Focusing method and system of interference microscope based on laser auxiliary device

Abstract

The invention provides a focusing method and system of an interference microscope based on a laser auxiliary device, and relates to the technical field of optical microscopic imaging, the method comprises the following steps: collecting an image at an initial position as an initial image, and determining a region of interest; controlling a motor to move from top to bottom at a first speed, synchronously acquiring a first image, and calculating a laser focusing factor based on the region of interest; determining an initial focus position according to a change curve of the laser focus factor along with the motor position; and setting a secondary focusing interval based on the initial focus position, in the secondary focusing interval, controlling the motor to move from top to bottom at a second speed, synchronously acquiring a second image, determining a final focus position through a differential sequence and Hilbert transform, and driving the motor to move to the final focus position to complete focusing. According to the invention, rapid and high-precision focusing without dependence on the texture of the imaging target is realized, the system cost and complexity are reduced, the stability and adaptability are improved, and the method is suitable for various complex imaging environments and materials.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscopic imaging, and in particular to a focusing method and system of an interference microscope based on a laser-assisted device. Background Art

[0002] In the evolution of microscopy technology, autofocus technology has always been the focus and difficulty of research. Currently, common autofocus technologies mainly include contrast detection autofocus and phase detection autofocus.

[0003] As for contrast detection autofocus, its principle is to determine the focus position by detecting the contrast of the image. When the image contrast reaches the maximum value, the focus is determined to be complete. However, this method has significant defects: first, it has a strong dependence on the texture features of the imaging target. For samples with unclear texture features such as smooth mirrors and transparent films, it is difficult to accurately capture the changes in contrast, which leads to focus failure or poor focus accuracy; second, the focusing speed is relatively slow. Since it is necessary to repeatedly search for the maximum contrast value in a large range, this leads to a huge amount of calculation and a long time consumption, which cannot meet the actual needs of rapid observation and measurement.

[0004] Phase detection autofocus uses phase difference to detect the focus position, which has certain advantages in focusing speed. However, it also has many shortcomings: on the one hand, the system structure is relatively complex, requiring a special phase detection sensor and a complex optical path design, which undoubtedly increases the manufacturing cost of the microscope and the complexity of the system, and hinders the miniaturization and integration of the microscope; on the other hand, it has high environmental requirements and is easily affected by external environmental factors (such as temperature, humidity, vibration, etc.), which causes deviations in the phase detection results, thereby affecting the focusing accuracy and stability.

[0005] In summary, the existing autofocus technology has many problems in terms of stability, focusing speed, accuracy, cost and system structure, which makes it difficult to meet the needs of modern scientific research and industrial production for high-precision, high-efficiency, low-cost and stable and reliable microscope focusing technology. Therefore, a new technical solution is urgently needed to effectively solve these problems in order to effectively improve the performance and practicality of microscopes. Summary of the invention

[0006] To this end, an embodiment of the present invention provides a focusing method and system for an interference microscope based on a laser-assisted device, which is used to solve the problems existing in the prior art of existing focusing technologies, such as strong dependence on the texture features of the imaging target, slow focusing speed, complex system structure, high environmental requirements, high cost, and poor stability.

[0007] In order to solve the above problems, an embodiment of the present invention provides a focusing method of an interference microscope based on a laser-assisted device, the method comprising:

[0008] S1: Acquire an image at a starting position as an initial image, wherein the starting position is the upper limit position of the motor movement, and determine a region of interest based on the initial image through threshold segmentation and closing operation;

[0009] S2: Control the motor to move downward at a first speed, synchronously capture a first image and calculate a laser focusing factor based on the region of interest;

[0010] S3: determining the initial focus position according to the variation curve of the laser focus factor with the motor position;

[0011] S4: Based on the initial focus position, a secondary focus interval is set. Within the secondary focus interval, the motor is controlled to move from top to bottom at a second speed, a second image is synchronously captured, a final focus position is determined through a differential sequence and a Hilbert transform, and the motor is driven to move to the final focus position to complete focusing.

[0012] Preferably, in step S1, determining the region of interest based on the initial image by threshold segmentation and closing operation includes:

[0013] S11: Count the grayscale histogram of the initial image image0, find the two maximum peaks of the grayscale histogram, and take the median gary;

[0014] S12: Perform threshold segmentation on the initial image image0 to find the region region0 where the gray value is higher than the median value gary;

[0015] S13: Perform a closing operation on region region0 to obtain the coordinates of the center point of the new region (x0, y0);

[0016] S14: Make a circular region region1 with a preset radius with the center point coordinate (x0, y0) as the center, and obtain the darker part of the circular region region1, that is, the region of interest region2.

[0017] Preferably, in step S2, the calculating of the laser focus factor based on the region of interest includes:

[0018] S21: Calculate the difference image imagedif between the first image imagei and the initial image image0;

[0019] S22: Calculate the average grayscale value of the region of interest region2 of the difference image imagedif as the laser focusing factor factori.

[0020] Preferably, in step S3, determining the initial focus position according to the curve of the laser focus factor changing with the motor position includes:

[0021] The image is drawn with the Z-axis position of the motor when it moves downward as the horizontal coordinate and the laser focusing factor factori as the vertical coordinate;

[0022] The Z-axis position corresponding to the center point of the pit in the image is determined as the initial focus position.

[0023] Preferably, in step S4, determining the final focus position by using the differential sequence and Hilbert transform includes:

[0024] S41: Calculate the average gray value of the region of interest region2 of the second image imagei2 to obtain a gray2 sequence;

[0025] S42: perform pairwise differences on the gray2 sequences to obtain a difference sequence gray2diff;

[0026] S43: performing Hilbert transform on the differential sequence gray2diff to obtain the gray2hi sequence;

[0027] S44: Calculate the center of gravity position of the gray2hi sequence as the final focus position Zresult.

[0028] Preferably, the setting range of the secondary focus interval is ±200 μm of the initial focus position.

[0029] Preferably, the second speed is lower than the first speed.

[0030] Preferably, the first speed is not higher than 1000 μm / s, and the second speed is not higher than 100 μm / s.

[0031] The embodiment of the present invention further provides a focusing system of an interference microscope based on a laser-assisted device, the system comprising:

[0032] A laser auxiliary device, used for projecting laser light to the objective lens and generating a reflected light spot;

[0033] A motor drive module, used to control the movement of the objective lens along the Z axis;

[0034] Camera, used to collect images in real time;

[0035] A processing module, used to execute the focusing method of an interference microscope based on a laser-assisted device as described in any one of claims 1 to 8.

[0036] An embodiment of the present invention further provides a computer storage medium storing a computer software product, wherein the computer software product includes a number of instructions for enabling a computer device to execute the above-mentioned focusing method of an interference microscope based on a laser-assisted device.

[0037] It can be seen from the above technical solutions that the present invention has the following beneficial effects:

[0038] (1) Wide applicability and high stability: Without relying on the surface texture features of the sample, the laser-assisted and dynamic region of interest extraction technology can adapt to high-reflectivity mirrors, low-reflectivity rough surfaces, and transparent materials (such as acrylic), and can still accurately focus under texture-free or extremely reflective conditions.

[0039] (2) High precision and high repeatability: A dual-stage focusing strategy (coarse focus + fine focus) is adopted, combined with Hilbert transform and center of gravity algorithm. The final focusing accuracy reaches ±10μm and the repeatability accuracy is ≤3μm, which is significantly better than traditional contrast or phase detection methods.

[0040] (3) High efficiency, low cost and compact design: Only a single fiber laser needs to be integrated into the original optical path of the interference microscope, and the hardware modification cost is low; the image processing algorithm is efficient (single frame calculation ≤3ms) and supports real-time focusing; the system structure is compact and suitable for space-constrained microscope platforms. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the implementation cases of the present invention or the technical solutions in the prior art, the following is a brief description of the drawings required for use in the embodiments. By referring to the drawings, the features and advantages of the present invention will be more clearly understood. The drawings are schematic and should not be understood as limiting the present invention in any way. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0042] Figure 1 It is a flow chart of a focusing method of an interference microscope based on a laser-assisted device provided in an embodiment;

[0043] Figure 2 Schematic diagram of the reflected light spot of the initial image image0 in the embodiment;

[0044] Figure 3 This is a schematic diagram of a region of interest region 2 in the embodiment;

[0045] Figure 4 Schematic diagram of the factori curve (pit shape) under the high reflectivity mirror material in embodiment (1);

[0046] Figure 5Schematic diagram of factori curve (pit morphology) under low reflectivity rough surface material in the embodiment;

[0047] Figure 6 Schematic diagram of factori curve (pit shape) under transparent acrylic material in the embodiment;

[0048] Figure 7 This is a diagram showing the effect of interference fringes after focusing is completed in the embodiment;

[0049] Figure 8 It is a block diagram of a focusing system of an interference microscope based on a laser-assisted device provided in an embodiment. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Embodiment 1

[0052] In order to solve the problems of existing focusing technology in the prior art, such as strong dependence on the texture features of the imaging target, slow focusing speed, complex system structure, high environmental requirements, high cost, poor stability, etc. Figure 1 The embodiment of the present invention provides a focusing method of an interference microscope based on a laser-assisted device, the method comprising:

[0053] S1: Acquire an image at the starting position as the initial image. The starting position is the upper limit position of the motor movement. According to the initial image, the region of interest is determined by threshold segmentation and closing operation.

[0054] S2: Control the motor to move downward at a first speed, synchronously acquire a first image and calculate a laser focusing factor based on the region of interest;

[0055] S3: determining the initial focus position according to the curve of the laser focus factor changing with the motor position;

[0056] S4: Based on the initial focus position, a secondary focus interval is set. Within the secondary focus interval, the motor is controlled to move from top to bottom at a second speed, and a second image is synchronously captured. The final focus position is determined by a differential sequence and a Hilbert transform, and the motor is driven to move to the final focus position to complete focusing.

[0057] It can be seen from the above technical scheme that the present invention proposes a focusing method for an interference microscope based on a laser-assisted device. First, the initial image is collected at the upper limit position of the motor, and the region of interest (ROI) is determined by threshold segmentation and closing operation, thereby avoiding dependence on the texture of the imaging target and providing an accurate analysis area for subsequent focusing. Secondly, the motor moves downward at a first speed, synchronously collects images and calculates the laser focusing factor, quickly determines the rough focus position, significantly improves the focusing speed and enhances stability. Then, the initial focus position is determined according to the laser focusing factor change curve to further improve the focusing adaptability. Finally, a secondary focusing interval is set near the initial focus position, and the motor moves at a second speed. The final focus position is accurately determined by the differential sequence and Hilbert transform to achieve high-precision focusing, with an accuracy of less than 10μm and a repeatability of about 3μm. This method achieves focusing based on the original optical path, does not require complex mechanical structure adjustment, reduces cost and complexity, improves the compactness and stability of the system, is suitable for a variety of complex imaging environments and materials, and has broad application prospects.

[0058] In step S1, an image at a starting position is collected as an initial image, and a region of interest (ROI) is determined based on the initial image through threshold segmentation and closing operations.

[0059] Specifically, first move the objective lens of the interference microscope to the upper limit position of the motor movement, and collect the image at the starting position as the initial image (recorded as image0). The image spot is obtained by the partial reflection of the laser irradiated to the objective lens, such as Figure 2 shown.

[0060] Then, based on the initial image, the region of interest is determined through threshold segmentation and closing operation, including:

[0061] S11: Count the grayscale histogram of the initial image image0, obtain the two maximum peaks of the grayscale histogram, and take the median value gary (for example, determine the threshold value by using a bimodal interpolation method).

[0062] S12: Perform threshold segmentation on the initial image image0, and find the region whose grayscale value is higher than the median value gary, which is recorded as region0.

[0063] S13: Perform a morphological closing operation (such as a 3×3 circular kernel) on region region0 to eliminate small holes and smooth the edges, and obtain the coordinates of the center point (x0, y0) of the new region.

[0064] S14: With the center point coordinate (x0, y0) as the center, make a circular area with a radius of about 80 pixels (recorded as region1), and obtain the darker part of the circular area region1 as the region of interest region2, such as Figure 3The region of interest region2 is a key region used to calculate the laser focusing factor in the subsequent focusing process.

[0065] In step S2, the motor is controlled to move from top to bottom at a first speed, and a first image is synchronously acquired and a laser focus factor is calculated based on the region of interest.

[0066] Specifically, the motor is controlled to move from top to bottom at a first speed (not higher than 1000 μm / s, 1000 μm / s is used in this embodiment), and the camera captures images at a maximum frame rate, and the first image captured each time is recorded as imagei.

[0067] Further, the laser focusing factor factori is calculated based on the region of interest, specifically including:

[0068] S21: Calculate the difference image imagedif between the first image imagei and the initial image image0;

[0069] S22: Calculate the average grayscale value of the region of interest region2 of the difference image imagedif as the laser focusing factor factori.

[0070] In step S3, the initial focus position is determined according to the variation curve of the laser focus factor factori with the motor position.

[0071] Specifically, a curve is drawn with the Z-axis position of the motor when it moves downward as the horizontal coordinate and the laser focusing factor factori as the vertical coordinate, such as Figure 4-6 For different materials (such as high reflectivity mirror, low reflectivity rough surface, transparent acrylic), the curve presents a pit shape, and the Z-axis position corresponding to the center of the pit is the initial focus position.

[0072] In step S4, a secondary focusing interval is set based on the initial focus position. Within the secondary focusing interval, the motor is controlled to move from top to bottom at a second speed, and a second image is synchronously captured. The final focus position is determined by a differential sequence and a Hilbert transform, and the motor is driven to move to the final focus position to complete focusing.

[0073] Specifically, with the initial focus position as the center, the setting range of the secondary focus interval is set to the initial focus position ±200μm, the motor is controlled to move at a low speed of ≤100μm / s (60μm / s is used in this embodiment), and the synchronous camera captures the second image imagei2 at the maximum frame rate.

[0074] Furthermore, the final focus position is determined by using the difference sequence and Hilbert transform, specifically including:

[0075] S41: Calculate the average gray value of the region of interest region2 of the second image imagei2 to obtain a gray2 sequence;

[0076] S42: perform pairwise differences on the gray2 sequences to obtain a difference sequence gray2diff;

[0077] S43: performing Hilbert transform on the differential sequence gray2diff to obtain the gray2hi sequence;

[0078] S44: Calculate the center of gravity position of the gray2hi sequence as the final focus position Zresult.

[0079] Finally, the motor is controlled to move to the final focus position Zresult to complete the focusing. After the focusing is completed, obvious interference fringes will appear in the imaging area of ​​the interference microscope, such as Figure 7 As shown, it indicates that the focus is successful.

[0080] Experimental verification:

[0081] (1) Focus accuracy:

[0082] The error of the coarse focus (first time) stage is ≤50μm, the final accuracy after fine focus (second time) is ≤±10μm, and the repeatability is ≤3μm (based on the standard deviation of 10 experiments).

[0083] (2) Computational efficiency:

[0084] The single-frame image processing time is ≤3ms (1932×1464 pixels), meeting the real-time focusing requirements.

[0085] (3) Applicable scenarios:

[0086] High reflectivity mirror material, low reflectivity rough surface material, transparent acrylic material ( Figure 4-6 ), strong adaptability.

[0087] Embodiment 2

[0088] like Figure 8 As shown, the present invention provides a focusing system for an interference microscope based on a laser-assisted device, the system comprising:

[0089] A laser auxiliary device, used for projecting laser light to the objective lens and generating a reflected light spot;

[0090] A motor drive module, used to control the movement of the objective lens along the Z axis;

[0091] Camera, used to collect images in real time;

[0092] The processing module is used to execute the focusing method of the interference microscope based on the laser-assisted device in the first embodiment.

[0093] Furthermore, the laser auxiliary device is used to project laser light to the objective lens and generate a reflected light spot for focusing. The device is integrated with the microscope system through a laser optical fiber, without the need for an additional complex structure, thereby reducing costs.

[0094] The motor drive module is used to control the movement of the objective lens along the Z axis. The speed and direction of the motor are controlled by the system to ensure the accuracy and stability of the focusing process.

[0095] The camera is used to collect images of the microscope imaging area in real time. The frame rate of the camera should be high enough to meet the needs of rapid image acquisition during the focusing process.

[0096] The processing module is used to perform the image processing and calculation steps in the above-mentioned focusing method. This module can be implemented based on an embedded system or computer software, and can quickly calculate the laser focusing factor, determine the focus position, and control the motor movement. In order to avoid redundancy, it will not be repeated here.

[0097] Embodiment 3

[0098] An embodiment of the present invention provides a computer storage medium storing a computer software product. The computer software product includes several instructions for enabling a computer device to execute the above-mentioned focusing method of an interference microscope based on a laser-assisted device.

[0099] Those skilled in the art will appreciate that the embodiments of the present application may be provided as methods, systems, or computer program products. Therefore, the present application may adopt the form of a complete hardware embodiment, a complete software embodiment, or an embodiment in combination with software and hardware. Moreover, the present application may adopt the form of a computer program product implemented in one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) that include computer-usable program code.

[0100] The present application is described with reference to the flowcharts and / or block diagrams of the methods, devices (systems), and computer program products according to the embodiments of the present application. It should be understood that each process and / or box in the flowchart and / or block diagram, as well as the combination of the processes and / or boxes in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to generate a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 A process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0101] These computer program instructions may also be stored in a computer-readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer-readable memory produce an article of manufacture comprising an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 These computer program instructions can also be loaded onto a computer or other programmable data processing device, so that a series of operation steps are executed on the computer or other programmable device to produce a computer-implemented process, so that the instructions executed on the computer or other programmable device provide for implementing the process in the process. Figure 1 A process or multiple processes and / or boxes Figure 1 The steps for the functions specified in one or more boxes.

[0102] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A focusing method for an interference microscope based on a laser-assisted device, characterized in that: include: S1: Acquire an image at a starting position as an initial image, wherein the starting position is the upper limit position of the motor movement, and determine a region of interest based on the initial image through threshold segmentation and closing operation; S2: Control the motor to move downward at a first speed, synchronously capture a first image and calculate a laser focusing factor based on the region of interest; S3: determining the initial focus position according to the variation curve of the laser focus factor with the motor position; S4: Based on the initial focus position, a secondary focus interval is set. Within the secondary focus interval, the motor is controlled to move from top to bottom at a second speed, a second image is synchronously captured, a final focus position is determined through a differential sequence and a Hilbert transform, and the motor is driven to move to the final focus position to complete focusing.

2. The focusing method of an interference microscope based on a laser-assisted device according to claim 1, characterized in that: In step S1, determining the region of interest based on the initial image by threshold segmentation and closing operation includes: S11: Count the grayscale histogram of the initial image image0, find the two maximum peaks of the grayscale histogram, and take the median gary; S12: Perform threshold segmentation on the initial image image0 to find the region region0 where the gray value is higher than the median value gary; S13: Perform a closing operation on region region0 to obtain the coordinates of the center point of the new region (x0, y0); S14: Make a circular region region1 with a preset radius with the center point coordinate (x0, y0) as the center, and obtain the darker part of the circular region region1, that is, the region of interest region2.

3. The focusing method of an interference microscope based on a laser-assisted device according to claim 1, characterized in that: In step S2, the calculating of the laser focus factor based on the region of interest includes: S21: Calculate the difference image imagedif between the first image imagei and the initial image image0; S22: Calculate the average grayscale value of the region of interest region2 of the difference image imagedif as the laser focusing factor factori.

4. The focusing method of an interference microscope based on a laser-assisted device according to claim 1, characterized in that: In step S3, determining the initial focus position according to the variation curve of the laser focus factor with the motor position includes: The image is drawn with the Z-axis position of the motor when it moves downward as the horizontal coordinate and the laser focusing factor factori as the vertical coordinate; The Z-axis position corresponding to the center point of the pit in the image is determined as the initial focus position.

5. The focusing method of an interference microscope based on a laser-assisted device according to claim 1, characterized in that: In step S4, determining the final focus position by using the differential sequence and Hilbert transform includes: S41: Calculate the average gray value of the region of interest region2 of the second image imagei2 to obtain a gray2 sequence; S42: perform pairwise differences on the gray2 sequences to obtain a difference sequence gray2diff; S43: performing Hilbert transform on the differential sequence gray2diff to obtain the gray2hi sequence; S44: Calculate the center of gravity position of the gray2hi sequence as the final focus position Zresult.

6. The focusing method of the primary focal position interference microscope based on the laser-assisted device according to claim 1, characterized in that: The setting range of the secondary focus interval is ±200 μm of the initial focus position.

7. The focusing method of an interference microscope based on a laser-assisted device according to claim 1, characterized in that: The second speed is lower than the first speed.

8. The focusing method of an interference microscope based on a laser-assisted device according to claim 1, characterized in that: The first speed is not higher than 1000 μm / s, and the second speed is not higher than 100 μm / s.

9. A focusing system for an interference microscope based on a laser-assisted device, characterized in that: include: A laser auxiliary device, used for projecting laser light to the objective lens and generating a reflected light spot; A motor drive module, used to control the movement of the objective lens along the Z axis; Camera, used to collect images in real time; A processing module, used to execute the focusing method of an interference microscope based on a laser-assisted device as described in any one of claims 1 to 8.

10. A computer storage medium, characterized in that: The computer storage medium stores a computer software product, which includes a number of instructions for enabling a computer device to execute the focusing method of an interference microscope based on a laser-assisted device as described in any one of claims 1 to 8.

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