat reflector and a reflecting prism; the field lens is selected from a plano-convex lens, a biconvex lens, or a cemented lens; the collimating lens assembly and the focusing lens assembly are selected from a plano-convex lens, a cemented lens, or a combination lens group.

10. The automatic focusing device according to claim 8, characterized in that, The first, second, and third reflectors bend the light path by 90°.

Images