CN110824689B - Full-automatic microscopic image depth of field expanding system and method thereof - Google Patents
Full-automatic microscopic image depth of field expanding system and method thereof Download PDFInfo
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- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
Abstract
The invention discloses a full-automatic microscopic image depth of field expanding system and a method thereof, wherein the system comprises an image acquisition part and an automatic control part, wherein the image acquisition part comprises a color camera, a cylindrical lens, an objective table and a light source; the automatic control part comprises a stepping motor, a guide rail, a sensor and intermediate equipment, the whole system collects images at different positions at equal intervals in the upper and lower ranges of the focus plane of each visual field, and the series of images are subjected to image fusion to obtain a result of expanded depth of field. According to the method, the field depth expansion is carried out in an image fusion-based mode, so that the clearest image of each field of view is obtained, and compared with the traditional field depth expansion mode, the luminous flux and the resolution of an optical system cannot be weakened; the image acquisition and the image fusion are full-automatic, and the realized fusion algorithm is simple and does not discard the image detail information.
Description
Technical Field
The invention belongs to the optical imaging technology, and particularly relates to a full-automatic microscopic image depth of field expanding system and a method thereof.
Background
The depth of field is one of the very important parameters of a microscope, and refers to the range of the object moving back and forth when the imaging is clear. Generally, the field depth range of a common optical microscope is very limited, and especially when the microscope needs to be observed under a high power microscope, the field depth is smaller, and the acquisition and observation of images by an observer are more difficult. With the technical improvement of computer digital image processing, images can be fused, denoised and the like, a field depth expanding microscope is developed at present, and the field depth expanding method mainly comprises two implementation modes: a non-image fusion mode; based on the fusion mode of the image. The non-image fusion mode is to use the optical imaging principle to reform the traditional microscope, and people originally propose a method of applying a ring aperture, wherein the method changes the shape of the aperture diaphragm of the microscope, and then propose an amplitude filtering method, wherein the method mainly changes the aperture form, the improved amplitude distribution meets the Gaussian distribution or the super-Gaussian distribution, and although the mode effectively obtains the depth of field expansion, the luminous flux and the resolution ratio of the system are weakened. The image fusion-based mode is to collect a plurality of images according to certain positions and fuse the images according to a certain fusion algorithm to obtain images (Zhao red wave, depth of field synthesis algorithm research and application thereof in super depth of field microscope design [ D ]. 2017). In general, the method of non-image fusion brings many disadvantages, and is not a perfect implementation scheme.
In the whole field depth expanding system, automatic focusing operation is firstly carried out, so that an object is focused on a focal plane, and then the object can enter a field depth area, and image acquisition is carried out on the upper part and the lower part of the focal plane. Therefore, the selection of a proper automatic focusing algorithm is particularly important for the whole system, the focusing plane image is clearer than the defocusing image, the detail information is richer, the gradient value is larger in the spatial domain, and the high-frequency component is more in the frequency domain. The sharpness evaluation function based on the frequency domain is mainly based on fourier transform, is mainly determined by high-frequency information in an image, and has high sensitivity but relatively large calculation amount. The definition evaluation function based on the spatial domain mainly comprises a square gradient function, a Tenengrad function, a Brenner function and a Reblur secondary fuzzy function, and the definition evaluation function based on the frequency domain is a Fourier transform function (Chen autumn, digital image automatic focusing technical research and system implementation [ D ]. Western Ann electronic technology university 2007).
The automatic focusing process is a process of controlling the microscope to move by a driving motor to obtain images under different planes and then searching the clearest image position according to a definition evaluation function, and the common focusing search method comprises hill climbing searchLaw, traversal search method, curve fitting method. (1) A mountain climbing search method: the hill climbing search method firstly requires that a focus evaluation function has good unimodal performance, namely that a focus evaluation function curve is required to be at the best focus clear position at the extreme point position, and monotonously increases on the left side of the extreme point and monotonously decreases on the right side of the extreme point. The mountain climbing search method is as shown in the figure, and the main idea is to rapidly reach the focusing position, namely the mountain peak position, by increasing the moving step length at the position far away from the focusing position under the condition that the focusing evaluation function has good unimodal shape, and to search the position of further refined focusing by reducing the step length by half when the moving position is close to the focusing position (Cheng jin, digital image processing autofocus technology research and realization thereof [ D]The university of west ampere electronic science and technology 2007). However, this method has high requirements on the unimodal type of the focus evaluation function, but some samples are easily affected by other factors when being calculated by the evaluation function, and a plurality of peak points may exist, so that the universality of the method is not good. (2) Curve fitting, in the mathematical domain, can be performed by selecting some relevant points. We assume that the focus evaluation function has good unimodal, and the function is a quadratic function, and a quadratic curve is used for fitting, as shown in the formula: f (z) ═ az2+ bz + c, the curve fitting method makes the focus evaluation function curve have good unimodal and an extreme point, and makes the function curve as a quadratic function to perform curve fitting. Selecting three points (x) near the extreme point1，y1)、(x2，y2)、(x3，y3) And performing quadratic fitting, wherein the requirement on the selected point is higher, and the fitting accuracy is higher the closer the selected peak point is to the peak point. (3) And the traversal search method is fixed at every certain distance for image acquisition by controlling the movement of the stepping motor, then each layer of image is calculated and evaluated, and simultaneously the image definition at each position is calculated. At the clearest position, the greater the sharpness of the image, and by comparing the magnitudes of the sharpness of the images, we can know the focus position of the image. The traversal search method is simple and easy to understand and has strong anti-interference capability, but because the method needs to calculate and evaluate each layer of image,the calculated amount is large, the moving distance is not clear, and a large amount of experiments are needed to summarize, if the distance is set too large, the focus plane may be skipped, which causes the defect of local search, and if the distance is set too small, the focusing precision is high, but because the distance is too small, the number of moving steps is too large, the calculated amount is too large, and therefore, the traversal search method has no universality.
Disclosure of Invention
The invention aims to provide a full-automatic microscopic image depth of field expanding system and a method thereof, which overcome the defect that the traditional depth of field expanding mode can weaken the luminous flux and the resolution of the system and obtain the clearest image.
The technical solution for realizing the invention is as follows: a full-automatic microscopic image depth of field expanding system and a method thereof comprise an image acquisition part and an automatic control part, wherein the image acquisition part comprises a color camera, a cylindrical lens, an objective table and a light source, wherein the color camera is fixed at the tail end of the cylindrical lens and is used for acquiring the imaging of a microscope in real time; the automatic control part comprises a stepping motor, a guide rail, a sensor and intermediate equipment, wherein the stepping motor is arranged on the guide rail, the sensor is arranged on the guide rail and used for limiting the motion stop position of the stepping motor on the guide rail, the cylindrical lens is fixed with the stepping motor through the intermediate equipment, the acquisition field of view is changed by moving the stepping motor back and forth and left and right, the image in the focusing range of the microscope is acquired by moving the stepping motor up and down, the whole system acquires the images at different positions at equal intervals in the upper and lower ranges of the focusing surface of each field of view, and the series of images are subjected to image fusion processing to obtain the result of expanded depth of field.
Compared with the prior art, the invention has the following remarkable advantages: (1) the field depth expansion is carried out by adopting an image fusion-based mode, so that the clearest image of each visual field is obtained, and compared with the traditional field depth expansion mode, the luminous flux and the resolution of the optical system cannot be weakened. (2) The definition is defined by solving the Tenengrad function value after the image is subjected to Gaussian filtering, so that automatic focusing is realized, and the definition is defined by directly solving the Tenengrad function value, so that the accuracy is higher. (3) The image acquisition and the image fusion are full-automatic, and the realized fusion algorithm is simple and does not discard the image detail information.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
Fig. 1 is a two-dimensional structure diagram of the fully automatic microscopic image depth of field expanding system of the present invention.
FIG. 2 is a flow chart of the method for expanding the depth of field of a fully automatic microscopic image according to the present invention.
FIG. 3 is a schematic diagram of a variable step-pitch traversal method used in the present invention.
Fig. 4 is a diagram of the effect of the image fusion algorithm used in the present invention after fusion.
Fig. 5 shows several images of the invention with the objective 6 moving up and down in a field of view in a focused position.
Fig. 6 is an image obtained after the image in fig. 5 is acquired and the depth of field is expanded by using an image fusion algorithm.
Fig. 7 shows several images of the invention with insect wings moved up and down the objective 6 in a focal position of the field of view.
Fig. 8 is an image obtained after the image in fig. 7 is acquired and the depth of field is expanded by using an image fusion algorithm.
Fig. 9 is a line graph of the present invention using gaussian filtering in combination with Tenengrad functions at different positions.
Fig. 10 is a line graph of the present invention based on fourier transform-based merit functions at different locations.
Fig. 11 is a line graph of the present invention at different positions based on the Tenengrad merit function.
FIG. 12 is a line graph of the present invention based on the squared gradient merit function at different locations.
Fig. 13 is a schematic diagram of the optical path of the system of the present invention.
Detailed Description
With reference to fig. 1 and 13, the full-automatic microscopic image depth of field expanding system of the present invention includes an image collecting part and an automatic control part, wherein the image collecting part includes a color camera 1, a tube lens 5, an objective lens 6, an objective table 7, and an illumination system (light source) 8, wherein the color camera 1 is fixed at the tail end of the tube lens 5 for collecting the imaging (microscopic image) of the microscope in real time, the objective lens 6 is installed below the front end of the tube lens 5, the objective table 7 is fixed at the position below the objective lens 6 in the vertical direction, the light source 8 is fixed on a base 9 below the objective table 7, and the distance from the objective table 7 is about 60-70 mm; the automatic control part comprises a stepping motor 2, a guide rail 3, a sensor 4 and an intermediate device 10, wherein the stepping motor 2 is arranged on the guide rail 3 and can move along the guide rail 3, the sensor 4 is arranged on the guide rail 3 and is used for limiting the motion stop position of the stepping motor 2 on the guide rail 3, the intermediate device 10 is a shell with a screw hole, the upper part of the shell is connected with a barrel mirror 5 through the screw hole, the shell is connected with the stepping motor 2 through the screw hole, and the barrel mirror 5 is fixed with the stepping motor 2 through the intermediate device 10. The color camera 1 and the stepping motor 2 are respectively connected with a computer, and the system expands the depth of field on the computer according to the following process. Therefore, the automatic control part can change the collection field of view by moving the stepping motor 2 forwards, backwards, leftwards and rightwards, can move the collection microscope to collect images within the focusing range by moving the stepping motor 2 upwards and downwards, the whole system collects the images at different positions at equal intervals within the upper and lower ranges of the focusing surface of each field of view, and the series of images are processed by an image fusion algorithm to obtain the result of expanded depth of field. As shown in fig. 13, which is an optical path diagram of the system, first, the illumination system 8 generates a light source, the light source generates light which is transmitted through an object, then passes through the objective lens and the tube lens, and then irradiates on the reflector, and finally forms an enlarged microscopic image on the image plane.
With reference to fig. 2, the fully automatic microscopic image depth of field expanding method of the present invention comprises the following steps:
the first step is as follows: the automatic focusing of the microscopic image, namely moving the stepping motor 2 for many times, driving the objective lens 6 to move and collect the image, traversing according to the mode, processing the collected image, firstly performing Gaussian filtering on the image, then solving a Tenengrad function value for the filtered image to obtain a definition focusing evaluation function value of the image, comparing the focusing evaluation function values of each image to obtain a maximum focusing evaluation function value, and returning to the position corresponding to the focusing evaluation function value.
The experimental comparison of the evaluation functions in the background art is shown in the figure: it was found that the Tenengrad merit function, the squared gradient function, and the fourier transform function all reflect the peak magnitude of the focus position, and data simulation was performed on these several merit functions, fig. 10 is a line graph based on the fourier transform merit function at different positions, fig. 11 is a line graph based on the Tenengrad merit function at different positions, and fig. 12 is a line graph based on the squared gradient merit function at different positions. Therefore, some small peak points appear at other out-of-focus positions due to the influence of external factors, and even a peak value larger than the focus position may appear, so that the value of the definition of the focus position relative to other positions in the depth of field range cannot be accurately reflected. The method comprises the steps of carrying out Gaussian filtering processing on images on the basis of the images, firstly carrying out Gaussian filtering on each image by using a Gaussian kernel with the sigma of 1.4 and the size of 5 multiplied by 5, then calculating a gradient value by using a Tenengrad function to obtain a focus evaluation function value, finding a gradient value at a focus position which can well reflect through the Gaussian filtering processing, and finding other out-of-focus positions which hardly have any peak value, wherein the step is shown in figure 9. The sharpness of the image is defined in this way in the present invention, and the calculation formula for the Tenengrad function to calculate each pixel is as follows.
First fx＝[f(x+1，y-1)+2f(x+1，y)+f(x+1，y+1)][f(x-1，y-1)+2f(x-1，y)+f(x-1，y+1)]。
Second fy＝[f(x-1，y+1)+2f(x，y+1)+f(x+1，y+1)]-[f(x-1，y-1)+2f(x，y-1)+f(x+1，y-1)]。
Computing
Wherein
The traversal method in the whole focusing process is improved in the prior scheme, the scheme avoids repeated searching results of a hill climbing search method, a traversal search method and a curve fitting method, avoids repeated back and forth movement of an objective lens, and realizes the automatic focusing scheme of the invention through the traversal method and a focusing evaluation function, and as shown in fig. 3, the specific traversal method comprises the following steps:
step one, traversing for the first time, wherein the stepping motor 2 moves to drive the objective lens 6 to move together, and the defocusing distance is estimated so that the objective lens 6 moves to the initial position a of the first traversal in the figure 31(Upper bound Iimit of FIG. 3) and determines the initial search field [ a [1，b1]，b1The position is the end position of the first traversal (Lower limit in fig. 3), the whole search area is traversed by a large step (which may be 0.04-0.08mm) from the Upper limit of the area, i.e., Upper limit in the graph, to the Lower limit, and a focus position 1(focus point1) is obtained, and the stepping motor 2 moves to move the objective lens 6 to the focus position 1.
Step two, the stepping motor 2 drives the objective lens 6 to move to a distance above the focusing position 1, and a second search domain [ a ] is set2，b2]，a2、b2Respectively, the start position and the end position of the second traversal. A second search is performed with a small step size (which may be half of the large step size) to get the focus position 2(focus point 2).
And step three, continuously reducing the moving step length, repeating the step two, and when the focusing is finished when the step length is smaller than the threshold (0.01mm), controlling the objective lens 6 to return to the focusing position (the finally obtained focusing position) by the stepping motor 2, wherein fig. 3 is a schematic diagram of the improved variable-step-pitch traversing method.
The second step is that: after the automatic focusing step is completed, the depth of field expansion can be carried out according to the focused position, and the depth of field expansion mode adopted by the invention is based on an image fusion method to obtain a target image. For each pixel point in the microscopic image, at the focus position, the information is the most clear, but the brightness is the least, as opposed to not at the focus position. In the invention, a microscopic imaging system is composed of the illumination system 8, the objective lens 6 and the tube lens 5, and after the microscopic imaging system images, the camera 1 collects images on an image surface, as shown in fig. 13 and fig. 1. The stepping motor 2 moves to drive the objective lens 6 to collect images in the set upper and lower ranges, after the series of images are collected through the camera 1, corresponding pixel values in all the images are compared, according to the characteristic that the brightness at the focusing position is minimum, the minimum pixel value at each pixel position is taken as the optimal pixel value, all the pixels are traversed in sequence, the pixel value of each pixel of the target image is obtained, the target image is the result after the depth of field is expanded, and the image contains the most sample information, so that the method can not lose the information of the image, and the calculated amount is small. The upper left side of fig. 4 is an image focused on the lens, but the camera is in an out-of-focus state, the lower left side of fig. 4 is an image focused on the camera, but the lens is in an out-of-focus state, and the large image on the right side is a result of fusing the two images by using the image fusion method, so that the clearest information of the focusing states of the two images is retained.
With reference to fig. 1 and fig. 2, in the fully automatic microscopic image depth of field expanding method of the present invention, two times of focusing are taken as an example to explain the specific steps:
in the first step, assuming that the total number of target images is represented by multiplying the number of rows row by the number of columns col, a row-direction movement step (distance between adjacent positions) row _ d, a column-direction movement step col _ d, the number of images to be fused in depth expansion is N, and an objective lens 6 movement step z _ d are set, and then a total displacement AF1_ dis and a movement step AF1_ d of primary focusing, a total displacement AF2_ dis and a movement step AF2_ d of secondary focusing are set, and AF1_ d is larger than AF2_ d.
And secondly, the stepping motor 2 firstly moves to the position of the first row and the first column of the shooting visual field according to the parameters set in the previous step, firstly carries out primary focusing, carries out step AF1_ d downward movement to acquire images through the color camera 1, carries out automatic focusing processing on the images until the total displacement of AF1_ dis is moved, calculates the position of the clearest image and returns the position of the clearest image, the process is equivalent to coarse focusing, then moves upwards by half of the total displacement of the secondary focusing for AF2_ dis/2, carries out step AF2_ d downward movement, carries out secondary focusing, obtains the position of the clearest image after the AF2_ dis displacement is moved, returns to the position of the clearest image obtained for the second time, and is the focusing point of the visual field position, and the secondary focusing is equivalent to fine focusing.
And thirdly, according to the position focused on the first row and the first column in the second step, starting to move upwards (N-1) z _ d/2 displacement according to the fused total number N and the movement step z _ d, starting to collect images from the position, then moving downwards by the stepping motor 2 according to the step size to move for total (N-1) z _ d displacement, collecting images once by the color camera 1 every time when moving one step size, and returning to the secondary focusing position in the previous step after collecting.
And fourthly, carrying out image fusion processing on a series of images acquired at the visual field position in the third step to finally obtain an image of the visual field position after the depth of field is expanded, namely a target image.
And fifthly, after the first target image is obtained after the previous step of processing, moving a step row _ d to a second row position of the first row along the row direction, namely a next view field position, firstly carrying out secondary focusing like the previous view field, carrying out image acquisition in an upper and lower range after focusing on a focusing plane, and carrying out image fusion after acquiring the images to obtain a second target image. In the following moving process, image acquisition and fusion in the field depth are carried out once by moving one step length, the image acquisition and fusion are carried out continuously in the row direction, the image acquisition and fusion are carried out until the image acquisition and fusion are carried out to the first col column, then the image acquisition and fusion are carried out to the second row, the first col column, the second row, the second col-2 column, the 2 nd column, the 1 st column … … in the row direction, then the image acquisition and fusion are carried out to the next row, the first col column, the 2 nd column, the 3 rd column, the … … th col-1 st column and the first col column in the row direction, the field of view is switched according to the method until the row col target image is obtained and the final field of view position is finished.
The method of the invention not only solves the defect that the traditional depth of field expanding mode can weaken the luminous flux and resolution of the system, but also automatically focuses by controlling the stepping motor to move the lens at each visual field position through an automatic focusing and traversing method, then automatically collects the upper and lower range images at the focused positions, fuses the series of images, and obtains the final depth of field expanding result, namely the clearest image.
Claims (2)
1. A full-automatic microscopic image depth of field expanding method is characterized by comprising the following steps:
the first step is as follows: the automatic focusing of the microscopic image is processed, namely the stepping motor (2) is moved for multiple times to drive the objective lens (6) to move and collect the image, traversal is carried out according to the mode, the collected image is processed, Gaussian filtering is firstly carried out on the image, then a Tenengrad function value is obtained on the filtered image, the definition focusing evaluation function value of the image is obtained, the focusing evaluation function value of each image is compared, a maximum focusing evaluation function value is obtained, and the maximum focusing evaluation function value returns to the position corresponding to the focusing evaluation function value;
the second step is that: after the automatic focusing step is completed, field depth expansion is carried out according to the focused position, a target image is obtained by image fusion, a microscopic imaging system is formed by an illumination system (8), an objective lens (6) and a cylindrical lens (5), after the microscopic imaging system is imaged, a color camera (1) collects images on an image surface, a stepping motor (2) moves to drive the objective lens (6) to collect the images in a set upper range and a set lower range, the series of images are collected by the color camera (1), then corresponding pixel values in all the images are compared, according to the characteristic of minimum brightness at the focused position, the minimum pixel value at each pixel position is taken as the optimal pixel value, all the pixels are traversed in sequence to obtain the pixel value of each pixel of the target image, and the target image is the result after the field depth expansion;
the color camera (1) is fixed at the tail end of the tube lens (5) and used for collecting images of the microscope in real time, the objective lens (6) is installed at the position below the front end of the tube lens (5), the tube lens (5) and the stepping motor (2) are fixed through intermediate equipment, the collection field of view is changed by moving the stepping motor (2) back and forth, left and right, and images in the focusing range of the microscope are collected by moving the stepping motor (2) up and down;
the specific steps of realizing the depth of field expansion by twice focusing are as follows:
firstly, assuming that the total number of target images is represented by multiplying the number of rows row by the number of columns col, setting a row direction movement step row _ d, a column direction movement step col _ d, the number of images to be fused in depth expansion is N, and an objective lens (6) movement step z _ d, and then setting a total displacement AF1_ dis and a movement step AF1_ d of primary focusing, a total displacement AF2_ dis and a movement step AF2_ d of secondary focusing, and AF1_ d is larger than AF2_ d;
secondly, the stepping motor (2) firstly moves to the position of the first row and the first column of the shooting visual field according to the parameters set in the previous step, firstly, the first focusing is carried out, the step AF1_ d moves downwards to collect images through the color camera (1), automatic focusing processing is carried out on the images until the total displacement of AF1_ dis is completed, the position of the clearest image can be calculated, the clearest image position is returned, the process is equivalent to coarse focusing, then, the half distance AF2_ dis/2 of the total displacement of the secondary focusing is moved upwards, the movement of the step AF2_ d is carried out downwards, the second focusing is carried out, the position of the clearest image can be obtained after the same movement of the AF2_ dis is completed, the position of the clearest image obtained in the second time is returned to be the focusing point of the visual field position, and the second focusing is equivalent to fine focusing;
thirdly, according to the position focused on the first row and the first column in the second step, moving upwards (N-1) × z _ d/2 displacement according to the fused total number N and the motion step z _ d, collecting images from the position, then moving downwards according to the motion step z _ d by the stepping motor (2) for total (N-1) × z _ d displacement, collecting images once by the color camera (1) every time one step is moved, and returning to the secondary focusing position in the previous step after the collection is finished;
fourthly, carrying out image fusion processing on a series of images acquired at the visual field position in the third step to finally obtain an image of the visual field position after the depth of field expansion, namely a target image;
fifthly, after the first target image is obtained after the previous step of processing, moving a step row _ d to a second row position of the first row along the row direction, namely a next visual field position, firstly carrying out secondary focusing like the previous visual field, carrying out image acquisition in an upper and lower range after focusing on a focusing plane, and carrying out image fusion after acquiring the images to obtain a second target image; in the following moving process, image acquisition and fusion in the field depth are carried out once by moving one step length, the image acquisition and fusion are carried out continuously in the row direction, the image acquisition and fusion are carried out until the image acquisition and fusion are carried out to the first col column, then the image acquisition and fusion are carried out to the second row, the first col column, the second row, the second col-2 column, the 2 nd column, the 1 st column … … in the row direction, then the image acquisition and fusion are carried out to the next row, the first col column, the 2 nd column, the 3 rd column, the … … th col-1 st column and the first col column in the row direction, the field of view is switched according to the method until the row col target image is obtained and the final field of view position is finished.
2. The method of claim 1, wherein the step of traversing is as follows:
firstly, traversing for the first time, wherein the stepping motor (2) moves to drive the objective lens (6) to move together, and the defocusing distance is estimated to enable the objective lens (6) to move to the initial position a of the first traversal1And determining an initial search field [ a ]1，b1]，b1Traversing the whole search area in large step length to obtain a first-traversed focusing position 1, and moving the stepping motor (2) to enable the objective lens (6) to move to the first-traversed focusing position 1;
step two, the stepping motor (2) drives the objective lens (6) to move to a distance above the first-traversed focusing position 1, and a second search domain [ a ] is set2，b2]，a2、b2Respectively carrying out second search on the initial position and the end position of the second traversal in small step length to obtain a focusing position 2 of the second traversal;
and step three, continuously reducing the moving step length, repeating the step two, and when the step length is smaller than the threshold value, finishing focusing, and controlling the objective lens (6) to return to the focusing position by the stepping motor (2).
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