CN119394993B - Rescanning confocal measurement system and measurement method based on dynamic focus weighted fusion - Google Patents
Rescanning confocal measurement system and measurement method based on dynamic focus weighted fusion Download PDFInfo
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Abstract
本发明涉及光学检测领域，尤其涉及一种基于动态焦点加权融合的重扫描共焦测量系统及测量方法，激光光源发射的激发光依次经二相色镜的反射、第一扫描摆镜的摆扫、第一扫描透镜的聚焦、像差校正透镜的像差校正、物镜的聚焦后入射至待测样品，待测样品激发出的发射光返回至二相色镜，经二相色镜的透射、第二扫描透镜的聚焦通过针孔，再经第三扫描透镜的准直、第二扫描摆镜的摆扫及三个分束器的分束，分别入射至两个焦前探测器和两个焦后探测器，共获得四幅离焦图像。本发明通过引入重扫描单元与图像融合方法，使得图像可以直接投射到四个不同焦平面的相机上，增强了图像的细节和清晰度，减少模糊和失焦区域，进而降低噪声影响，提升图像的整体质量。
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. 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. 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 is 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
The invention belongs to the technical field of optical detection, and particularly relates to a rescanning confocal measurement system and a measurement method based on dynamic focus weighted fusion.
Background
The traditional confocal microscope has a plurality of advantages, so that the confocal microscope is widely applied to the fields of material science, biomedicine, semiconductor manufacturing and the like, and can inhibit scattered light from a non-focal plane due to the high resolution and optical slicing capability, so that the transverse resolution and axial resolution of an image can be remarkably improved, and the confocal microscope is particularly outstanding in researching the three-dimensional structure and surface morphology of a sample. In addition, the confocal microscope can eliminate the interference of out-of-focus light, so that the generated image has higher contrast and is suitable for researching the microstructure in a complex sample. The real-time imaging function enables the confocal microscope to rapidly generate images of dynamic processes, such as living cell imaging, and the characteristic of reducing sample damage is also particularly suitable for living imaging. The confocal microscope can also be compatible with various marking technologies, is widely applied to marking and detection of cells and molecules, and further improves the application value of the confocal microscope in biological research.
In confocal rescanning techniques, there are still some problems in the lateral scanning process. First, there may be non-uniformity in signal intensity, particularly when the scan area is large, the imaging brightness may change, affecting the overall contrast and detail performance of the image. Second, lateral distortion may occur during scanning, especially when the imaging system is unable to completely correct the optical distortion, resulting in geometric distortion of the image. In addition, single focus imaging often only presents a part of details of a sample, especially a sample with a large depth, and when out-of-focus image fusion is not performed, blurring and loss of hierarchical information are easily caused. Lack of out-of-focus image fusion may also result in reduced image contrast, especially when the focus position deviates, and the details of certain areas may be distorted.
Disclosure of Invention
In view of the above, the invention aims to provide a dynamic focus weighted fusion-based rescanning confocal measurement system and a measurement method, so as to solve the problems of insufficient image quality and insufficient image details in the conventional confocal rescanning technology.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
A rescanning confocal measurement system based on dynamic focus weighted fusion comprises a laser light source, a dichroic mirror, a first scanning swing mirror, a first scanning lens, an aberration correction lens, an objective lens, a piezoceramic objective lens driver, a platform, a second scanning lens, a pinhole, a third scanning lens, a second scanning swing 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 measured is placed on a platform, the objective lens is placed on a piezoelectric ceramic objective lens driver, and the objective lens is driven by the piezoelectric ceramic objective lens driver to axially move, so that the measurement of different tangential planes of the sample to be measured is realized;
The second scanning lens, the pinhole, the third scanning lens and the second scanning swing mirror are sequentially arranged in the transmission direction of the two-phase 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 respectively arranged in the reflection direction and the transmission direction of the first beam splitter, the first-focus front detector and the second-focus front detector are arranged in the reflection direction and the transmission direction of the second beam splitter, and the first-focus rear detector and the second-focus rear detector are arranged in the reflection direction and the transmission direction of the third beam splitter.
Further, the first pre-focus detector comprises a fourth scanning lens and a first camera, the second pre-focus detector comprises a fifth scanning lens and a second camera, the first post-focus detector comprises a sixth scanning lens and a third camera, and the second post-focus detector comprises a seventh scanning lens and a fourth camera.
Further, any one of the fourth to seventh scanning lenses satisfies the following relationship with the first, second, third, first and second scanning swing mirrors:
Wherein, Shows relative mechanical magnification of a rescanning confocal measurement system based on dynamic focus weighted fusion,Representing the mechanical magnification of the rescanning confocal measurement system,Indicating the optical magnification of the rescanning confocal measurement system,Representing the focal length of the first scanning lens,Representing the focal length of the second scanning lens,Representing the focal length of the third scanning lens,Represents the focal length of any one of the fourth to seventh scan lenses,Representing the amplitude of the second scanning swing mirror,Representing the amplitude of the first scanning swing mirror.
Further, the first to fourth cameras are CMOS cameras or CCD cameras.
The rescanning confocal measurement method based on dynamic focus weighted fusion is realized by the rescanning confocal measurement system based on dynamic focus weighted fusion, and comprises the following steps:
The method comprises the steps that S1, excitation light emitted by a laser light source is sequentially reflected by a dichroic mirror, swept by a first scanning swing mirror, focused by an aberration correction lens, focused by an objective lens and then is incident on a sample to be tested, emission light excited by the sample to be tested returns to the dichroic mirror along an original excitation light path, is transmitted by the dichroic mirror, focused by a second scanning lens and then is incident on a pinhole, is collimated by a third scanning lens and swept by a second scanning swing mirror and then is incident on a first beam splitter, is divided into reflected light and transmitted light by the first beam splitter, the reflected light is divided into reflected light before focus and transmitted light before focus by the second beam splitter, the reflected light before focus is incident on a first detector before focus, the transmitted light is divided into reflected light after focus and transmitted light after focus by a third beam splitter, the reflected light after focus is incident on a first detector after focus, and the transmitted light after focus is incident on a second detector after focus, and four focus images are obtained;
s2, calculating the transverse intensity distribution of each defocused image, wherein the transverse intensity distribution of each defocused image The calculation formula of (2) is as follows:
In the formula, The intensity of the emitted light excited for the sample to be measured; an object plane position for a rescanning confocal measurement system; The position of the first focal front detector, the second focal front detector, the first focal rear detector or the second focal rear detector relative to the laser light source; The axial position of the sample to be measured; is a defocus diffusion function; scanning the sample to be measured; Is the point spread function of the excitation light; A point spread function for the emitted light; Is a relative mechanical magnification; is the transfer function of the pinhole;
s3, fusing the four defocused images by using a weighted average method according to the transverse intensity distribution of the four defocused images to obtain a fused focus image, wherein the fusion formula of the defocused images is as follows:
In the formula, The fused focus image; a weight for each out-of-focus image; a focal length weighting function for each out-of-focus image; Sharpness for each out-of-focus image; Defocus amount for each camera; Is a constant; A gradient in the lateral direction for each out-of-focus image; A gradient along the axial direction for each out-of-focus image; Is the total number of pixels in each out-of-focus image.
Compared with the prior art, the invention has the following beneficial effects:
The invention obviously improves the integral quality of imaging by introducing a rescanning unit and a defocusing 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 definition of the images can be enhanced, so that details of each depth level can be clearly presented, and the problem of blurring caused by single-focus imaging is avoided. The 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 true and accurate. The fusion of the multi-focal images also enhances the depth penetration of the images, making the observation of multi-layer structures or deep samples clearer. In addition, the resolution and sensitivity of the imaging system are improved through defocusing image fusion, and the imaging effect can be remarkably improved particularly in a low-light or high-noise environment. Therefore, the defocused image fusion not only improves the visual quality of the image, but also provides powerful technical support for accurate analysis and three-dimensional reconstruction.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute an undue limitation on the invention. In the drawings:
FIG. 1 is a schematic diagram of a rescanning confocal measurement system based on dynamic focus weighted fusion according to an embodiment of the invention;
FIG. 2 is a schematic diagram of a separation process of light spots and a separation process of signal intensity distribution in a rescanning process according to an embodiment of the invention;
FIG. 3 is a schematic view of the lateral intensity of a confocal microscope according to an inventive embodiment of the present invention;
FIG. 4 is a schematic view of the lateral intensity of a rescanning confocal measurement system according to an inventive embodiment of the invention;
fig. 5 is a graph showing FWHM of a rescanning confocal measurement system and confocal microscope as a function of pinhole radius according to an inventive embodiment of the present invention.
The reference numerals illustrate a stage 1, a sample 2 to be measured, a piezoceramic 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 fourth camera 16, a fourth scanning lens 17, a first camera 18, a fifth scanning lens 19, a second camera 20, a sixth scanning lens 21, a third camera 22, and a seventh scanning lens 23.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
In the description of the invention, it should be understood that the terms "center," "longitudinal," "transverse," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like indicate orientations or positional relationships that are based on the orientation or positional relationships shown in the drawings, merely to facilitate describing the invention and simplify the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be configured and operate in a particular orientation, and therefore should not be construed as limiting the invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first", "a second", etc. may explicitly or implicitly include one or more such feature. In the description of the invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In the description of the present invention, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected, mechanically connected, electrically connected, directly connected, indirectly connected via an intervening medium, or in communication between two elements. The specific meaning of the above terms in the creation of the present invention can be understood by those of ordinary skill in the art in a specific case.
The invention will be described in detail below with reference to the drawings in connection with embodiments.
As shown in fig. 1, the rescanning confocal measurement system based on dynamic focus weighted fusion provided by the inventive embodiment includes a laser light source, a stage 1, a piezoceramic 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.
The sample 2 to be measured is placed on the platform 1, the objective lens 5 is placed on the piezoelectric ceramic type objective lens driver 3, and the objective lens 5 is driven by the piezoelectric ceramic type objective lens driver 3 to axially move the sample 2 to be measured, so that the measurement of different tangential planes of the sample 2 to be measured is realized.
The two-phase mirror 8 is arranged in the outgoing direction of the laser light source, the first scanning lens 6, the aberration correcting lens 4 and the objective lens 5 are sequentially arranged between the first scanning swing mirror 7 and the sample 2 to be measured, the first scanning swing mirror 7 is arranged in the reflection direction of the two-phase mirror 8, the second scanning lens 9, the pinhole 10, the third scanning lens 11 and the second scanning swing mirror 12 are sequentially arranged in the transmission direction of the two-phase mirror 8, the first light splitter 13 is arranged in the reflection direction of the second scanning swing mirror 12, the second light splitter 14 is arranged in the reflection direction of the first light splitter 13, the third light splitter 13 is arranged in the transmission direction of the first light splitter 13, the first pre-focus detector is arranged in the reflection direction of the second light splitter 14, the second pre-focus detector is arranged in the transmission direction of the second light splitter 14, the first post-focus detector is arranged in the reflection direction of the third light splitter 15, and the second post-focus detector is arranged in the transmission direction of the third light splitter 15.
The first pre-focus detector comprises a fourth scanning lens 17 and a first camera 18, the second pre-focus detector comprises a fifth scanning lens 19 and a second camera 20, the first post-focus detector comprises a sixth scanning lens 21 and a third camera 22, and the second post-focus detector comprises a seventh scanning lens 23 and a fourth camera 16. Defocus is achieved by adjusting the positions of the first camera 18, the second camera 20, the third camera 22, the fourth camera 16. The first camera 18, the second camera 20, the third camera 22, and the fourth camera 16 use a CCD camera or a CMOS camera.
The working principle of the dynamic focus weighted fusion-based rescanning confocal measurement system is that excitation light emitted by a laser light source is sequentially reflected by a dichroic mirror 8, swept by a first scanning oscillating mirror 7, focused by a first scanning lens 6, aberration corrected by an aberration correction lens 4 and focused by an objective lens 5 and then is incident to a sample 2 to be measured, emission light excited by the sample 2 to be measured returns to the dichroic mirror 8 along an original excitation light path, is transmitted by the dichroic mirror 8 and focused by a second scanning lens 9 and then is incident to a pinhole 10, stray light is filtered through the pinhole 10 and then is collimated by a third scanning lens 11, swept by a second scanning oscillating mirror 12 and then is incident to a first beam splitter 13, reflected light is divided into reflected light and transmitted light by the first beam splitter 14, the reflected light is divided into reflected light before focus and transmitted light before focus by a second beam splitter 14, the reflected light before focus is received by a second camera 20, the transmitted light before focus is received by a first camera 18, the transmitted light is divided into reflected light after focus, the reflected light after focus is transmitted by a third beam splitter, the reflected light after focus is reflected by a fourth scanning lens 21, the reflected after focus is received by a third camera 18, the focus lens is driven by a fourth lens 23, and finally is transmitted by a fourth scanning lens 23, and finally the focus is fused by a fourth scanning lens 2, the image is driven by a fourth scanning lens 23, the focus is fused, and the image is finally obtained, and the image is subjected to the weighted and the image is fused.
As an example of the present invention, the sample 2 to be measured is a fluorescent material to be measured, and the emitted light is fluorescence of the excited fluorescent material to be measured.
The laser light source, the aberration correcting lens 4, the objective lens 5 and the two-phase mirror 8 form a standard confocal microscope, the first scanning lens 6, the first scanning oscillating mirror 7, the two-phase mirror 8, the second scanning lens 9, the pinhole 10, the third scanning lens 11 and the second scanning oscillating mirror 12 form a rescanning unit, and the first scanning oscillating mirror 7 and the second scanning oscillating mirror 12 have double scanning functions, namely, the oscillating scanning of excitation light and the oscillating scanning of emission light are realized.
The first scanning swing mirror 7 guides excitation light onto the sample 2 to be measured in a scanning manner and guides emission light excited by the sample 2 to be measured to the pinhole 10, and the second scanning swing 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.
The first scanning swing mirror 7 and the second scanning swing mirror 12 perform synchronous scanning movement under the same frequency and phase, but the angular amplitude of the scanning movement of the first scanning swing mirror 7 and the second scanning swing mirror 12 can be independently adjusted.
Taking the defocused image obtained by the first pre-focusing detector as an example, the other three defocused images are known in the same way. One novelty of the present invention is to control the amplitude of the first 7 and second 12 scanning pendulums so that an additional mechanical magnification is introduced over the optical magnification of a conventional confocal microscope. The mechanical magnification is determined by the amplitude of the second scanning oscillating mirror 12Amplitude of the first scanning swing mirror 7And focal lengths of the first scanning lens 6 and the fourth scanning lens 17、And (3) determining.
Mechanical magnification of rescanning confocal measurement systemThe method comprises the following steps:
when the first scanning swing mirror 7 and the second scanning swing mirror 12 are arranged at fixed positions, the optical magnification of the rescanning confocal measurement system The method comprises the following steps:
Wherein, The focal length of the first scanning lens 6 is indicated,The focal length of the second scanning lens 9 is indicated,The focal length of the third scanning lens 11 is indicated,The focal length of the fourth scanning lens 17 is shown.
When the amplitude of the second scanning swing mirror 12 exactly compensates the amplitude of the first scanning swing mirror 7, and the sample image to be measured on the chip of the first camera 18 does not move during scanning. Relative mechanical magnification of rescanning confocal measurement systemThe definition is as follows:
when (when) When the rescanning confocal measurement system has exactly the same resolution as a conventional confocal microscope, both the lateral and axial resolution of the obtained defocused image depend on the diameter of the pinhole 10, which is the same as a conventional confocal microscope. Mechanical magnification of the rescanning confocal measurement system if the angular amplitude of the second scanning swing mirror 12 is doubledWill 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 first camera 18 chip depends only on the optical magnification. However, when the second scanning oscillating mirror 12 moves at double amplitude relative to the first scanning oscillating mirror 7, this additional "scanning" is shown in FIG. 2 below, the light moves at a constant speed on the first camera 18 chip, and can therefore be moved at a constant speedThe coefficients of the multiples are smeared out of the molecular image. Therefore, the distance between two molecules is doubled due to the double scanning of the secondary scanning, and the resolution of the defocused image is improvedMultiple times.
Resolution of a rescanning confocal measurement system with relative mechanical magnificationSemi-quantitative analysis was performed for the improvement of (c). Spot widthCan be expressed as relative mechanical magnificationIs a function of:
In the formula, In order to emit a width of the light spot,Is the width of the excitation light spot.
At the position ofIn the time-course of which the first and second contact surfaces,I.e. the resolution of the rescanning confocal measurement system is determined only by the excitation light. If the widths of the excitation light spot and the emission light spot are equal, i.eAt this time, the first and second electrodes are connected,ThenResolution of the rescanning confocal measurement system may be improvedMultiple times.
The axial and lateral resolution of a conventional confocal microscope is determined by the diameter of the pinhole 10. In the rescanning confocal measurement system provided by the invention, the pinhole 10 only affects the axial resolution, not the lateral resolution. The maximum axial resolution of a conventional confocal microscope is obtained by setting the diameter of the pinhole 10 to one Airy Unit (AU). However, for lateral resolution, conventional confocal microscopy and rescanning confocal measurement systems have completely different responses to the diameter of pinhole 10. The lateral resolution in a rescanning confocal measurement system is independent of the diameter of the pinhole 10, whereas in a conventional confocal microscope the lateral resolution is only increased when the pinhole size is less than 1 airy spot unit, but results in a loss of the detected fluorescent signal.
According to the invention, the transverse resolution is improved by utilizing a pure physical mode, the information of the sample to be detected is firstly obtained through scanning, then the information of the sample to be detected is projected onto the camera of the detector through rescanning, the movement speed of rescanning is faster than that of scanning, and the information of the sample to be detected can be more clearly distinguished on an image.
Transverse intensity distribution of rescanning confocal measurement systemThe method comprises the following steps:
wherein i=1, 2, 3, 4, For the intensity of the emitted light of the sample to be measured,For scanning the object plane position of the confocal measurement system,For the axial position of the sample to be measured,The positions of the first focal front detector, the second focal front detector, the first focal rear detector and the second focal rear detector relative to the laser light source are shown.
Off-focus diffusion functionThe method comprises the following steps:
In the formula, Scanning the sample to be measured; Is the point spread function of the excitation light; A point spread function for the emitted light; is the transfer function of the pinhole 10.
For comparison with a confocal microscope, the transverse intensity distribution of the confocal microscope is obtainedThe method comprises the following steps:
wherein, the defocus diffusion function is:
In the formula, Is the position of the pinhole 10; As a result of the position of the first camera 18, Is thatIs a fourier transform of the above.
As shown in fig. 3 and 4, by simulation analysis, pinholes of different diameters R are provided, the width of a conventional confocal microscope is gradually narrowed at the time of small-diameter pinholes, and a rescanning confocal microscope can achieve the same degree of width narrowing at any diameter pinhole.
The main advantage of the present invention introduced in the rescanning unit is to provide lateral resolution, maintaining good signal-to-noise ratio even for medium or large pinhole sizes. FWHM (Full WIDTH AT HALF Maxima, full width at half maximum) refers to the width at which the function value of the point spread function reaches half maximum, and can reflect the resolution of the rescanning confocal measurement system at the time of imaging. In brief, the smaller the FWHM, the higher the resolution of the rescanning confocal measurement system and the sharper the image. FIG. 5 shows the FWHM of a conventional confocal microscope and a rescanning confocal measurement system as the pinhole radius increases, in the case of a large diameter pinhole, the FWHM of a conventional confocal microscope is that of a rescanning confocal measurement systemMultiple times.
In the invention, the transverse resolution of the rescanning confocal measurement system of the large-diameter pinhole is equal to that of a traditional confocal microscope of the small-diameter pinhole. Therefore, the rescanning confocal measurement system greatly improves the lateral resolution while maintaining the sectioning capability (axial resolution) of a conventional confocal microscope.
In order to dynamically adjust the weighting of the defocused images based on the focal length, the focus images are reconstructed through the images obtained at the four different defocused positions, four defocused images are obtained through the first camera to the fourth camera, and one focus image is generated through the four defocused images in a weighted average mode, so that the focus images are more accurate and reliable, and are more suitable for human eye observation or further processing by a computer.
To achieve focus-based weighting, a focus weighting function is designed that is associated with the focus difference. The greater the defocus amount, the smaller the weight of the defocus image should be. Focal length weighting functionCan be expressed as:
In the formula, The constant is smaller, and zero dividing errors when the focal length difference is zero are avoided; An amount of defocus for the first camera 18; is the defocus amount of the second camera 20; A defocus amount for the third camera 22; is the defocus amount of the fourth camera 16.
The sharpness of the defocused image is closely related to the edge information of the defocused image, and generally the richer the edge information is, the sharper the defocused image is. The sharpness of the out-of-focus image can be evaluated by calculating the gradient (edge information) of the out-of-focus image. The gradient reflects the rate of change of the out-of-focus image in space, with sharp out-of-focus images typically having a higher gradient value. Sharpness of out-of-focus imageThis can be expressed by the standard deviation of the gradient:
Wherein, Is the total number of pixels in the out-of-focus image; A gradient in the lateral direction for each out-of-focus image; a gradient in the axial direction for each out-of-focus image.
Since the sharpness of an out-of-focus image is directly related to the focal length, an out-of-focus image with high sharpness should be weighted more heavily. Considering focus and sharpness in combination, the final weight of an out-of-focus image can be expressed as:
The four defocused images are fused by using a weighted average method, and the fused focus image can be obtained by the following formula:
Wherein, The combined impact of focal length and sharpness is expressed for the weight of each out-of-focus image.
Through the defocused image fusion, the overall quality of imaging is remarkably improved, and the method has important advantages in three-dimensional imaging and observation of complex samples. By fusing images from different focal planes together, the definition of the images can be enhanced, so that details of each depth level can be clearly presented, and the problem of blurring caused by single-focus imaging is avoided. The 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 true and accurate. The fusion of the multi-focal images also enhances the depth penetration of the images, making the observation of multi-layer structures or deep samples clearer. In addition, the resolution and sensitivity of the imaging system are improved through defocusing image fusion, and the imaging effect can be remarkably improved particularly in a low-light or high-noise environment. Therefore, the defocused image fusion not only improves the visual quality of the image, but also provides powerful technical support for accurate analysis and three-dimensional reconstruction.
It should be appreciated that various forms of the flows shown above may be used to reorder, add, or delete steps. For example, the steps described in the present disclosure may be performed in parallel, sequentially, or in a different order, provided that the desired results of the technical solutions of the present disclosure are achieved, and are not limited herein.
The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives are possible, depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.
Claims (3)
1. A dynamic focus weighted fusion-based rescanning confocal measurement system is characterized by comprising a laser light source, a dichroic mirror, a first scanning swing 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 swing 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 measured is placed on a platform, the objective lens is placed on a piezoelectric ceramic objective lens driver, and the objective lens is driven by the piezoelectric ceramic objective lens driver to axially move, so that the measurement of different tangential planes of the sample to be measured is realized;
The second scanning lens, the pinhole, the third scanning lens and the second scanning swing mirror are sequentially arranged in the transmission direction of the two-phase 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 respectively arranged in the reflection direction and the transmission direction of the first beam splitter, the first front-focus detector and the second front-focus detector are arranged in the reflection direction and the transmission direction of the second beam splitter, and the first rear-focus detector and the second rear-focus detector are arranged in the reflection direction and the transmission direction of the third beam splitter;
The first pre-focus detector comprises a fourth scanning lens and a first camera, the second pre-focus detector comprises a fifth scanning lens and a second camera, the first post-focus detector comprises a sixth scanning lens and a third camera, and the second post-focus detector comprises 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 swing mirror and the second scanning swing mirror satisfy the following relations:
Wherein, Shows relative mechanical magnification of a rescanning confocal measurement system based on dynamic focus weighted fusion,Representing the mechanical magnification of the rescanning confocal measurement system,Indicating the optical magnification of the rescanning confocal measurement system,Representing the focal length of the first scanning lens,Representing the focal length of the second scanning lens,Representing the focal length of the third scanning lens,Represents the focal length of any one of the fourth to seventh scan lenses,Representing the amplitude of the second scanning swing mirror,Representing the amplitude of the first scanning swing mirror.
2. The dynamic focus weighted fusion-based rescan confocal measurement system of claim 1 wherein the first to fourth cameras are CMOS cameras or CCD cameras.
3. A dynamic focus weighted fusion-based rescanning confocal measurement method implemented by the dynamic focus weighted fusion-based rescanning confocal measurement system according to claim 1 or 2, comprising the steps of:
The method comprises the steps that S1, excitation light emitted by a laser light source is sequentially reflected by a dichroic mirror, swept by a first scanning swing mirror, focused by an aberration correction lens, focused by an objective lens and then is incident on a sample to be tested, emission light excited by the sample to be tested returns to the dichroic mirror along an original excitation light path, is transmitted by the dichroic mirror, focused by a second scanning lens and then is incident on a pinhole, is collimated by a third scanning lens and swept by a second scanning swing mirror and then is incident on a first beam splitter, is divided into reflected light and transmitted light by the first beam splitter, the reflected light is divided into reflected light before focus and transmitted light before focus by the second beam splitter, the reflected light before focus is incident on a first detector before focus, the transmitted light is divided into reflected light after focus and transmitted light after focus by a third beam splitter, the reflected light after focus is incident on a first detector after focus, and the transmitted light after focus is incident on a second detector after focus, and four focus images are obtained;
s2, calculating the transverse intensity distribution of each defocused image, wherein the transverse intensity distribution of each defocused image The calculation formula of (2) is as follows:
In the formula, The intensity of the emitted light excited for the sample to be measured; an object plane position for a rescanning confocal measurement system; The position of the first focal front detector, the second focal front detector, the first focal rear detector or the second focal rear detector relative to the laser light source; The axial position of the sample to be measured; is a defocus diffusion function; scanning the sample to be measured; Is the point spread function of the excitation light; A point spread function for the emitted light; Is a relative mechanical magnification; is the transfer function of the pinhole;
s3, fusing the four defocused images by using a weighted average method according to the transverse intensity distribution of the four defocused images to obtain a fused focus image, wherein the fusion formula of the defocused images is as follows:
In the formula, The fused focus image; a weight for each out-of-focus image; a focal length weighting function for each out-of-focus image; Sharpness for each out-of-focus image; Defocus amount for each camera; Is a constant; A gradient in the lateral direction for each out-of-focus image; A gradient along the longitudinal direction for each out-of-focus image; Is the total number of pixels in each out-of-focus image.
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