WO2019075680A1 - 视觉辅助的傅里叶叠层成像位置标定方法及装置 - Google Patents

视觉辅助的傅里叶叠层成像位置标定方法及装置 Download PDF

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WO2019075680A1
WO2019075680A1 PCT/CN2017/106786 CN2017106786W WO2019075680A1 WO 2019075680 A1 WO2019075680 A1 WO 2019075680A1 CN 2017106786 W CN2017106786 W CN 2017106786W WO 2019075680 A1 WO2019075680 A1 WO 2019075680A1
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spatial
image
coordinates
dimensional coordinates
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刘晓利
李成
汤其剑
彭翔
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Shenzhen University
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    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/80—Analysis of captured images to determine intrinsic or extrinsic camera parameters, i.e. camera calibration

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  • the invention belongs to the field of microscopic imaging technology, and in particular relates to a visual aided Fourier laminate imaging position calibration method and device.
  • Microscopes have made it possible for humans to observe smaller objects since the day of their invention.
  • the direct change is that the field of view that the observer can observe becomes smaller.
  • the commonly used method is to realize a large-scale scanning by using a precision electric platform and combined with an image processing method, but this method requires very precise scanning and the operation process is very complicated. How to get large field of view and high resolution images at the same time has been plaguing many researchers engaged in microscopy.
  • the breakthrough in this regard should belong to the Fourier stacked imaging technology first proposed and successfully achieved large field of view and super-resolution imaging.
  • a typical Fourier stacked imaging system is implemented on the basis of a conventional microscope device, replacing the microscope's own illumination source with a programmable LED array, and controlling the sample by controlling the brightness and darkness of each LED on the LED array. Lighting in different directions.
  • the image obtained by the LED illumination sample at each position is the imaging result limited by the numerical aperture of the objective lens, and the image has the high frequency information loss of the sample, which we call a low resolution image.
  • Fourier stacked imaging technology is to obtain a plurality of low-resolution images containing different angle information of samples by sequentially lighting each LED on the illumination array, and then adopting synthetic aperture and phase recovery method in the Fourier domain of the image. To recover high resolution information from the sample.
  • the resolution of the reconstructed high-resolution image depends on the sum of the objective numerical aperture and the numerical aperture of the illumination, so from this perspective, Fourier stacked imaging technology can break the limitation of the numerical aperture of the traditional microscope objective, and finally realize the big vision. Field and high resolution imaging.
  • the implementation of Fourier stacked imaging technology is to find the spatial frequency corresponding to each different position LED in the high-resolution spectrum to be recovered, and take the corresponding position in the high-resolution spectrum with the numerical aperture of the objective lens as the radius.
  • the spectral aperture is replaced by a low-resolution image acquired at the corresponding LED position to achieve phase recovery.
  • the quality of the final restored high-resolution image is greatly affected by the positioning error of the spatial LED array, which is represented by the spatial frequency corresponding to each LED in the conventional Fourier stack.
  • the spatial frequencies at other locations are calculated based on this. Therefore, once there is a positional deviation between the center LED and the center of the micro-system, the position of the LED space of the entire illumination area is deviated, so that the center position of each sub-aperture spectrum in the high-resolution spectrum to be recovered is not Accurate, its phase recovery is also difficult to get an accurate result. Therefore, in order to obtain better recovery quality results, it is necessary to solve the problem of spatial position deviation of the LED array in the Fourier stack.
  • the invention provides a visual aided Fourier laminate imaging position calibration method and device, which aims to solve the problem that the LED array and the microscopic system center in the traditional Fourier stacked imaging optical path are difficult to precisely adjust and align, and the high resolution is to be restored.
  • the problem of degraded image quality is a problem that the LED array and the microscopic system center in the traditional Fourier stacked imaging optical path are difficult to precisely adjust and align, and the high resolution is to be restored.
  • the invention provides a visually assisted Fourier stacked imaging position calibration method, which is applied to a Fourier stacked imaging position calibration system, the calibration system comprising: a leaf stack imaging device and a vision assisted binocular camera, the Fourier stacked imaging device comprising a microscopic image acquisition camera, a microscope, a stage, a resolution plate with circular landmarks, and an LED array, a resolution plate is disposed on the stage, the microscopic image acquisition camera is located directly above the stage, the LED array is located directly below the stage, and is in a plane opposite to the stage Parallelly, the visual aid binocular camera comprises: a vision assisted left camera and a visual aid right camera, the visual assist left camera, the visual aid right camera being located directly in front of the Fourier stacked imaging device, the method comprising :
  • Step S1 using the microscopic image acquisition camera to acquire an image of the resolution plate with the circular marker points, and extracting the center of the circular marker point in the image of the resolution plate by using a circular marker point extraction algorithm.
  • Step S2 acquiring an image by using the microscopic image acquisition camera to obtain image pixel coordinates of a microscopic field center point of the image, and using a coordinate measuring device to measure the resolution plate on which the circular marker point is attached.
  • the actual physical size coordinates of each set of elements of the first level, and the actual physical state of the center of the microscopic field of view and the center of the circular mark point is calculated by establishing a relationship between the image coordinate system and the actual physical size coordinate system. Size coordinate
  • Step S3 using the vision-assisted binocular camera to capture an image when the LED array is not placed under the resolution panel, thereby obtaining each LED in the LED array in the visual aid binocular Image coordinates in the camera, the first spatial three-dimensional coordinates of each LED of the LED array are calculated from the image coordinates of each LED and the calibration parameters of the visual aid binocular camera pre-calibrated;
  • Step S4 using the vision assisting camera to acquire an image of the resolution plate with the circular marker point in the focus position and an image of the LED array placed under the resolution panel, and combined with the pre-calibrated
  • the calibration parameter of the vision assisted binocular camera calculates a spatial three-dimensional coordinate of the center of the circular marker point in the focus position and a second spatial three-dimensional coordinate of the unoccluded LED array in the focus position;
  • Step S5 calculating a spatial three-dimensional coordinate of the center of the microscopic field of view according to the spatial three-dimensional coordinates of the center of the circular marker point and the relationship between the center of the microscopic field of view and the actual physical size coordinate of the center of the circular marker point;
  • the position coordinates of each LED in the high-resolution spectrum to be recovered are calculated by combining the spatial three-dimensional coordinates of the microscopic field center and the calibration result of the LED array in the illumination area.
  • the present invention also provides a visually assisted Fourier stacked imaging position calibration device for use in a Fourier stacked imaging position calibration system, the calibration system comprising: a superimposed stacked imaging device and a visual aided binocular camera, the Fourier stacked imaging device comprising a microscopic image acquisition camera, a microscope, a stage, a resolution plate with circular landmarks, and an LED array
  • the resolution plate is placed on the stage
  • the microscopic image acquisition camera is located directly above the stage
  • the LED array is located directly below the stage and is opposite to the stage Parallel to the plane
  • the visual aid binocular camera comprises: a vision assisted left camera and a visual aid right camera, the vision assisted left camera, the visual aid right camera being located directly in front of the Fourier stack imaging device
  • the device include:
  • An image pixel coordinate extraction module configured to acquire an image of the resolution plate with a circular marker point by using the microscopic image acquisition camera, and extract an image circle of the resolution plate according to a circular marker point extraction algorithm Image pixel coordinates of the center of the mark point;
  • An actual physical size coordinate calculation module configured to acquire an image by using the microscopic image acquisition camera to obtain image pixel coordinates of a microscopic field center point of the image, and measure the circular marker point by using a three coordinate device Calculating the microscopic field center point and the circular mark by establishing a relationship between the image coordinate system and the actual physical size coordinate system on the actual physical size coordinates of each set of elements in each stage of the resolution board.
  • a first calculation module configured to acquire, by the visual aid binocular camera, an image when the LED array is not placed under the resolution panel, thereby obtaining each LED in the LED array Image coordinates in the visual aid binocular camera, the first spatial three-dimensional coordinates of each LED of the LED array are calculated from the image coordinates of each LED and the calibration parameters of the visual aid binocular camera pre-calibrated;
  • a second calculation module configured to acquire, by the vision assisting camera, an image of the resolution plate with the circular mark point attached to the focus position and an image of the LED array placed under the resolution plate at this time, And calculating, in combination with the calibration parameters of the visually assisted binocular camera, which are pre-calibrated, the spatial three-dimensional coordinates of the center of the circular marker point in the focus position and the second spatial three-dimensional coordinates of the unoccluded LED array in the focus position;
  • a calibration module configured to calculate a spatial three-dimensional space of the center of the microscopic field of view according to a spatial three-dimensional coordinate of a center of the circular marker point and an actual physical size coordinate of a center of the microscopic field of view and a center of a circular marker point coordinate;
  • the position coordinates of each LED in the high-resolution spectrum to be recovered are calculated by combining the spatial three-dimensional coordinates of the microscopic field center and the calibration result of the LED array in the illumination area.
  • the present invention has the beneficial effects that the present invention provides a visually assisted Fourier stacked imaging position calibration method and apparatus, the method comprising: passing the traditional Fourier laminate imaging calculation before recovering The visual aided binocular system is built to collect the image of the LED array not placed under the resolution plate, and the first spatial three-dimensional coordinates of the LED array are calculated by combining the camera calibration parameters; and then the circular mark is attached on the resolution plate.
  • the point method finds the spatial three-dimensional coordinates of the center of the microscopic field of view; then combines the three-dimensional coordinates of the first space and finds the spatial three-dimensional coordinates of the LED array when the system is in the focus position by rotating translation transformation, and uses the spatial three-dimensional coordinates of the center of the microscopic field of view.
  • FIG. 1 is a schematic diagram showing the hardware structure of a Fourier stacked imaging position calibration system according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram of a vision assisted Fourier stacked imaging position calibration method according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of a calculation model of an image coordinate system and an actual physical size coordinate system according to an embodiment of the present invention
  • FIG. 4 is a schematic diagram showing the comparison between the spatial position of the LED array and the visually assisted calibration of the spatial position of the LED array in the conventional Fourier stacked imaging according to an embodiment of the present invention
  • FIG. 5(a) is a schematic diagram showing the result of reconstructing a resolution plate by a conventional Fourier stacked imaging method according to an embodiment of the present invention
  • FIG. 5(b) is a schematic diagram showing the result of reconstructing the resolution plate after visually assisted position calibration according to an embodiment of the present invention
  • FIG. 6(a) is a schematic diagram showing the results of reconstructing human blood cells by conventional Fourier laminate imaging according to an embodiment of the present invention
  • FIG. 6(b) is a schematic diagram showing the result of reconstructing human blood cells after visual aided position calibration according to an embodiment of the present invention
  • FIG. 7 is a schematic block diagram of a vision assisted Fourier stacked imaging position calibration apparatus according to an embodiment of the present invention.
  • the present invention provides a visually assisted Fourier stacked imaging position calibration method, which is applied to a Fourier stacked imaging position calibration system, as shown in FIG.
  • the calibration system comprises: a Fourier stacked imaging device 1 and a visual aid binocular camera 2, the Fourier stacked imaging device 1 comprising a microscopic image acquisition camera 11, a microscope 12 (the microscopic image acquisition camera described in the figure) 11 below, a lens 121 and an objective lens 122), a stage (not shown), a resolution plate 13 to which a circular mark is attached, and an LED array 14 are sequentially disposed, and the resolution plate 13 is placed in the On the stage, the microscopic image acquisition camera 11 is located directly above the stage, and the LED array 14 is located directly below the stage and parallel to the plane of the stage, the vision
  • the auxiliary binocular camera 2 includes a visual assist left camera and a visual assist right camera, the visual assist left camera and the visual assist right camera being located directly in front of the Fourier stacked imaging device.
  • the microscope 12 is of the type Olympus BX43, and is equipped with an Olympus plan apochromatic objective lens, the objective lens magnification is 4 times, the numerical aperture is 0.1; and the microscope 12 is equipped with an SCMOS camera.
  • the model is PCO.edge5.5, the resolution is 2560*2048, and the pixel size is 6.5um*6.5um.
  • the visual aid binocular camera 2 uses a Basler camera, model piA2400-17gc, and the resolution is 2448*2050.
  • the pixel size is 3.45um*3.45um; the visual aid binocular camera uses a Computar lens, model number M1214-MP2.
  • the resolution board 13 adopts a USAF1951 resolution board.
  • the Fourier stacked imaging position calibration method includes:
  • Step S1 using the microscopic image acquisition camera to acquire an image of the resolution plate with the circular marker points, and extracting the center of the circular marker point in the image of the resolution plate by using a circular marker point extraction algorithm.
  • Step S2 acquiring an image by using the microscopic image acquisition camera to obtain image pixel coordinates of a microscopic field center point of the image, and using a coordinate measuring device to measure the resolution plate on which the circular marker point is attached.
  • the actual physical size coordinates of each set of elements of the first level, and the actual physical state of the center of the microscopic field of view and the center of the circular mark point is calculated by establishing a relationship between the image coordinate system and the actual physical size coordinate system. Size coordinate
  • the microscopic field center point refers to the field of view center of the image acquired by the microscopic image acquisition camera.
  • FIG. 3 is a schematic diagram of a calculation model of an image coordinate system and an actual physical size coordinate system. Specifically, the relationship between the established image coordinate system and the actual physical size coordinate system is:
  • x 0 x center +(u 0 -u center ) ⁇ dx
  • y 0 y center +(v 0 -v center ) ⁇ dy
  • x circle x center +(u circle -u center ) ⁇ dx
  • y circle y center +(v circle -v center ) ⁇ dy
  • (x 0 , y 0 ) represents the actual physical size coordinate of the center of the microscopic field of view
  • (x circle , y circle ) represents the actual physical size coordinate of the center of the circular marker point
  • (u center , v center ) indicates the selected
  • (x center , y center ) represents the physical size coordinate of the selected image coordinate system
  • the physical size coordinate is obtained by a coordinate coordinate device
  • (u 0 , v 0 ) represents the microscopic field of view.
  • the image pixel coordinates of the center point (u circle , v circle ) represent the image pixel coordinates of the center of the circular marker point, and d x and d y are the unit physical dimensions in the x and y directions of the actual physical size coordinate system, respectively.
  • Step S3 using the vision-assisted binocular camera to capture an image when the LED array is not placed under the resolution panel, thereby obtaining each LED in the LED array in the visual aid binocular Image coordinates in the camera, image coordinates from each LED and pre-calibrated view
  • a calibration parameter of the auxiliary binocular camera calculates a first spatial three-dimensional coordinate of each LED of the LED array;
  • the LED array is not placed under the resolution plate, so the LED array is not blocked by the stage, and the visual auxiliary binocular camera can collect the complete LED array when it is lit. image.
  • the pre-calibration process of the vision-assisted binocular camera is: placing a target under the vision-assisted binocular camera, and acquiring the target image in several postures by using the vision-assisted binocular camera, and according to The captured target image calculates internal parameters and external parameters of the visual aid binocular camera, and the visual assisted binocular camera is calibrated to obtain a visually assisted left camera, and the visual assisted right camera has an effective focal length of f l and f r , and the rotation matrix between the vision assisted left camera and the visual aid right camera And translation matrix
  • the parameters r 1 , r 2 , r 3 , r 4 , r 5 , r 6 , r 7 , r 8 , r 9 , t x , t y , t z can all be calibrated from the visual aid binocular camera In the camera calibration process, the MATLAB Camera Calibration Toolbox (MATLAB) is used.
  • MATLAB MATLAB Camera Calibration Toolbox
  • the formula for calculating the three-dimensional coordinates of the first space is:
  • the calibration parameters of the vision assisted binocular camera include: the effective focal length f l of the visual assisted left camera, and the effective focal length f of the visual assisted right camera r , further comprising a rotation matrix between the visual aided left camera and the visually assisted right camera Translation matrix
  • Step S4 using the vision assisting camera to acquire an image of the resolution plate with the circular marker point in the focus position and an image of the LED array placed under the resolution panel, and combined with the pre-calibrated
  • the calibration parameter of the vision assisted binocular camera calculates a spatial three-dimensional coordinate of the center of the circular marker point in the focus position and a second spatial three-dimensional coordinate of the unoccluded LED array in the focus position;
  • an image of the center of the circular marker point on the resolution panel in the vision assisted binocular camera is obtained by acquiring an image of the resolution plate with the circular marker point in the focus position. Coordinate; calculating the spatial three-dimensional coordinates of the center of the circular marker point in the focus position by the image coordinates of the center of the circular marker point and the calibration parameters of the visually-assisted binocular camera pre-calibrated. At the same time, by collecting an image of the LED array placed under the resolution plate at this time, image coordinates of the LED array in the LED array that are not blocked by the stage are obtained, and the LED array is unoccluded by the LED array. The image coordinates and the calibration parameters of the visually assisted binocular camera, which are pre-calibrated, calculate a second spatial three-dimensional coordinate of the unblocked LED array at the focus position.
  • the vision assisting camera can only observe a part of the LED array, and a part of the image can only obtain a spatial three-dimensional coordinate of a part of the LED array;
  • all spatial three-dimensional coordinates of the LED array can be obtained; then, in order to obtain all the LED arrays placed under the resolution plate
  • the spatial three-dimensional coordinates need to convert the spatial three-dimensional coordinates of the LED array not placed under the resolution plate to the position posture of step S4, and the corresponding relationship is to find the coordinate relationship of the corresponding LEDs in S3 and S4, and rotate and translate. Find the transformation relationship.
  • the spatial three-dimensional coordinates of the LED array in the focus position are obtained by combining the first spatial three-dimensional coordinates and the second spatial three-dimensional coordinates, including:
  • Step S5 calculating a spatial three-dimensional coordinate of the center of the microscopic field of view according to the spatial three-dimensional coordinates of the center of the circular marker point and the relationship between the center of the microscopic field of view and the actual physical size coordinate of the center of the circular marker point;
  • the position coordinates of each LED in the high-resolution spectrum to be recovered are calculated by combining the spatial three-dimensional coordinates of the microscopic field center and the calibration result of the LED array in the illumination area.
  • each LED of the illumination region to the focus position fitting plane Ax 0 +By 0 +Cz 0 + is calculated.
  • a height z of D 0 and an abscissa x and an ordinate y projected to the fitting plane, completing a spatial position (x, y, z) calibration of the LED array with respect to a focus position of the resolution panel;
  • the invention provides a visual aided Fourier stacked imaging position calibration method for performing Fourier stacked imaging position calibration, because the calibration result is obtained by visual aid calculation, thus reducing the experimental optical path adjustment pair The exact difficulty; at the same time, the accurate position of each LED in the high-resolution spectrum to be recovered can be calculated by the calibration result, and the influence of the position deviation on the recovery result is avoided.
  • the invention also provides a visual aided Fourier stacked imaging position calibration device, the Fuli The leaf laminate imaging position calibration device is applied to a Fourier stacked imaging position calibration system, the calibration system comprising: a Fourier laminate imaging device 1 and a vision assisted binocular camera 2, the Fourier stacked imaging device 1 includes a microscopic image acquisition camera 11, a microscope 12 (the lens 121 and the objective lens 122 are sequentially shown below the microscopic image acquisition camera 11 in the figure), a stage (not shown), and a circular mark Point resolution plate 13 and LED array 14, said resolution plate 13 being placed on said stage, said microscopic image acquisition camera 11 being located directly above said stage, said LED array 14 being located Directly below the stage and parallel to the plane of the stage, the visual aid binocular camera 2 comprises: a visual assist left camera and a visual aid right camera, the visual assist left camera and the visual assist right camera are located Directly in front of the Fourier laminate imaging device, as shown in FIG. 7, the device includes:
  • An image pixel coordinate extraction module 1 is configured to acquire an image of the resolution plate with the circular marker points by using the microscopic image acquisition camera, and extract the image of the resolution plate by using a circular marker point extraction algorithm. Image pixel coordinates of the center of the circle mark point;
  • the circular marker point extraction algorithm is specifically: detecting a circular marker point edge image coordinate (x, y) by using a canny operator in image processing and fitting an elliptic equation.
  • x 2 +2Bxy+Cy 2 +2Dx+2Ey+F 0, wherein the parameters B, C, D, E, and F are obtained by fitting, and an image of the center of the circular marker point in the image of the resolution plate is calculated
  • the formula for pixel coordinates (x, y) is:
  • the actual physical size coordinate calculation module 2 is configured to acquire an image by using the microscopic image acquisition camera to obtain image pixel coordinates of a microscopic field center point of the image, and measure the rounded mark by using a three coordinate device. Calculating the microscopic field center point and the circle by establishing a relationship between the image coordinate system and the actual physical size coordinate system on the actual physical size coordinate of each group of elements in the resolution board of the point. The actual physical size coordinates of the center of the marker point;
  • the relationship between the established image coordinate system and the actual physical size coordinate system is:
  • x 0 x center +(u 0 -u center ) ⁇ dx
  • y 0 y center +(v 0 -v center ) ⁇ dy
  • x circle x center +(u circle -u center ) ⁇ dx
  • y circle y center +(v circle -v center ) ⁇ dy
  • (x 0 , y 0 ) represents the actual physical size coordinate of the center of the microscopic field of view
  • (x circle , y circle ) represents the actual physical size coordinate of the center of the circular marker point
  • (u center , v center ) indicates the selected
  • the center image coordinate of the image coordinate system (x center , y center ) represents the physical size coordinate of the selected image coordinate system
  • (u 0 , v 0 ) represents the image pixel coordinates of the microscopic field center point
  • (u circle , v circle ) represents the image pixel coordinates of the center of the circular marker point
  • d x and d y are the unit physical dimensions in the x and y directions of the actual physical size coordinate system, respectively.
  • a first calculation module 3 configured to acquire, by using the visual aid binocular camera, an image when the LED array is not placed under the resolution panel, thereby obtaining each LED in the LED array Calculating the image coordinates in the visual aid binocular camera, calculating the first spatial three-dimensional coordinates of each LED of the LED array from the image coordinates of each LED and the calibration parameters of the visually assisted binocular camera pre-calibrated;
  • the formula for calculating the three-dimensional coordinates of the first space is:
  • the calibration parameters of the visual aid binocular camera include: the effective focal length f l of the visual aid left camera, the effective focal length f r of the visual aid right camera, and Rotation matrix between the vision assisted left camera and the vision assisted right camera Translation matrix
  • a second calculation module 4 configured to acquire, by using the vision assisting camera, an image of the resolution plate with the circular marker point in the focus position and a diagram of the LED array placed under the resolution panel at this time Calculating, according to the calibration parameters of the visually-assisted binocular camera, which is pre-calibrated, the spatial three-dimensional coordinates of the center of the circular marker point in the focus position and the second spatial three-dimensional coordinates of the unoccluded LED array in the focus position;
  • the formula for calculating the spatial three-dimensional coordinates of the LED array at the focus position is:
  • (x, y, z) represents a first spatial three-dimensional coordinate
  • (X, Y, Z) represents a second spatial three-dimensional coordinate
  • the rotation matrix R 2 and the translation matrix T 2 may utilize the first calculation module and the In the two position states of the second calculation module, the first spatial three-dimensional coordinates corresponding to the illumination LED array and the second spatial three-dimensional coordinates are calculated and fitted.
  • a calibration module 5 configured to calculate a space of the center of the microscopic field of view according to a spatial three-dimensional coordinate of a center of the circular marker point and an actual physical size coordinate of a center of the microscopic field of view and a center of a circular marker point 3D coordinates; and using the spatial three-dimensional coordinates of the center of the microscopic field of view combined with the spatial three-dimensional coordinates of the LED array at the focus position to calculate the height z of each of the LEDs in the illumination region to the focus position and the LED projection to the projection Combining the horizontal and vertical coordinates x, y of the plane, completes the spatial position (x, y, z) calibration of the LED array with respect to the resolution plate; and simultaneously combines the spatial three-dimensional coordinates of the microscopic field center with the illumination area
  • the calibration result of the LED array calculates the position coordinates of each LED in the high resolution spectrum to be recovered.

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Abstract

本发明适用于显微成像技术领域，提供了一种视觉辅助的傅里叶叠层成像位置标定方法及装置，在传统傅里叶叠层成像计算恢复之前，通过视觉辅助双目系统采集未置于分辨率板下的LED阵列点亮时的图像，结合相机标定参数计算出其第一空间三维坐标；再通过在分辨率板上贴圆形标志点的方法找到显微视场中心的空间三维坐标；然后结合第一空间三维坐标并通过旋转平移变换找到系统处于聚焦位置时LED阵列的空间三维坐标，利用显微视场中心的空间三维坐标结合LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度以及LED投影到拟合平面的横纵坐标，完成空间位置标定；本发明可避免位置偏差给恢复结果带来的影响。

Description

视觉辅助的傅里叶叠层成像位置标定方法及装置 技术领域
本发明属于显微成像技术领域，尤其涉及一种视觉辅助的傅里叶叠层成像位置标定方法及装置。
背景技术
显微镜自发明之日起，为人类观察更小的物体提供了可能。在使用显微镜观察样本时，通常都是在低倍物镜下找到样本所在的位置，进而旋转转换器，使用较高倍数的物镜来观察样本的清晰细节。当将低倍镜转换成高倍镜后，带来的直接变化就是观察者所能够观察的视场变小。为了解决视场大小和分辨率之间的矛盾关系，常用的方法就是借用精密电动平台实现大范围扫描并结合图像处理方法来实现，但是这种方法需要十分精密的扫描且操作过程非常复杂。如何同时得到大视场和高分辨率的图像一直困扰着许多从事显微研究的科研工作者。在这方面的突破性进展应当属于有人首次提出的傅里叶叠层成像技术并成功实现了大视场和超分辨成像。
典型的傅里叶叠层成像系统是在普通显微镜装置的基础上实现的，用一个可编程的LED阵列取代显微镜自身的照明光源，通过控制LED阵列上每一个LED的亮暗来实现对样本的不同方向照明。通常情况下，每一个位置处的LED照射样本得到的图像是受物镜数值孔径限制的成像结果，其图像存在着样本的高频信息丢失，我们称之为低分辨率图像。傅里叶叠层成像技术就是通过依次点亮照明阵列上每一个LED来得到很多张包含样本不同角度信息的低分辨率图像，然后在图像的傅里叶域中采用合成孔径以及相位恢复的方法来恢复样本的高分辨率信息。最终重建的高分辨率图像的分辨率大小取决于物镜数值孔径和照明数值孔径之和，所以从这个角度出发，傅里叶叠层成像技术能够打破传统显微镜物镜数值孔径的限制，最终实现大视场和高分辨率成像。
傅里叶叠层成像技术的实现是通过在待恢复的高分辨率频谱中找到每一个不同位置LED对应的空间频率，以物镜的数值孔径为半径在高分辨率频谱中取相应位置下的子频谱孔径，用对应LED位置下采集的低分辨率图像做振幅替换，实现相位恢复。
但在传统的傅里叶叠层成像中，最终恢复高分辨图像的质量很大程度受到空间LED阵列定位误差的影响，具体表现为在传统傅里叶叠层中每一个LED对应的空间频率是通过选定一个中心LED与显微系统中心对准后，其它位置的空间频率以此为基准计算出来的。所以一旦中心LED与显微系统中心存在位置偏差，就会导致整个照明区域的LED空间位置都存在偏差，这样对应在待恢复的高分辨率频谱中的每一个子孔径频谱的中心位置也就不准确，其相位恢复也很难得到一个准确的结果。因此，为了得到恢复质量较好的结果，就必须解决傅里叶叠层中LED阵列的空间位置偏差问题。
发明内容
本发明提供一种视觉辅助的傅里叶叠层成像位置标定方法及装置，旨在解决传统傅里叶叠层成像光路中LED阵列与显微系统中心难以精确调节对准，待恢复高分辨率图像质量下降的问题。
本发明提供了一种视觉辅助的傅里叶叠层成像位置标定方法，所述傅里叶叠层成像位置标定方法运用于傅里叶叠层成像位置标定系统，所述标定系统包括：傅里叶叠层成像装置和视觉辅助双目相机，所述傅里叶叠层成像装置包括显微图像采集相机、显微镜、载物台、贴有圆形标志点的分辨率板和LED阵列，所述分辨率板置于所述载物台上，所述显微图像采集相机位于所述载物台正上方，所述LED阵列位于所述载物台正下方，并与所述载物台所在平面平行，所述视觉辅助双目相机包括：视觉辅助左相机和视觉辅助右相机，所述视觉辅助左相机、视觉辅助右相机位于所述傅里叶叠层成像装置的正前方，所述方法包括：
步骤S1，利用所述显微图像采集相机采集所述贴有圆形标志点的分辨率板的图像，并结合圆形标志点提取算法提取所述分辨率板的图像中圆形标志点的圆心的图像像素坐标；
步骤S2，利用所述显微图像采集相机采集图像，得到所述图像的显微视场中心点的图像像素坐标，利用三坐标仪测出所述贴有圆形标志点的分辨率板上每一级每一组元素的实际物理尺寸坐标，通过建立图像坐标系与实际物理尺寸坐标系之间的关系，计算出所述显微视场中心点和所述圆形标志点的圆心的实际物理尺寸坐标；
步骤S3，利用所述视觉辅助双目相机采集未置于所述分辨率板下的所述LED阵列点亮时的图像，从而得到所述LED阵列中的每一个LED在所述视觉辅助双目相机中的图像坐标，由每一个LED的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出所述LED阵列的每一个LED的第一空间三维坐标；
步骤S4，利用所述视觉辅助相机采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像以及此时置于所述分辨率板下的LED阵列的图像，并结合预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时的空间三维坐标以及未被遮挡的LED阵列在聚焦位置时的第二空间三维坐标；
结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标；
步骤S5，根据所述圆形标志点圆心的空间三维坐标以及所述显微视场中心和圆形标志点的圆心的实际物理尺寸坐标的关系计算所述显微视场中心的空间三维坐标；
并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度z以及LED投影到拟合平面的横纵坐标x、y，完成所述LED阵列相对所述分辨率板的空间位置(x,y,z)标定；
同时结合所述显微视场中心的空间三维坐标和照明区域中LED阵列的标定结果计算每一个LED在待恢复高分辨率频谱中的位置坐标。
本发明还提供了一种视觉辅助的傅里叶叠层成像位置标定装置，所述傅里叶叠层成像位置标定装置运用于傅里叶叠层成像位置标定系统，所述标定系统包括：傅里叶叠层成像装置和视觉辅助双目相机，所述傅里叶叠层成像装置包括显微图像采集相机、显微镜、载物台、贴有圆形标志点的分辨率板和LED阵列，所述分辨率板置于所述载物台上，所述显微图像采集相机位于所述载物台正上方，所述LED阵列位于所述载物台正下方，并与所述载物台所在平面平行，所述视觉辅助双目相机包括：视觉辅助左相机和视觉辅助右相机，所述视觉辅助左相机、视觉辅助右相机位于所述傅里叶叠层成像装置的正前方，所述装置包括：
图像像素坐标提取模块，用于利用所述显微图像采集相机采集所述贴有圆形标志点的分辨率板的图像，并结合圆形标志点提取算法提取所述分辨率板的图像中圆形标志点的圆心的图像像素坐标；
实际物理尺寸坐标计算模块，用于利用所述显微图像采集相机采集图像，得到所述图像的显微视场中心点的图像像素坐标，利用三坐标仪测出所述贴有圆形标志点的分辨率板上每一级每一组元素的实际物理尺寸坐标，通过建立图像坐标系与实际物理尺寸坐标系之间的关系，计算出所述显微视场中心点和所述圆形标志点的圆心的实际物理尺寸坐标；
第一计算模块，用于利用所述视觉辅助双目相机采集未置于所述分辨率板下的所述LED阵列点亮时的图像，从而得到所述LED阵列中的每一个LED在所述视觉辅助双目相机中的图像坐标，由每一个LED的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出所述LED阵列的每一个LED的第一空间三维坐标；
第二计算模块，用于利用所述视觉辅助相机采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像以及此时置于所述分辨率板下的LED阵列的图像， 并结合预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时的空间三维坐标以及未被遮挡的LED阵列在聚焦位置时的第二空间三维坐标；
结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标；
标定模块，用于根据所述圆形标志点圆心的空间三维坐标以及所述显微视场中心和圆形标志点的圆心的实际物理尺寸坐标的关系计算所述显微视场中心的空间三维坐标；
并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度z以及LED投影到拟合平面的横纵坐标x、y，完成所述LED阵列相对所述分辨率板的空间位置(x,y,z)标定；
同时结合所述显微视场中心的空间三维坐标和照明区域中LED阵列的标定结果计算每一个LED在待恢复高分辨率频谱中的位置坐标。
本发明与现有技术相比，有益效果在于：本发明提供了一种视觉辅助的傅里叶叠层成像位置标定方法及装置，方法包括：在传统傅里叶叠层成像计算恢复之前，通过搭建的视觉辅助双目系统采集未置于分辨率板下的LED阵列点亮时的图像，结合相机标定参数计算出LED阵列的第一空间三维坐标；再通过在分辨率板上贴圆形标志点的方法找到显微视场中心的空间三维坐标；然后结合第一空间三维坐标并通过旋转平移变换找到系统处于聚焦位置时LED阵列的空间三维坐标，并利用显微视场中心的空间三维坐标结合LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度以及LED投影到拟合平面的横纵坐标，完成LED阵列相对分辨率板的空间位置标定；并进一步计算出每一个LED在待恢复高分辨率频谱中的位置坐标；本发明与现有技术相比，一方面，采用视觉辅助傅里叶叠层成像进行位置标定之后，不需要再反复调节照明LED阵列的中心与显微系统的中心进行对准，相应每一 个LED空间位置坐标都可以通过计算精确得到，大大减小了实验上光路调节的难度；另一方面，采用视觉辅助进行位置标定的方法能够减少传统傅里叶叠层成像中存在的位置偏差，这样就能在待恢复的高分辨率频谱中找到相应LED准确的空间频率坐标，进而能够得到比传统恢复方法更准确的恢复结果。
附图说明
图1是本发明实施例提供的傅里叶叠层成像位置标定系统的硬件结构示意图；
图2是本发明实施例提供的一种视觉辅助的傅里叶叠层成像位置标定方法的示意图；
图3是本发明实施例提供的图像坐标系与实际物理尺寸坐标系计算模型的示意图；
图4是本发明实施例提供的传统傅里叶叠层成像中LED阵列空间位置和经视觉辅助标定LED阵列空间位置的对比示意图；
图5(a)是本发明实施例提供的用传统傅里叶叠层成像方法重建分辨率板结果的示意图；
图5(b)是本发明实施例提供的经过视觉辅助位置标定之后重建分辨率板结果的示意图；
图6(a)是本发明实施例提供的用传统傅里叶叠层成像重建人血细胞结果的示意图；
图6(b)是本发明实施例提供的经过视觉辅助位置标定之后重建人血细胞结果的示意图；
图7是本发明实施例提供的一种视觉辅助的傅里叶叠层成像位置标定装置的模块示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提供了一种视觉辅助的傅里叶叠层成像位置标定方法，所述傅里叶叠层成像位置标定方法运用于傅里叶叠层成像位置标定系统，如图1所示，所述标定系统包括：傅里叶叠层成像装置1和视觉辅助双目相机2，所述傅里叶叠层成像装置1包括显微图像采集相机11、显微镜12(图中所述显微图像采集相机11下方依次示出了透镜121和物镜122)、载物台(图中未示出)、贴有圆形标志点的分辨率板13和LED阵列14，所述分辨率板13置于所述载物台上，所述显微图像采集相机11位于所述载物台正上方，所述LED阵列14位于所述载物台正下方，并与所述载物台所在平面平行，所述视觉辅助双目相机2包括：视觉辅助左相机和视觉辅助右相机，所述视觉辅助左相机、视觉辅助右相机位于所述傅里叶叠层成像装置的正前方。
具体地，本发明实施例中，所述显微镜12采用的型号为Olympus BX43，搭配Olympus平场复消色差物镜，物镜放大倍数为4倍，数值孔径为0.1；所述显微镜12上搭载SCMOS相机，型号为PCO.edge5.5，分辨率大小为2560*2048，像素尺寸为6.5um*6.5um；所述视觉辅助双目相机2采用Basler相机，型号为piA2400-17gc，分辨率大小为2448*2050，像素尺寸为3.45um*3.45um；所述视觉辅助双目相机采用Computar镜头，型号为M1214-MP2。
具体地，本发明实施例中，所述分辨率板13采用USAF1951分辨率板。
具体地，如图2所示，所述傅里叶叠层成像位置标定方法包括：
步骤S1，利用所述显微图像采集相机采集所述贴有圆形标志点的分辨率板的图像，并结合圆形标志点提取算法提取所述分辨率板的图像中圆形标志点的圆心的图像像素坐标；
具体地，所述步骤S1中，所述圆形标志点提取算法具体为：利用图像处理中canny算子检测圆形标志点边缘图像坐标(x,y)并拟合出椭圆方程 x2+2Bxy+Cy2+2Dx+2Ey+F＝0，其中参数B、C、D、E和F由拟合得出，计算所述分辨率板的图像中圆形标志点的圆心的图像像素坐标(x,y)公式为：
Figure PCTCN2017106786-appb-000001
Figure PCTCN2017106786-appb-000002
步骤S2，利用所述显微图像采集相机采集图像，得到所述图像的显微视场中心点的图像像素坐标，利用三坐标仪测出所述贴有圆形标志点的分辨率板上每一级每一组元素的实际物理尺寸坐标，通过建立图像坐标系与实际物理尺寸坐标系之间的关系，计算出所述显微视场中心点和所述圆形标志点的圆心的实际物理尺寸坐标；
具体地，所述显微视场中心点指的是所述显微图像采集相机采集的图像的视场中心。
图3为图像坐标系与实际物理尺寸坐标系计算模型的示意图，具体地，建立的图像坐标系与实际物理尺寸坐标系之间的关系为：
x0＝xcenter+(u0-ucenter)·dx
y0＝ycenter+(v0-vcenter)·dy
xcircle＝xcenter+(ucircle-ucenter)·dx
ycircle＝ycenter+(vcircle-vcenter)·dy
其中，(x0,y0)表示显微视场中心的实际物理尺寸坐标，(xcircle,ycircle)表示圆形标志点圆心的实际物理尺寸坐标，(ucenter,vcenter)表示选定的图像坐标系的中心图像坐标，(xcenter,ycenter)表示选定的图像坐标系的物理尺寸坐标，该物理尺寸坐标通过三坐标仪得到，(u0,v0)表示显微视场中心点的图像像素坐标，(ucircle,vcircle)表示圆形标志点圆心的图像像素坐标，dx、dy分别为实际物理尺寸坐标系中x、y方向上的单位物理尺寸。
步骤S3，利用所述视觉辅助双目相机采集未置于所述分辨率板下的所述LED阵列点亮时的图像，从而得到所述LED阵列中的每一个LED在所述视觉辅助双目相机中的图像坐标，由每一个LED的图像坐标以及预先标定的所述视 觉辅助双目相机的标定参数计算出所述LED阵列的每一个LED的第一空间三维坐标；
具体地，此时所述LED阵列未置于所述分辨率板下，所以，所述LED阵列没有被载物台遮挡，所述视觉辅助双目相机可以采集到完整的LED阵列点亮时的图像。
具体地，所述视觉辅助双目相机的预先标定过程为，将标靶置于所述视觉辅助双目相机下方，利用所述视觉辅助双目相机采集几个姿态下的标靶图像，并根据采集的所述标靶图像计算所述视觉辅助双目相机的内部参数和外部参数，所述视觉辅助双目相机经过标定之后得到视觉辅助左相机、视觉辅助右相机的有效焦距分别为fl和fr，以及所述视觉辅助左相机、视觉辅助右相机之间的旋转矩阵
Figure PCTCN2017106786-appb-000003
和平移矩阵
Figure PCTCN2017106786-appb-000004
其中，参数r1、r2、r3、r4、r5、r6、r7、r8、r9、tx、ty、tz都可以从所述视觉辅助双目相机标定结果中得到，相机标定过程采用的是MATLAB相机标定工具箱(Camera Calibration Toolbox for MATLAB)。
具体地，所述步骤S3中，计算所述第一空间三维坐标的公式为：
x＝z Xl/fl
y＝z Yl/fl
Figure PCTCN2017106786-appb-000005
其中，(x,y,z)表示每一个LED的第一空间三维坐标，(Xl,Yl)表示每一个LED在所述视觉辅助左相机中的图像坐标，(Xr,Yr)表示每一个LED在所述视觉辅助右相机中的图像坐标；由前述可知，所述视觉辅助双目相机的标定参数包括：视觉辅助左相机的有效焦距fl，视觉辅助右相机的有效焦距fr，还包括所述视 觉辅助左相机和视觉辅助右相机之间的旋转矩阵
Figure PCTCN2017106786-appb-000006
平移矩阵
Figure PCTCN2017106786-appb-000007
步骤S4，利用所述视觉辅助相机采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像以及此时置于所述分辨率板下的LED阵列的图像，并结合预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时的空间三维坐标以及未被遮挡的LED阵列在聚焦位置时的第二空间三维坐标；
结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标；
具体地，通过采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像，从而得到所述分辨率板上的圆形标志点的圆心在所述视觉辅助双目相机中的图像坐标；由所述圆形标志点的圆心的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时的空间三维坐标。同时，通过采集此时置于所述分辨率板下的LED阵列的图像，从而得到所述LED阵列中未被载物台遮挡的LED阵列的图像坐标，由未被遮挡的所述LED阵列的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出未被遮挡的所述LED阵列在聚焦位置时的第二空间三维坐标。
具体地，由于所述LED阵列置于所述分辨率板下时，所述视觉辅助相机只能观察到所述LED阵列的一部分，一部分图像只能得到LED阵列中一部分的空间三维坐标；而所述LED阵列在步骤S3位置姿态下即未置于所述分辨率板下时，可以得到所述LED阵列所有的空间三维坐标；那么，为了得到置于所述分辨率板下的LED阵列的全部的空间三维坐标，需要将未置于所述分辨率板下的LED阵列的空间三维坐标转换到步骤S4位置姿态下，其对应关系就是找到S3与S4中对应LED的坐标关系，经旋转和平移求出变换关系。
具体地，所述步骤S4中，所述结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标，包括：
利用所述第一空间三维坐标和所述第二空间三维坐标计算拟合得到旋转矩阵R2和平移矩阵T2，并利用如下公式计算得到LED阵列在聚焦位置时的空间三维坐标：
(X,Y,Z)T＝R2·(x,y,z)T+T2
其中，(x,y,z)表示第一空间三维坐标，(X,Y,Z)表示第二空间三维坐标。
步骤S5，根据所述圆形标志点圆心的空间三维坐标以及所述显微视场中心和圆形标志点的圆心的实际物理尺寸坐标的关系计算所述显微视场中心的空间三维坐标；
并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度z以及LED投影到拟合平面的横纵坐标x、y，完成所述LED阵列相对所述分辨率板的空间位置(x,y,z)标定；
同时结合所述显微视场中心的空间三维坐标和照明区域中LED阵列的标定结果计算每一个LED在待恢复高分辨率频谱中的位置坐标。
具体地，所述步骤S5中，根据显微视场中心到临近的圆形标志点圆心(x1,y1,z1)和(x2,y2,z2)的实际物理长度d1、d2以及两者的共面方程Ax+By+Cz+D＝0，计算出显微视场中心的空间三维坐标(x0,y0,z0)；
(x0-x1)2+(y0-y1)2+(z0-z1)2＝d1 2
(x0-x2)2+(y0-y2)2+(z0-z2)2＝d2 2
Ax0+By0+Cz0+D＝0
其中，d1、d2根据计算出的显微视场中心和圆形标志点的圆心的实际物理尺寸坐标计算而得；显微视场中心和圆形标志点所在的平面Ax0+By0+Cz0+D＝0通过所述圆形标志点圆心的空间三维坐标拟合得出；通过解上面三个方程可以 计算出显微视场中心的空间三维坐标(x0,y0,z0)。
进一步地，利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面Ax0+By0+Cz0+D＝0的高度z以及投影到所述拟合平面的横坐标x和纵坐标y，完成所述LED阵列相对所述分辨率板聚焦位置时的空间位置(x,y,z)标定；
进一步地，以显微视场中心所在平面建立坐标系，计算每一个LED对应在待恢复高分辨率频谱中的空间频率，
Figure PCTCN2017106786-appb-000008
Figure PCTCN2017106786-appb-000009
其中，(xm,n,ym,n,zm,n)为照明区域中第m行第n列的LED用视觉辅助标定的结果，(um,n,vm,n)对应第m行第n列的LED在待恢复高频分辨率频谱中的位置坐标。
本发明提供了一种视觉辅助的傅里叶叠层成像位置标定方法，进行傅里叶叠层成像位置标定，因为标定结果是通过视觉辅助计算得出，这样就减小了实验光路上调节对准的难度；同时，通过标定结果可以计算出每一个LED在待恢复高分辨率频谱中的准确位置，避免了位置偏差给恢复结果带来的影响。
为了测试本发明提出的一种视觉辅助的傅里叶叠层成像位置标定方法的实用性，我们分别用USAF1951分辨率板和人血细胞进行傅里叶叠层实验，图4“+”表示经视觉辅助标定LED阵列空间位置，“*”表示传统傅里叶叠层成像中LED阵列空间位置；图5(a)表示用传统傅里叶叠层成像方法重建分辨率板结果，图5(b)表示经过视觉辅助位置标定之后重建分辨率板结果；图6(a)表示用传统傅里叶叠层成像重建人血细胞结果，图6(b)表示经过视觉辅助位置标定之后重建人血细胞结果。从两组实验对比中可以看出，经过视觉辅助的傅里叶叠层成像位置标定之后的重建结果能够有效避免传统方法中空间位置存在偏差的问题，不需要反复调节光路对准，并同时提高了传统傅里叶叠层成像的恢复质量。
本发明还提供了一种视觉辅助的傅里叶叠层成像位置标定装置，所述傅里 叶叠层成像位置标定装置运用于傅里叶叠层成像位置标定系统，所述标定系统包括：傅里叶叠层成像装置1和视觉辅助双目相机2，所述傅里叶叠层成像装置1包括显微图像采集相机11、显微镜12(图中所述显微图像采集相机11下方依次示出了透镜121和物镜122)、载物台(图中未示出)、贴有圆形标志点的分辨率板13和LED阵列14，所述分辨率板13置于所述载物台上，所述显微图像采集相机11位于所述载物台正上方，所述LED阵列14位于所述载物台正下方，并与所述载物台所在平面平行，所述视觉辅助双目相机2包括：视觉辅助左相机和视觉辅助右相机，所述视觉辅助左相机、视觉辅助右相机位于所述傅里叶叠层成像装置的正前方，如图7所示，所述装置包括：
图像像素坐标提取模块1，用于利用所述显微图像采集相机采集所述贴有圆形标志点的分辨率板的图像，并结合圆形标志点提取算法提取所述分辨率板的图像中圆形标志点的圆心的图像像素坐标；
具体地，所述图像像素坐标提取模块1中，所述圆形标志点提取算法具体为：利用图像处理中canny算子检测圆形标志点边缘图像坐标(x,y)并拟合出椭圆方程x2+2Bxy+Cy2+2Dx+2Ey+F＝0，其中参数B、C、D、E和F由拟合得出，计算所述分辨率板的图像中圆形标志点的圆心的图像像素坐标(x,y)公式为：
Figure PCTCN2017106786-appb-000010
Figure PCTCN2017106786-appb-000011
实际物理尺寸坐标计算模块2，用于利用所述显微图像采集相机采集图像，得到所述图像的显微视场中心点的图像像素坐标，利用三坐标仪测出所述贴有圆形标志点的分辨率板上每一级每一组元素的实际物理尺寸坐标，通过建立图像坐标系与实际物理尺寸坐标系之间的关系，计算出所述显微视场中心点和所述圆形标志点的圆心的实际物理尺寸坐标；
具体地，所述图像像素坐标提取模块2中，建立的图像坐标系与实际物理尺寸坐标系之间的关系为：
x0＝xcenter+(u0-ucenter)·dx
y0＝ycenter+(v0-vcenter)·dy
xcircle＝xcenter+(ucircle-ucenter)·dx
ycircle＝ycenter+(vcircle-vcenter)·dy
其中，(x0,y0)表示显微视场中心的实际物理尺寸坐标，(xcircle,ycircle)表示圆形标志点圆心的实际物理尺寸坐标，(ucenter,vcenter)表示选定的图像坐标系的中心图像坐标，(xcenter,ycenter)表示选定的图像坐标系的物理尺寸坐标，(u0,v0)表示显微视场中心点的图像像素坐标，(ucircle,vcircle)表示圆形标志点圆心的图像像素坐标，dx、dy分别为实际物理尺寸坐标系中x、y方向上的单位物理尺寸。
第一计算模块3，用于利用所述视觉辅助双目相机采集未置于所述分辨率板下的所述LED阵列点亮时的图像，从而得到所述LED阵列中的每一个LED在所述视觉辅助双目相机中的图像坐标，由每一个LED的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出所述LED阵列的每一个LED的第一空间三维坐标；
具体地，所述第一计算模块3中，计算所述第一空间三维坐标的公式为：
x＝z Xl/fl
y＝z Yl/fl
Figure PCTCN2017106786-appb-000012
其中，(x,y,z)表示每一个LED的第一空间三维坐标，(Xl,Yl)表示每一个LED在所述视觉辅助左相机中的图像坐标，(Xr,Yr)表示每一个LED在所述视觉辅助右相机中的图像坐标，所述视觉辅助双目相机的标定参数包括：视觉辅助左相机的有效焦距fl，视觉辅助右相机的有效焦距fr，还包括所述视觉辅助左相机和视觉辅助右相机之间的旋转矩阵
Figure PCTCN2017106786-appb-000013
平移矩阵
Figure PCTCN2017106786-appb-000014
第二计算模块4，用于利用所述视觉辅助相机采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像以及此时置于所述分辨率板下的LED阵列的图 像，并结合预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时的空间三维坐标以及未被遮挡的LED阵列在聚焦位置时的第二空间三维坐标；
结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标；
所述第二计算模块4中，计算LED阵列在聚焦位置时的空间三维坐标的公式为：
(X,Y,Z)T＝R2·(x,y,z)T+T2
其中，(x,y,z)表示第一空间三维坐标，(X,Y,Z)表示第二空间三维坐标，旋转矩阵R2和平移矩阵T2可以利用所述第一计算模块和所述第二计算模块的两个位置状态下，照明LED阵列对应的第一空间三维坐标和第二空间三维坐标计算拟合得到。
标定模块5，用于根据所述圆形标志点圆心的空间三维坐标以及所述显微视场中心和圆形标志点的圆心的实际物理尺寸坐标的关系计算所述显微视场中心的空间三维坐标；并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度z以及LED投影到拟合平面的横纵坐标x、y，完成所述LED阵列相对所述分辨率板的空间位置(x,y,z)标定；同时结合所述显微视场中心的空间三维坐标和照明区域中LED阵列的标定结果计算每一个LED在待恢复高分辨率频谱中的位置坐标。
具体地，所述标定模块5，用于根据显微视场中心到临近的圆形标志点圆心(x1,y1,z1)和(x2,y2,z2)的实际物理长度d1、d2以及两者的共面方程Ax+By+Cz+D＝0，计算出显微视场中心的空间三维坐标(x0,y0,z0)，
(x0-x1)2+(y0-y1)2+(z0-z1)2＝d1 2
(x0-x2)2+(y0-y2)2+(z0-z2)2＝d2 2
Ax0+By0+Cz0+D＝0
其中，d1、d2根据计算出的显微视场中心和圆形标志点的圆心的实际物理尺寸坐标计算而得；显微视场中心和圆形标志点所在的平面Ax0+By0+Cz0+D＝0通过所述圆形标志点圆心的空间三维坐标拟合得出；
并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面Ax0+By0+Cz0+D＝0的高度z以及投影到所述拟合平面的横坐标x和纵坐标y，完成所述LED阵列相对所述分辨率板聚焦位置时的空间位置(x,y,z)标定；
然后，以显微视场中心所在平面建立坐标系，计算每一个LED对应在待恢复高分辨率频谱中的位置坐标，
Figure PCTCN2017106786-appb-000015
Figure PCTCN2017106786-appb-000016
其中，(xm,n,ym,n,zm,n)为照明区域中第m行第n列的LED用视觉辅助标定的结果，(um,n,vm,n)对应第m行第n列的LED在待恢复高频分辨率频谱中的位置坐标。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种视觉辅助的傅里叶叠层成像位置标定方法，其特征在于，所述傅里叶叠层成像位置标定方法运用于傅里叶叠层成像位置标定系统，所述标定系统包括：傅里叶叠层成像装置和视觉辅助双目相机，所述傅里叶叠层成像装置包括显微图像采集相机、显微镜、载物台、贴有圆形标志点的分辨率板和LED阵列，所述分辨率板置于所述载物台上，所述显微图像采集相机位于所述载物台正上方，所述LED阵列位于所述载物台正下方，并与所述载物台所在平面平行，所述视觉辅助双目相机包括：视觉辅助左相机和视觉辅助右相机，所述视觉辅助左相机、视觉辅助右相机位于所述傅里叶叠层成像装置的正前方，所述方法包括：
    步骤S1，利用所述显微图像采集相机采集所述贴有圆形标志点的分辨率板的图像，并结合圆形标志点提取算法提取所述分辨率板的图像中圆形标志点的圆心的图像像素坐标；
    步骤S2，利用所述显微图像采集相机采集图像，得到所述图像的显微视场中心点的图像像素坐标，利用三坐标仪测出所述贴有圆形标志点的分辨率板上每一级每一组元素的实际物理尺寸坐标，通过建立图像坐标系与实际物理尺寸坐标系之间的关系，计算出所述显微视场中心点和所述圆形标志点的圆心的实际物理尺寸坐标；
    步骤S3，利用所述视觉辅助双目相机采集未置于所述分辨率板下的所述LED阵列点亮时的图像，从而得到所述LED阵列中的每一个LED在所述视觉辅助双目相机中的图像坐标，由每一个LED的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出所述LED阵列的每一个LED的第一空间三维坐标；
    步骤S4，利用所述视觉辅助相机采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像以及此时置于所述分辨率板下的LED阵列的图像，并结合预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时 的空间三维坐标以及未被遮挡的LED阵列在聚焦位置时的第二空间三维坐标；
    结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标；
    步骤S5，根据所述圆形标志点圆心的空间三维坐标以及所述显微视场中心和圆形标志点的圆心的实际物理尺寸坐标的关系计算所述显微视场中心的空间三维坐标；
    并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度z以及LED投影到拟合平面的横纵坐标x、y，完成所述LED阵列相对所述分辨率板的空间位置(x,y,z)标定；
    同时结合所述显微视场中心的空间三维坐标和照明区域中LED阵列的标定结果计算每一个LED在待恢复高分辨率频谱中的位置坐标。
  2. 如权利要求1所述的傅里叶叠层成像位置标定方法，其特征在于，所述步骤S1中，所述圆形标志点提取算法具体为：利用图像处理中canny算子检测圆形标志点边缘图像坐标(x,y)并拟合出椭圆方程x2+2Bxy+Cy2+2Dx+2Ey+F＝0，其中参数B、C、D、E和F由拟合得出，计算所述分辨率板的图像中圆形标志点的圆心的图像像素坐标(x,y)公式为：
    Figure PCTCN2017106786-appb-100001
    Figure PCTCN2017106786-appb-100002
  3. 如权利要求1所述的傅里叶叠层成像位置标定方法，其特征在于，所述步骤S2中，建立的图像坐标系与实际物理尺寸坐标系之间的关系为：
    x0＝xcenter+(u0-ucenter)·dx
    y0＝ycenter+(v0-vcenter)·dy
    xcircle＝xcenter+(ucircle-ucenter)·dx
    ycircle＝ycenter+(vcircle-vcenter)·dy
    其中，(x0,y0)表示显微视场中心的实际物理尺寸坐标，(xcircle,ycircle)表示圆形标志点圆心的实际物理尺寸坐标，(ucenter,vcenter)表示选定的图像坐标系的中心 图像坐标，(xcenter,ycenter)表示选定的图像坐标系的物理尺寸坐标，(u0,v0)表示显微视场中心点的图像像素坐标，(ucircle,vcircle)表示圆形标志点圆心的图像像素坐标，dx、dy分别为实际物理尺寸坐标系中x、y方向上的单位物理尺寸。
  4. 如权利要求1所述的傅里叶叠层成像位置标定方法，其特征在于，所述步骤S3中，计算所述第一空间三维坐标的公式为：
    x＝z Xl/fl
    y＝z Yl/fl
    Figure PCTCN2017106786-appb-100003
    其中，(x,y,z)表示每一个LED的第一空间三维坐标，(Xl,Yl)表示每一个LED在所述视觉辅助左相机中的图像坐标，(Xr,Yr)表示每一个LED在所述视觉辅助右相机中的图像坐标，所述视觉辅助双目相机的标定参数包括：视觉辅助左相机的有效焦距fl，视觉辅助右相机的有效焦距fr，还包括所述视觉辅助左相机和视觉辅助右相机之间的旋转矩阵
    Figure PCTCN2017106786-appb-100004
    平移矩阵
    Figure PCTCN2017106786-appb-100005
    所述步骤S4中，所述结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标，包括：
    利用所述第一空间三维坐标和所述第二空间三维坐标计算拟合得到旋转矩阵R2和平移矩阵T2，并利用如下公式计算得到LED阵列在聚焦位置时的空间三维坐标：
    (X,Y,Z)T＝R2·(x,y,z)T+T2
    其中，(x,y,z)表示第一空间三维坐标，(X,Y,Z)表示第二空间三维坐标。
  5. 如权利要求1所述的傅里叶叠层成像位置标定方法，其特征在于，所述步骤S5中，根据显微视场中心到临近的圆形标志点圆心(x1,y1,z1)和(x2,y2,z2)的 实际物理长度d1、d2以及两者的共面方程Ax+By+Cz+D＝0，计算出显微视场中心的空间三维坐标(x0,y0,z0)，
    (x0-x1)2+(y0-y1)2+(z0-z1)2＝d1 2
    (x0-x2)2+(y0-y2)2+(z0-z2)2＝d2 2
    Ax0+By0+Cz0+D＝0
    其中，d1、d2根据计算出的显微视场中心和圆形标志点的圆心的实际物理尺寸坐标计算而得；显微视场中心和圆形标志点所在的平面Ax0+By0+Cz0+D＝0通过所述圆形标志点圆心的空间三维坐标拟合得出；
    利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面Ax0+By0+Cz0+D＝0的高度z以及投影到所述拟合平面的横坐标x和纵坐标y，完成所述LED阵列相对所述分辨率板聚焦位置时的空间位置(x,y,z)标定；
    以显微视场中心所在平面建立坐标系，计算每一个LED对应在待恢复高分辨率频谱中的空间频率，
    Figure PCTCN2017106786-appb-100006
    Figure PCTCN2017106786-appb-100007
    其中，(xm,n,ym,n,zm,n)为照明区域中第m行第n列的LED用视觉辅助标定的结果，(um,n,vm,n)对应第m行第n列的LED在待恢复高频分辨率频谱中的位置坐标。
  6. 一种视觉辅助的傅里叶叠层成像位置标定装置，其特征在于，所述傅里叶叠层成像位置标定装置运用于傅里叶叠层成像位置标定系统，所述标定系统包括：傅里叶叠层成像装置和视觉辅助双目相机，所述傅里叶叠层成像装置包括显微图像采集相机、显微镜、载物台、贴有圆形标志点的分辨率板和LED阵列，所述分辨率板置于所述载物台上，所述显微图像采集相机位于所述载物台正上方，所述LED阵列位于所述载物台正下方，并与所述载物台所在平面平行，所述视觉辅助双目相机包括：视觉辅助左相机和视觉辅助右相机，所述视觉辅 助左相机、视觉辅助右相机位于所述傅里叶叠层成像装置的正前方，所述装置包括：
    图像像素坐标提取模块，用于利用所述显微图像采集相机采集所述贴有圆形标志点的分辨率板的图像，并结合圆形标志点提取算法提取所述分辨率板的图像中圆形标志点的圆心的图像像素坐标；
    实际物理尺寸坐标计算模块，用于利用所述显微图像采集相机采集图像，得到所述图像的显微视场中心点的图像像素坐标，利用三坐标仪测出所述贴有圆形标志点的分辨率板上每一级每一组元素的实际物理尺寸坐标，通过建立图像坐标系与实际物理尺寸坐标系之间的关系，计算出所述显微视场中心点和所述圆形标志点的圆心的实际物理尺寸坐标；
    第一计算模块，用于利用所述视觉辅助双目相机采集未置于所述分辨率板下的所述LED阵列点亮时的图像，从而得到所述LED阵列中的每一个LED在所述视觉辅助双目相机中的图像坐标，由每一个LED的图像坐标以及预先标定的所述视觉辅助双目相机的标定参数计算出所述LED阵列的每一个LED的第一空间三维坐标；
    第二计算模块，用于利用所述视觉辅助相机采集所述贴有圆形标志点的分辨率板在聚焦位置时的图像以及此时置于所述分辨率板下的LED阵列的图像，并结合预先标定的所述视觉辅助双目相机的标定参数计算出圆形标志点圆心在聚焦位置时的空间三维坐标以及未被遮挡的LED阵列在聚焦位置时的第二空间三维坐标；
    结合所述第一空间三维坐标和所述第二空间三维坐标得到LED阵列在聚焦位置时的空间三维坐标；
    标定模块，用于根据所述圆形标志点圆心的空间三维坐标以及所述显微视场中心和圆形标志点的圆心的实际物理尺寸坐标的关系计算所述显微视场中心的空间三维坐标；
    并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置 时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面的高度z以及LED投影到拟合平面的横纵坐标x、y，完成所述LED阵列相对所述分辨率板的空间位置(x,y,z)标定；
    同时结合所述显微视场中心的空间三维坐标和照明区域中LED阵列的标定结果计算每一个LED在待恢复高分辨率频谱中的位置坐标。
  7. 如权利要求6所述的傅里叶叠层成像位置标定装置，其特征在于，所述图像像素坐标提取模块中，所述圆形标志点提取算法具体为：利用图像处理中canny算子检测圆形标志点边缘图像坐标(x,y)并拟合出椭圆方程x2+2Bxy+Cy2+2Dx+2Ey+F＝0，其中参数B、C、D、E和F由拟合得出，计算所述分辨率板的图像中圆形标志点的圆心的图像像素坐标(x,y)公式为：
    Figure PCTCN2017106786-appb-100008
    Figure PCTCN2017106786-appb-100009
  8. 如权利要求6所述的傅里叶叠层成像位置标定装置，其特征在于，所述图像像素坐标提取模块中，建立的图像坐标系与实际物理尺寸坐标系之间的关系为：
    x0＝xcenter+(u0-ucenter)·dx
    y0＝ycenter+(v0-vcenter)·dy
    xcircle＝xcenter+(ucircle-ucenter)·dx
    ycircle＝ycenter+(vcircle-vcenter)·dy
    其中，(x0,y0)表示显微视场中心的实际物理尺寸坐标，(xcircle,ycircle)表示圆形标志点圆心的实际物理尺寸坐标，(ucenter,vcenter)表示选定的图像坐标系的中心图像坐标，(xcenter,ycenter)表示选定的图像坐标系的物理尺寸坐标，(u0,v0)表示显微视场中心点的图像像素坐标，(ucircle,vcircle)表示圆形标志点圆心的图像像素坐标，dx、dy分别为实际物理尺寸坐标系中x、y方向上的单位物理尺寸。
  9. 如权利要求6所述的傅里叶叠层成像位置标定装置，其特征在于，所述第一计算模块中，计算所述第一空间三维坐标的公式为：
    x＝z Xl/fl
    y＝z Yl/fl
    Figure PCTCN2017106786-appb-100010
    其中，(x,y,z)表示每一个LED的第一空间三维坐标，(Xl,Yl)表示每一个LED在所述视觉辅助左相机中的图像坐标，(Xr,Yr)表示每一个LED在所述视觉辅助右相机中的图像坐标，所述视觉辅助双目相机的标定参数包括：视觉辅助左相机的有效焦距fl，视觉辅助右相机的有效焦距fr，还包括所述视觉辅助左相机和视觉辅助右相机之间的旋转矩阵
    Figure PCTCN2017106786-appb-100011
    平移矩阵
    Figure PCTCN2017106786-appb-100012
    所述第二计算模块中，计算LED阵列在聚焦位置时的空间三维坐标的公式为：
    (X,Y,Z)T＝R2·(x,y,z)T+T2
    其中，(x,y,z)表示第一空间三维坐标，(X,Y,Z)表示第二空间三维坐标，旋转矩阵R2和平移矩阵T2为利用所述第一空间三维坐标和所述第二空间三维坐标计算拟合得到。
  10. 如权利要求6所述的傅里叶叠层成像位置标定装置，其特征在于，所述标定模块，用于根据显微视场中心到临近的圆形标志点圆心(x1,y1,z1)和(x2,y2,z2)的实际物理长度d1、d2以及两者的共面方程Ax+By+Cz+D＝0，计算出显微视场中心的空间三维坐标(x0,y0,z0)，
    (x0-x1)2+(y0-y1)2+(z0-z1)2＝d1 2
    (x0-x2)2+(y0-y2)2+(z0-z2)2＝d2 2
    Ax0+By0+Cz0+D＝0
    其中，d1、d2根据计算出的显微视场中心和圆形标志点的圆心的实际物理尺寸坐标计算而得；显微视场中心和圆形标志点所在的平面Ax0+By0+Cz0+D＝0通过所述圆形标志点圆心的空间三维坐标拟合得出；
    并利用所述显微视场中心的空间三维坐标结合所述LED阵列在聚焦位置时的空间三维坐标计算出照明区域每一个LED到聚焦位置拟合平面Ax0+By0+Cz0+D＝0的高度z以及投影到所述拟合平面的横坐标x和纵坐标y，完成所述LED阵列相对所述分辨率板聚焦位置时的空间位置(x,y,z)标定；
    然后，以显微视场中心所在平面建立坐标系，计算每一个LED对应在待恢复高分辨率频谱中的位置坐标，
    Figure PCTCN2017106786-appb-100013
    Figure PCTCN2017106786-appb-100014
    其中，(xm,n,ym,n,zm,n)为照明区域中第m行第n列的LED用视觉辅助标定的结果，(um,n,vm,n)对应第m行第n列的LED在待恢复高频分辨率频谱中的位置坐标。
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