CN103308452B - Optical projection tomography image capturing method based on depth-of-field fusion - Google Patents
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Abstract
The invention discloses an optical projection tomography image capturing method based on depth-of-field fusion. The method comprises the following steps: step one, moving the relative position of a sample and a microscopic device under the same rotation angle, acquiring two-dimensional image sequences on different focusing positions; step two, selecting the clearest points of pixel positions in the two-dimensional image sequence, synthesizing to generate a panorama deep image. The different focusing position images acquired by optical projection tomography system can be fused through the method disclosed by the invention, the obtained panorama deep image is used for three-dimensional reconstruction, and the resolution of the three-dimensional image is improved.
Description
Technical field
The present invention relates to a kind of optical projection tomography technology field, particularly relate to a kind of optical projection fault imaging image acquiring method merged based on the depth of field.
Background technology
Optical projection fault imaging (Optical Projection Tomography, OPT) technology can realize structure and the molecular specificity imaging of 1-10 mm-scale biological specimen.Thinner and the relative transparent of sample in OPT imaging process, visible ray is negligible through scattering effect during sample, and sample is absorption to photon main manifestations, therefore can think that light linearly propagates across sample.During OPT scanning, sample is fixed on a high-precision rotary platform, the LED of employing high stable or laser instrument are as irradiation source, be radiated on sample, highly sensitive electron multiplication formula ccd detector is transferred to after being amplified by microscope equipment through the light signal of sample, by ccd detector collection and imaging, system often gathers a width two-dimension projection, and universal stage rotates a fixed angle, piece image under system acquisition, the data for projection within the scope of gathering 360 ° altogether successively.Therefore, optical projection computed tomography (SPECT) system scanning collection to data be under a series of different angles light through the two-dimensional projection image of sample.Use filtered back-projection method to rebuild all projected images, can 3-D view be obtained.Two-dimension projection whether clear, is directly connected to the height of 3-D view resolution.
Optical projection tomography technology belongs to micro-imaging.In microoptic imaging, low-power microscope operating distance is long, and the depth of field is larger, and because micro objective focal depth range is little, along with the increase of enlargement factor, the depth of field can corresponding reduction.Only have those structures near focussing plane or its to be only visible, structure be the simplest even if this makes, the object of three dimensional depth relatively flat also can not in piece image focusing effect clear.And in optical projection fault imaging, sample thickness is generally 2 ~ 3mm, after high multiple amplifies, depth of field problem becomes particularly serious.And when observing sample, requiring that microscope imaging should have higher resolution to have the enough depth of field again, this is the contradiction of traditional optical hardware.But for optical projection fault imaging, this is again the problem having to solve, so depth of field problem is the bottleneck that restriction optical projection fault imaging obtains high-definition picture.
Summary of the invention
For solving the problem, the invention provides a kind of optical projection fault imaging image acquiring method merged based on the depth of field.The method comprises:
Step 1, by under the same anglec of rotation, the relative position of mobile sample and microscope equipment, gathers the two-dimensional image sequence on different focal position;
Step 2, in described two-dimensional image sequence, choose each location of pixels and synthesize the most clearly in this two-dimensional image sequence, generate full depth map.
The present invention can realize the different focal position image co-registration that optical projection computed tomography (SPECT) system gathers, and the full depth image obtained, for three-dimensional reconstruction, improves the resolution of 3-D view.
Accompanying drawing explanation
Fig. 1 is the principle schematic based on imaging system data acquisition in the optical projection fault imaging image acquiring method of depth of field fusion in the embodiment of the present invention;
Fig. 2 is the flow chart of steps based on Images uniting in the optical projection fault imaging image acquiring method of depth of field fusion in the embodiment of the present invention;
Fig. 3 is the specific experiment result figure based on the optical projection fault imaging image acquiring method of depth of field fusion in the embodiment of the present invention.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly understand, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.Although herein can providing package containing the demonstration of the parameter of particular value, should be appreciated that, parameter without the need to definitely equaling corresponding value, but is similar to described value in acceptable error margin or design constraint.
The invention discloses a kind of optical projection fault imaging image acquiring method merged based on the depth of field.The present invention specially for optical projection fault imaging under a certain fixing anglec of rotation, different focal position image carries out depth of field fusion, the pixel value choosing each location of pixels most articulation point on image sequence carries out image co-registration as the gray-scale value of this pixel on composograph, generate full depth map, thus improve the resolution of 3-D view.
Said method disclosed by the invention is divided into two key steps: data acquisition, image co-registration.Wherein, for utilizing, optical projection fault imaging gathers under a certain fixing anglec of rotation data acquisition step, the two-dimensional sequence image of different focal position; Image co-registration step, for synthesizing the most clearly in this two-dimensional image sequence according to location of pixels each in accessed two-dimensional image sequence, generates full depth map, for three-dimensional reconstruction, thus improves three-dimensional reconstruction body resolution.
The detailed step of said method disclosed by the invention is as follows:
Step S1: by under the same anglec of rotation, the relative position of mobile sample and microscope equipment, collects the two-dimensional image sequence on different focal position.
Optical projection tomography technology belongs to micro-imaging, when sample-size is very little, needs the microscope equipment adopting high-amplification-factor, carries out optical signal amplification.And during high multiple amplification, the depth of field becomes very little, and this will cause collecting each position image very clearly under a certain fixed angle thereupon.But when reality uses optical projection computed tomography (SPECT) system, require that imaging should have high resolution to have the enough depth of field again, this is the contradiction of traditional optical hardware.Effective ways of head it off utilize digital image processing techniques, carries out depth of field fusion to image sequence.
Fig. 1 shows imaging system in the present invention and carries out the principle schematic of data acquisition.As shown in Figure 1, microscope equipment and sample rotation center are on the same axis.By moving radially the relative position of sample and microscope equipment, and image data, all clear part of sample is included in collected image sequence.Horizontal moving stage step pitch is selected to move translation stage by one direction, makes sample image on the detector by fuzzy to clear, then to fuzzy.In whole process, after first time is fuzzy, the position of appearance first focus is the starting point of this image sequence acquisition, progressively equidistant mobile sample, after making image again fuzzy, a front focus position is terminal, records moved step number and step pitch, repeat the data acquisition of next angle, until gathered all data within the scope of 360 ° under different rotary angle.Wherein, before collection image, need adjustment sample position to be in the imaging region of detector, between collected two-dimensional image sequence, Y-axis and Z axis position do not change, and the sharpness of the full depth image that step pitch (position namely in the X-direction) impact between them finally generates; Horizontal moving stage move through computer software control, thus step pitch can be obtained accurately and uniform two-dimensional image sequence.
Step S2: in described two-dimensional image sequence, the pixel value choosing each location of pixels most articulation point on image sequence carries out image co-registration as the gray-scale value of this pixel on composograph, generates full depth map.The two-dimensional sequence image of multiple the different focal positions under a certain angle namely obtained for step S1, carries out depth of field fusion.
Fig. 2 shows the flow chart of steps of in the present invention, the sequence image of different focal position being carried out to depth of field fusion.As shown in Figure 2, this step specifically comprises:
The first step, according to the size of institute's acquisition sequence image, target setting composograph picture element matrix M
0with the size of the matrix T for intermediate data process, its size equals the pixel size of gathered image, and by M
0null matrix is initialized as with T, meanwhile, initialization comparison diagram sequence i=1;
Second step, inputs figure number of plies N to be compared, i.e. the image of the different focal position of a total N width;
3rd step, judge whether image to be compared has completed and compare, namely whether i equals N, compares if do not complete, then i=i+1, and turns to the 4th step, otherwise turns to the 6th step;
4th step, by image pixel matrix M to be compared
iwith target composograph picture element matrix M
0compare, generate intermediary matrix T, in T, whole element is 0 or 1,1 representative M on relevant position
ielement be greater than M
0in element; 0 representative M on relevant position
ielement be not more than M
0in element; Wherein, image pixel matrix M to be compared
iin the element pixel value that is image to be compared in gathered sequence image.
5th step, according to the intermediary matrix T more fresh target composograph picture element matrix M generated
0, namely in intermediary matrix, element is the position of 1, uses M
iin element substitution M
0the element of relevant position, uses image M
iin pixel value alternate image M
0the pixel value of relevant position, so far, completes the fusion of a tomographic image; Turn to the 3rd step to continue to judge, compare if do not complete, then repeat the 4th, the 5th step, otherwise turn to the 6th step;
6th step, completes image co-registration, exports target composograph picture element matrix M
0;
7th step, the depth of field of a certain angle merges and terminates, M
0corresponding image is the depth of field fused images under this angle.
Fig. 3 shows in the present invention and utilizes said method to carry out the result figure of imaging experiment to fruit bat pupa live body.Adopt live body fruit bat pupa to carry out the optical projection fault imaging of depth of field fusion treatment in experiment, the two dimensional image under gathering 360 ° altogether, the image of each angle acquisition 6 different focal positions, namely horizontal moving stage moves 5 times.
This experimental selection translation stage stepping 0.25 millimeter, mobile 5 times, gathers 6 images altogether.Its result as shown in Fig. 31,2,3,4,5,6, the two dimensional image collected under representing asynchronous distance respectively.Visible, in image sequence image by fuzzy to clear again to fuzzy, and often open and have part clearly.The image collected is preserved in order successively, is convenient to image co-registration.Wherein 7 be depicted as this angle under use the depth of field to merge the full depth image obtained, as seen from the figure, the clear position in this image collection Fig. 3 in 1 ~ 6 width figure.This will obviously improve the deficiency of single image, improves sharpness and the packets of information content of result images, for further scientific research analysis provides more sample information.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (12)
1., based on the optical projection fault imaging image acquiring method that the depth of field merges, comprising:
Step 1, by under the same anglec of rotation, the relative position of mobile sample and microscope equipment, gathers the two-dimensional image sequence on different focal position;
Step 2, in described two-dimensional image sequence, choose each location of pixels and synthesize the most clearly in this two-dimensional image sequence, generate full depth map;
Wherein, step 2 specifically comprises:
The first step, according to the size of image in gathered two-dimensional image sequence, target setting composograph picture element matrix M
0with the size of the matrix T for intermediate data process, its size equals the pixel size of gathered image, and by M
0null matrix is initialized as with T, meanwhile, initialization movement images sequence number i=1;
Second step, inputs picture number N to be compared;
3rd step, judge whether image to be compared has completed and compare, namely whether i equals N, compares if do not complete, then i=i+1, and turns to the 4th step, otherwise turns to the 6th step;
4th step, by image pixel matrix M to be compared
iwith target composograph picture element matrix M
0compare, generate intermediary matrix T, in T, whole element is 0 or 1,1 representative M on relevant position
ielement be greater than M
0in element; 0 representative M on relevant position
ielement be not more than M
0in element; Wherein, image pixel matrix M to be compared
iin element be the pixel value of image to be compared in described two-dimensional image sequence;
5th step, according to the intermediary matrix T more fresh target composograph picture element matrix M generated
0, namely in intermediary matrix, element is the position of 1, uses M
iin element substitution M
0the element of relevant position, uses image M
iin pixel value alternate image M
0the pixel value of relevant position, so far, completes the fusion of a tomographic image; Turn to the 3rd step to continue to judge, compare if do not complete, then repeat the 4th, the 5th step, otherwise turn to the 6th step;
6th step, completes image co-registration, exports target composograph picture element matrix M
0;
7th step, the depth of field of a certain angle merges and terminates, M
0corresponding image is the depth of field fused images under this angle.
2. by method according to claim 1, it is characterized in that, the method gathers the two-dimensional image sequence within the scope of 360 ° under different rotary angle, after it first gathers the two-dimensional image sequence of the different focal positions under a certain angle, then rotate the two-dimensional image sequence gathering the different focal positions of this next angle to next angle.
3. by method according to claim 1, it is characterized in that, the relative position of described mobile sample and microscope equipment is that the position of moving sample in the X-axis direction by horizontal moving stage realizes.
4. by method according to claim 3, it is characterized in that, before gathering image, adjustment sample position is in the imaging region of detector.
5., by method according to claim 1, it is characterized in that, between collected two-dimensional image sequence, Y-axis and Z axis position do not change, and the sharpness of the full depth image that the step pitch impact between them finally generates.
6. by method according to claim 1, it is characterized in that, the computing machine that moves through of horizontal moving stage controls, thus can obtain step pitch accurately and uniform two-dimensional image sequence.
7. by method according to claim 1, it is characterized in that, the two dimensional image in step 2 on different focal position, according to the pixel value of every width image, to be chosen on this position the pixel value of pixel value as composograph of most articulation point.
8. by method according to claim 7, it is characterized in that, in step 2, image co-registration is by realizing by the respective pixel values of amplitude ratio compared with width image every in two-dimensional image sequence.
9. by method according to claim 8, it is characterized in that, during by amplitude ratio comparatively, first initialized target composograph picture element matrix, intermediary matrix and two-dimensional image sequence sequence number to be compared, utilize target composograph picture element matrix and image pixel matrix to be compared to generate intermediary matrix, and realized by the element pixel value in intermediary matrix more fresh target composograph picture element matrix.
10. by method according to claim 9, it is characterized in that, intermediary matrix is 0,1 matrix, if the element pixel value in image pixel matrix to be compared is greater than the respective element pixel value of target composograph picture element matrix, then the respective element of intermediary matrix is set to 1, otherwise is 0.
11. by method according to claim 9, it is characterized in that, intermediary matrix is used for the renewal of element pixel value in target composograph picture element matrix, is wherein that the element pixel value of the target composograph pixel corresponding to element position of 1 replaces with corresponding element pixel value in image pixel matrix to be compared by element value in intermediary matrix.
12. by method according to claim 9, and it is characterized in that, after completeer last piece image, the image that the target composograph picture element matrix obtained is corresponding is the full depth map finally obtained.
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| CN111242880B (en) * | 2019-12-30 | 2023-05-02 | 广州市明美光电技术有限公司 | Multi-depth-of-field image superposition method, equipment and medium for microscope |
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