Rescanning confocal measurement system and measurement method based on dynamic focus weighted fusion

Abstract

The present invention relates to the field of optical detection, and in particular to a rescanning confocal measurement system and a measurement method based on dynamic focus weighted fusion, wherein the excitation light emitted by a laser light source is sequentially reflected by a dichroic mirror, swung by a first scanning oscillating mirror, focused by a first scanning lens, aberration corrected by an aberration correction lens, and focused by an objective lens before being incident on a sample to be tested, and the emission light excited by the sample to be tested returns to the dichroic mirror, is transmitted by the dichroic mirror, focused by a second scanning lens, passes through a pinhole, and then collimated by a third scanning lens, swung by a second scanning oscillating mirror, and split by three beam splitters, and is incident on two pre-focus detectors and two post-focus detectors, respectively, to obtain a total of four defocused images. The present invention introduces a rescanning unit and an image fusion method, so that images can be directly projected onto cameras with four different focal planes, thereby enhancing the details and clarity of the image, reducing blur and out-of-focus areas, thereby reducing the impact of noise, and improving the overall quality of the image.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical detection, and in particular relates to a rescanning confocal measurement system and a measurement method based on dynamic focus weighted fusion. Background Art

[0002] Traditional confocal microscopes have many advantages, making them widely used in fields such as materials science, biomedicine, and semiconductor manufacturing. Confocal microscopes have high resolution and optical sectioning capabilities, and can suppress scattered light from non-focal planes. Therefore, they can significantly improve the lateral and axial resolution of images, and are particularly outstanding when studying the three-dimensional structure and surface morphology of samples. In addition, because confocal microscopes can eliminate the interference of out-of-focus light, the generated images have higher contrast and are suitable for studying subtle structures in complex samples. The real-time imaging function enables confocal microscopes to quickly generate images of dynamic processes, such as living cell imaging, and the feature of reducing sample damage is also particularly suitable for in vivo imaging. Confocal microscopes are also compatible with a variety of labeling technologies and are widely used in the labeling and detection of cells and molecules, further enhancing their application value in biological research.

[0003] In the confocal rescanning technology, there are still some problems in the lateral scanning process. First, the signal intensity may be uneven, especially when the scanning area is large, the imaging brightness may change, affecting the overall contrast and detail performance of the image. Secondly, lateral distortion may occur during the scanning process, especially when the imaging system cannot completely correct the optical distortion, resulting in geometric distortion of the image. In addition, single-focus imaging can often only present part of the details of the sample, especially for samples with greater depth. When defocused image fusion is not performed, blurring and loss of hierarchical information are likely to occur. The lack of defocused image fusion may also lead to a reduction in image contrast, especially when the focus position deviates, the details of certain areas will be distorted. Summary of the invention

[0004] In view of this, the present invention aims to provide a rescanning confocal measurement system and a measurement method based on dynamic focus weighted fusion to solve the problems of insufficient image quality and image details in the existing confocal rescanning technology.

[0005] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0006] A rescanning confocal measurement system based on dynamic focus weighted fusion includes a laser light source, a dichroic mirror, a first scanning oscillating mirror, a first scanning lens, an aberration correction lens, an objective lens, a piezoelectric ceramic objective lens driver, a platform, a second scanning lens, a pinhole, a third scanning lens, a second scanning oscillating mirror, a first beam splitter, a second beam splitter, a third beam splitter, a first pre-focus detector, a second pre-focus detector, a first post-focus detector, and a second post-focus detector; wherein,

[0007] The sample to be tested is placed on the platform, and the objective lens is placed on the piezoelectric ceramic objective lens driver. The piezoelectric ceramic objective lens driver drives the objective lens to move axially to achieve measurement of different sections of the sample to be tested.

[0008] The dichroic mirror is arranged in the emitting direction of the laser light source, the first scanning lens, the aberration correction lens, and the objective lens are arranged in sequence between the first scanning oscillating mirror and the sample to be measured, and the first scanning oscillating mirror is arranged in the reflecting direction of the dichroic mirror; the second scanning lens, the pinhole, the third scanning lens, and the second scanning oscillating mirror are arranged in sequence in the transmitting direction of the dichroic mirror, the first beam splitter is arranged in the reflecting direction of the second scanning oscillating mirror, the second beam splitter and the third beam splitter are arranged in the reflecting direction and the transmitting direction of the first beam splitter respectively, the first pre-focus detector and the second pre-focus detector are arranged in the reflecting direction and the transmitting direction of the second beam splitter, and the first post-focus detector and the second post-focus detector are arranged in the reflecting direction and the transmitting direction of the third beam splitter.

[0009] Furthermore, the first pre-focus detector includes a fourth scanning lens and a first camera, the second pre-focus detector includes a fifth scanning lens and a second camera, the first post-focus detector includes a sixth scanning lens and a third camera, and the second post-focus detector includes a seventh scanning lens and a fourth camera.

[0010] Further, any one of the fourth scanning lens to the seventh scanning lens and the first scanning lens, the second scanning lens, the third scanning lens, the first scanning oscillating mirror, and the second scanning oscillating mirror satisfy the following relationship:

[0011]

[0012]

[0013]

[0014] in, represents the relative mechanical magnification of the rescanning confocal measurement system based on dynamic focus weighted fusion, represents the mechanical magnification of the rescanning confocal measurement system, represents the optical magnification of the rescanning confocal measurement system, represents the focal length of the first scanning lens, represents the focal length of the second scanning lens, represents the focal length of the third scanning lens, represents the focal length of any one of the fourth scanning lens to the seventh scanning lens, represents the amplitude of the second scanning oscillating mirror, Represents the amplitude of the first scanning oscillating mirror.

[0015] Furthermore, the first camera to the fourth camera are CMOS cameras or CCD cameras.

[0016] A rescanning confocal measurement method based on dynamic focus weighted fusion is implemented using the above-mentioned rescanning confocal measurement system based on dynamic focus weighted fusion, and includes the following steps:

[0017] S1: The excitation light emitted by the laser light source is sequentially reflected by the dichroic mirror, swung by the first scanning oscillating mirror, focused by the first scanning lens, aberration corrected by the aberration correction lens, and focused by the objective lens before being incident on the sample to be tested. The emission light excited by the sample to be tested returns to the dichroic mirror along the original excitation light path, is transmitted by the dichroic mirror, focused by the second scanning lens, and is incident on the pinhole. After passing through the pinhole, it is collimated by the third scanning lens and swung by the second scanning oscillating mirror and is incident on the first beam splitter. The light is divided into reflected light and transmitted light by the first beam splitter. The reflected light is divided into pre-focus reflected light and pre-focus transmitted light by the second beam splitter. The pre-focus reflected light is incident on the second pre-focus detector. The pre-focus transmitted light is incident on the first pre-focus detector. The transmitted light is divided into post-focus reflected light and post-focus transmitted light by the third beam splitter. The post-focus reflected light is incident on the first post-focus detector. The post-focus transmitted light is incident on the second post-focus detector. A total of four defocused images are obtained.

[0018] S2: Calculate the lateral intensity distribution of each defocused image; wherein the lateral intensity distribution of each defocused image The calculation formula is as follows:

[0019]

[0020]

[0021] In the formula, is the intensity of the emitted light excited by the sample to be tested; The object plane position of the rescanning confocal measurement system; is the position of the first pre-focus detector, the second pre-focus detector, the first post-focus detector or the second post-focus detector relative to the laser light source; is the axial position of the sample to be tested; is the out-of-focus spread function; is the scanning position of the sample to be tested; is the point spread function of the excitation light; is the point spread function of the emitted light; is the relative mechanical magnification; is the transfer function of the pinhole;

[0022] S3: According to the lateral intensity distribution of the four defocused images, the four defocused images are fused using the weighted average method to obtain a fused focus image; wherein the fusion formula of the defocused images is as follows:

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029] In the formula, is the fused focus image; is the weight of each defocused image; is the focal length weighting function for each out-of-focus image; is the clarity of each out-of-focus image; is the defocus amount for each camera; is a constant; is the gradient of each defocused image along the horizontal direction; is the gradient of each defocused image along the axial direction; is the total number of pixels in each out-of-focus image.

[0030] Compared with the prior art, the invention can achieve the following beneficial effects:

[0031] The present invention significantly improves the overall quality of imaging by introducing a rescanning unit and a defocused image fusion method, and has important advantages in three-dimensional imaging and observation of complex samples. By fusing images from different focal planes together, the clarity of the image can be enhanced, so that the details of each depth level can be clearly presented, avoiding the blur problem caused by single-focus imaging. This fusion not only improves the contrast and detail performance of the image, but also effectively reduces out-of-focus noise, improves the signal-to-noise ratio, and ensures that the image is more realistic and accurate. The fusion of multi-focus images also enhances the depth penetration ability of the image, making the observation of multi-level structures or deep samples clearer. In addition, the fusion of defocused images also improves the resolution and sensitivity of the imaging system, especially in low light or high noise environments, and can significantly improve the imaging effect. Therefore, the fusion of defocused images not only improves the visual quality of the image, but also provides strong technical support for precise analysis and three-dimensional reconstruction. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings:

[0033] Figure 1 It is a structural schematic diagram of a rescanning confocal measurement system based on dynamic focus weighted fusion according to an embodiment of the invention;

[0034] Figure 2 It is a schematic diagram of a light spot separation process and a signal intensity distribution separation process in a rescanning process according to an embodiment of the invention;

[0035] Figure 3 is a schematic diagram of the lateral intensity of a confocal microscope according to an embodiment of the invention;

[0036] Figure 4 is a schematic diagram of the lateral intensity of a rescanning confocal measurement system according to an embodiment of the invention;

[0037] Figure 5 Schematic diagram of the change of FWHM of the rescanning confocal measurement system and the confocal microscope with the pinhole radius according to the embodiment of the invention.

[0038] Explanation of the accompanying drawings: platform 1, sample to be tested 2, piezoelectric ceramic objective lens driver 3, aberration correction lens 4, objective lens 5, first scanning lens 6, first scanning oscillating mirror 7, dichroic mirror 8, second scanning lens 9, pinhole 10, third scanning lens 11, second scanning oscillating mirror 12, first beam splitter 13, second beam splitter 14, third beam splitter 15, fourth camera 16, fourth scanning lens 17, first camera 18, fifth scanning lens 19, second camera 20, sixth scanning lens 21, third camera 22, seventh scanning lens 23. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the invention more clear, the invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described here are only used to explain the invention and do not constitute a limitation of the invention.

[0040] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0041] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0042] In the description of the invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installation", "connection" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the invention can be understood according to specific circumstances.

[0043] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0044] like Figure 1 As shown, the rescanning confocal measurement system based on dynamic focus weighted fusion provided by the embodiment of the present invention includes a laser light source, a platform 1, a piezoelectric ceramic objective lens driver 3, an aberration correction lens 4, an objective lens 5, a first scanning lens 6, a first scanning swing mirror 7, a dichroic mirror 8, a second scanning lens 9, a pinhole 10, a third scanning lens 11, a second scanning swing mirror 12, a first beam splitter 13, a second beam splitter 14, a third beam splitter 15, a first pre-focus detector, a second pre-focus detector, a first post-focus detector and a second post-focus detector.

[0045] The sample 2 to be tested is placed on the platform 1, and the objective lens 5 is placed on the piezoelectric ceramic objective lens driver 3. The piezoelectric ceramic objective lens driver 3 drives the objective lens 5 to axially move the sample 2 to be tested, thereby achieving measurement of different sections of the sample 2 to be tested.

[0046] The dichroic mirror 8 is arranged in the emission direction of the laser light source, the first scanning lens 6, the aberration correction lens 4, and the objective lens 5 are arranged in sequence between the first scanning oscillating mirror 7 and the sample 2 to be measured, and the first scanning oscillating mirror 7 is arranged in the reflection direction of the dichroic mirror 8; the second scanning lens 9, the pinhole 10, the third scanning lens 11, and the second scanning oscillating mirror 12 are arranged in sequence in the transmission direction of the dichroic mirror 8, the first beam splitter 13 is arranged in the reflection direction of the second scanning oscillating mirror 12, the second beam splitter 14 is arranged in the reflection direction of the first beam splitter 13, and the third beam splitter 13 is arranged in the transmission direction of the first beam splitter 13, the first pre-focus detector is arranged in the reflection direction of the second beam splitter 14, and the second pre-focus detector is arranged in the transmission direction of the second beam splitter 14, the first post-focus detector is arranged in the reflection direction of the third beam splitter 15, and the second post-focus detector is arranged in the transmission direction of the third beam splitter 15.

[0047] The first pre-focus detector includes a fourth scanning lens 17 and a first camera 18, the second pre-focus detector includes a fifth scanning lens 19 and a second camera 20, the first post-focus detector includes a sixth scanning lens 21 and a third camera 22, and the second post-focus detector includes a seventh scanning lens 23 and a fourth camera 16. Defocusing is achieved by adjusting the positions of the first camera 18, the second camera 20, the third camera 22, and the fourth camera 16. The first camera 18, the second camera 20, the third camera 22, and the fourth camera 16 are CCD cameras or CMOS cameras.

[0048] The working principle of the rescanning confocal measurement system based on dynamic focus weighted fusion is as follows: the excitation light emitted by the laser light source is sequentially reflected by the dichroic mirror 8, swung by the first scanning oscillating mirror 7, focused by the first scanning lens 6, aberration corrected by the aberration correction lens 4, and focused by the objective lens 5 before being incident on the sample to be measured 2; the emission light excited by the sample to be measured 2 returns to the dichroic mirror 8 along the original excitation light path, is transmitted by the dichroic mirror 8, focused by the second scanning lens 9, and is incident on the pinhole 10; after the stray light is filtered out by the pinhole 10, it is collimated by the third scanning lens 11, swung by the second scanning oscillating mirror 12, and is incident on the first beam splitter 13; the first beam splitter 13 divides the emitted light into reflected light and transmitted light; the reflected light is incident on the second beam splitter 14 after being collimated by the third scanning lens 11 and swung by the second scanning oscillating mirror 12; the reflected light is then incident on the first beam splitter 13 The beam splitter 14 is used to divide the reflected light before focus and the transmitted light before focus. The reflected light before focus is received by the second camera 20 after being converged by the fifth scanning lens 19. The transmitted light before focus is received by the first camera 18 after being converged by the fourth scanning lens 17. The transmitted light is divided into the reflected light after focus and the transmitted light after focus by the third beam splitter. The reflected light after focus is received by the third camera 22 after being converged by the sixth scanning lens 21. The transmitted light after focus is received by the fourth camera 16 after being converged by the seventh scanning lens 23. A total of four defocused images are obtained. The four defocused images are weightedly fused to finally obtain a fused focus image. Then, the piezoelectric ceramic objective lens driver 3 is controlled to move the objective lens 5 axially to measure the next section of the sample 2 to be measured.

[0049] As an example of the present invention, the sample 2 to be tested is a fluorescent material to be tested, and the emitted light is the fluorescence of the excited fluorescent material to be tested.

[0050] The laser light source, the aberration correction lens 4, the objective lens 5, and the dichroic mirror 8 constitute a standard confocal microscope. The first scanning lens 6, the first scanning oscillating mirror 7, the dichroic mirror 8, the second scanning lens 9, the pinhole 10, the third scanning lens 11, and the second scanning oscillating mirror 12 constitute a heavy scanning unit. The first scanning oscillating mirror 7 and the second scanning oscillating mirror 12 have a dual scanning function, namely, realizing the oscillating scanning of the excitation light and the oscillating scanning of the emission light.

[0051] The first scanning oscillating mirror 7 guides the excitation light to the sample to be tested 2 in a scanning manner, and guides the emission light excited by the sample to be tested 2 to the pinhole 10, and the second scanning oscillating mirror 12 guides the emission light passing through the pinhole 10 to the first camera 18, the second camera 20, the third camera 22, and the fourth camera 16.

[0052] The first scanning oscillating mirror 7 and the second scanning oscillating mirror 12 perform synchronous scanning motion at the same frequency and phase, but the angular amplitudes of the scanning motions of the first scanning oscillating mirror 7 and the second scanning oscillating mirror 12 can be adjusted independently.

[0053] Taking the defocused image obtained by the first pre-focus detector as an example, the other three defocused images can be understood in the same way. A novelty of the present invention is to control the amplitude of the first scanning oscillating mirror 7 and the second scanning oscillating mirror 12, so as to introduce an additional mechanical magnification on top of the optical magnification of the traditional confocal microscope. The mechanical magnification is determined by the amplitude of the second scanning oscillating mirror 12. , the amplitude of the first scanning oscillating mirror 7 and the focal lengths of the first scanning lens 6 and the fourth scanning lens 17 , decided.

[0054] Mechanical magnification of the rescanning confocal measurement system for:

[0055]

[0056] When the first scanning oscillating mirror 7 and the second scanning oscillating mirror 12 are set to a fixed position, the optical magnification of the rescanning confocal measurement system for:

[0057]

[0058] in, represents the focal length of the first scanning lens 6, represents the focal length of the second scanning lens 9, represents the focal length of the third scanning lens 11, represents the focal length of the fourth scanning lens 17.

[0059] When the amplitude of the second scanning oscillating mirror 12 just compensates the amplitude of the first scanning oscillating mirror 7, and during the scanning process, the image of the sample to be measured on the chip of the first camera 18 does not move. Relative mechanical magnification of the rescanning confocal measurement system Defined as:

[0060]

[0061] when The rescanning confocal measurement system has exactly the same resolution as a conventional confocal microscope, and the lateral and axial resolutions of the obtained defocused image depend on the diameter of the pinhole 10, which is the same as a conventional confocal microscope. If the angular amplitude of the second scanning oscillating mirror 12 is doubled, the mechanical magnification of the rescanning confocal measurement system is will double, at this time Although the size of the diffraction-limited spot of the emitted light that produces the out-of-focus image on the chip of the first camera 18 depends only on the optical magnification However, when the second scanning mirror 12 moves with double amplitude relative to the first scanning mirror 7, this additional "scan" is as follows Figure 2 As shown, the light moves at a constant speed on the first camera 18 chip, so it can be Therefore, the resolution of the out-of-focus image is improved by doubling the distance between the two molecules due to the double scan of the secondary scan. times.

[0062] Resolution of the re-scanning confocal measurement system varies with relative mechanical magnification The improvement of the spot width was semi-quantitatively analyzed. Can be expressed as relative mechanical magnification Function:

[0063]

[0064] In the formula, is the width of the emitted light spot, is the width of the excitation light spot.

[0065] exist hour, , that is, the resolution of the rescanning confocal measurement system is determined only by the excitation light. If the width of the excitation light spot and the emission light spot are equal, that is ,at this time, ,but , the resolution of the rescanning confocal measurement system can be improved times.

[0066] The axial resolution and lateral resolution of a conventional confocal microscope are determined by the diameter of the pinhole 10. In the rescanning confocal measurement system provided by the present invention, the pinhole 10 only affects the axial resolution but not the lateral resolution. In a conventional confocal microscope, the maximum axial resolution is obtained by setting the diameter of the pinhole 10 to one Airy disk unit (AU). However, with respect to the lateral resolution, the conventional confocal microscope and the rescanning confocal measurement system have completely different responses to the diameter of the pinhole 10. The lateral resolution in the rescanning confocal measurement system is independent of the diameter of the pinhole 10, while in a conventional confocal microscope, the lateral resolution is only improved when the pinhole size is less than 1 Airy disk unit, but this results in the loss of the detected fluorescence signal.

[0067] The present invention utilizes a purely physical method to improve the lateral resolution. The information of the sample to be tested is first acquired by scanning, and then the information of the sample to be tested is projected onto the camera of the detector by rescanning. The movement speed of rescanning is faster than scanning, and the information of the sample to be tested can be more clearly distinguished on the image.

[0068] Transverse intensity distribution of rescanning confocal measurement system for:

[0069]

[0070] Where i=1, 2, 3, 4, is the emission light intensity of the sample to be tested, is the object plane position of the rescanning confocal measurement system, is the axial position of the sample to be tested, are the positions of the first pre-focus detector, the second pre-focus detector, the first post-focus detector, and the second post-focus detector relative to the laser light source.

[0071] Defocus spread function for:

[0072]

[0073] In the formula, is the scanning position of the sample to be tested; is the point spread function of the excitation light; is the point spread function of the emitted light; is the transfer function of the pinhole 10.

[0074] In order to compare with the confocal microscope, the lateral intensity distribution of the confocal microscope is obtained. for:

[0075]

[0076] Among them, the out-of-focus spread function is:

[0077]

[0078] In the formula, is the position of the pinhole 10; is the position of the first camera 18, yes The Fourier transform form of .

[0079] like Figure 3 and Figure 4 As shown, through simulation analysis, pinholes with different diameters R are set. The width of the traditional confocal microscope gradually narrows when the pinhole diameter is small, and the rescanning confocal microscope can achieve the same degree of width narrowing in the case of pinholes of any diameter.

[0080] The main advantage of introducing the rescanning unit in the present invention is that it provides lateral resolution and maintains a good signal-to-noise ratio even with medium or large pinhole sizes. FWHM (Full Width at Half Maxima) refers to the width when the function value of the point spread function reaches half of its maximum value, which can reflect the resolution capability of the rescanning confocal measurement system during imaging. In simple terms, the smaller the FWHM, the higher the resolution of the rescanning confocal measurement system and the clearer the image. Figure 5 The FWHM of the conventional confocal microscope and the rescanning confocal measurement system are shown as the pinhole radius increases. In the case of a large diameter pinhole, the FWHM of the conventional confocal microscope is higher than that of the rescanning confocal measurement system. times.

[0081] In the present invention, the lateral resolution of the rescanning confocal measurement system with a large diameter pinhole is equal to the lateral resolution of the conventional confocal microscope with a small diameter pinhole. Therefore, the rescanning confocal measurement system significantly improves the lateral resolution while maintaining the slicing capability (axial resolution) of the conventional confocal microscope.

[0082] In order to dynamically adjust the weight of the defocused image based on the focal length, the focus image is reconstructed by images obtained at four different defocus positions, four defocused images are obtained by the first camera to the fourth camera, and a focus image is generated by weighted averaging of the four defocused images. This focus image is more accurate, more reliable, and more suitable for human eye observation or further processing by a computer.

[0083] In order to achieve focal length-based weighting, a focal length weighting function is designed, which is associated with the focal length difference. The larger the defocus amount, the smaller the weight of the defocused image should be. Focal length weighting function It can be expressed as:

[0084]

[0085]

[0086] In the formula, is a small constant to avoid division by zero error when the focal length difference is zero; is the defocus amount of the first camera 18; is the defocus amount of the second camera 20; is the defocus amount of the third camera 22; is the defocus amount of the fourth camera 16 .

[0087] The clarity of a defocused image is closely related to the edge information of the defocused image. Generally, the richer the edge information, the clearer the defocused image. The clarity of a defocused image can be evaluated by calculating the gradient (edge ​​information) of the defocused image. The gradient reflects the rate of change of the defocused image in space, and a clear defocused image usually has a higher gradient value. It can be expressed by the standard deviation of the gradient:

[0088]

[0089]

[0090]

[0091] in, is the total number of pixels in the out-of-focus image; is the gradient of each defocused image along the horizontal direction; is the gradient of each defocused image along the axial direction.

[0092] Since the clarity of the out-of-focus image is directly related to the focal length, the out-of-focus image with high clarity should have a larger weight. Taking the focal length and clarity into consideration, the final weight of the out-of-focus image can be expressed as:

[0093]

[0094] The four defocused images are fused using the weighted average method, and the fused focus image can be obtained by the following formula:

[0095]

[0096] in, is the weight of each out-of-focus image, indicating the combined effect of focus and clarity.

[0097] Through the above-mentioned defocused image fusion, the overall quality of imaging is significantly improved, especially in three-dimensional imaging and observation of complex samples. By fusing images from different focal planes together, the clarity of the image can be enhanced, so that the details of each depth level can be clearly presented, avoiding the blur problem caused by single-focus imaging. This fusion not only improves the contrast and detail performance of the image, but also effectively reduces out-of-focus noise, improves the signal-to-noise ratio, and ensures that the image is more realistic and accurate. The fusion of multi-focus images also enhances the depth penetration ability of the image, making the observation of multi-level structures or deep samples clearer. In addition, defocused image fusion also improves the resolution and sensitivity of the imaging system, especially in low light or high noise environments, which can significantly improve the imaging effect. Therefore, defocused image fusion not only improves the visual quality of the image, but also provides strong technical support for precise analysis and three-dimensional reconstruction.

[0098] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0099] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rescanning confocal measurement system based on dynamic focus weighted fusion, characterized in that: It includes a laser light source, a dichroic mirror, a first scanning oscillating mirror, a first scanning lens, an aberration correction lens, an objective lens, a piezoelectric ceramic objective lens driver, a platform, a second scanning lens, a pinhole, a third scanning lens, a second scanning oscillating mirror, a first beam splitter, a second beam splitter, a third beam splitter, a first pre-focus detector, a second pre-focus detector, a first post-focus detector, and a second post-focus detector; wherein, The sample to be tested is placed on the platform, and the objective lens is placed on the piezoelectric ceramic objective lens driver. The piezoelectric ceramic objective lens driver drives the objective lens to move axially to achieve measurement of different sections of the sample to be tested. The dichroic mirror is arranged in the emission direction of the laser light source, the first scanning lens, the aberration correction lens, and the objective lens are arranged in sequence between the first scanning swing mirror and the sample to be measured, and the first scanning swing mirror is arranged in the reflection direction of the dichroic mirror; the second scanning lens, the pinhole, the third scanning lens, and the second scanning swing mirror are arranged in sequence in the transmission direction of the dichroic mirror, the first beam splitter is arranged in the reflection direction of the second scanning swing mirror, the second beam splitter and the third beam splitter are arranged in the reflection direction and the transmission direction of the first beam splitter respectively, the first pre-focus detector and the second pre-focus detector are arranged in the reflection direction and the transmission direction of the second beam splitter, and the first post-focus detector and the second post-focus detector are arranged in the reflection direction and the transmission direction of the third beam splitter; The first pre-focus detector includes a fourth scanning lens and a first camera, the second pre-focus detector includes a fifth scanning lens and a second camera, the first post-focus detector includes a sixth scanning lens and a third camera, and the second post-focus detector includes a seventh scanning lens and a fourth camera; Any one of the fourth scanning lens to the seventh scanning lens and the first scanning lens, the second scanning lens, the third scanning lens, the first scanning oscillating mirror, and the second scanning oscillating mirror satisfy the following relationship: in, represents the relative mechanical magnification of the rescanning confocal measurement system based on dynamic focus weighted fusion, represents the mechanical magnification of the rescanning confocal measurement system, represents the optical magnification of the rescanning confocal measurement system, represents the focal length of the first scanning lens, represents the focal length of the second scanning lens, represents the focal length of the third scanning lens, represents the focal length of any one of the fourth scanning lens to the seventh scanning lens, represents the amplitude of the second scanning oscillating mirror, Represents the amplitude of the first scanning oscillating mirror.

2. The rescanning confocal measurement system based on dynamic focus weighted fusion according to claim 1, characterized in that: The first to fourth cameras are CMOS cameras or CCD cameras.

3. A rescanning confocal measurement method based on dynamic focus weighted fusion, implemented by using the rescanning confocal measurement system based on dynamic focus weighted fusion according to claim 1 or 2, characterized in that: The steps include: S1: The excitation light emitted by the laser light source is sequentially reflected by the dichroic mirror, swung by the first scanning oscillating mirror, focused by the first scanning lens, aberration corrected by the aberration correction lens, and focused by the objective lens before being incident on the sample to be tested. The emission light excited by the sample to be tested returns to the dichroic mirror along the original excitation light path, is transmitted by the dichroic mirror, focused by the second scanning lens, and is incident on the pinhole. After passing through the pinhole, it is collimated by the third scanning lens and swung by the second scanning oscillating mirror and is incident on the first beam splitter. The light is divided into reflected light and transmitted light by the first beam splitter. The reflected light is divided into pre-focus reflected light and pre-focus transmitted light by the second beam splitter. The pre-focus reflected light is incident on the second pre-focus detector. The pre-focus transmitted light is incident on the first pre-focus detector. The transmitted light is divided into post-focus reflected light and post-focus transmitted light by the third beam splitter. The post-focus reflected light is incident on the first post-focus detector. The post-focus transmitted light is incident on the second post-focus detector. A total of four defocused images are obtained. S2: Calculate the lateral intensity distribution of each defocused image; wherein the lateral intensity distribution of each defocused image The calculation formula is as follows: In the formula, is the intensity of the emitted light excited by the sample to be tested; The object plane position of the rescanning confocal measurement system; is the position of the first pre-focus detector, the second pre-focus detector, the first post-focus detector or the second post-focus detector relative to the laser light source; is the axial position of the sample to be tested; is the out-of-focus spread function; is the scanning position of the sample to be tested; is the point spread function of the excitation light; is the point spread function of the emitted light; is the relative mechanical magnification; is the transfer function of the pinhole; S3: According to the lateral intensity distribution of the four defocused images, the four defocused images are fused using the weighted average method to obtain a fused focus image; wherein the fusion formula of the defocused images is as follows: In the formula, is the fused focus image; is the weight of each defocused image; is the focal length weighting function for each out-of-focus image; is the clarity of each out-of-focus image; is the defocus amount for each camera; is a constant; is the gradient of each defocused image along the horizontal direction; is the vertical gradient of each defocused image; is the total number of pixels in each out-of-focus image.

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