CN110136118A - A kind of method for cell count based on contours extract - Google Patents
A kind of method for cell count based on contours extract Download PDFInfo
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- CN110136118A CN110136118A CN201910403659.4A CN201910403659A CN110136118A CN 110136118 A CN110136118 A CN 110136118A CN 201910403659 A CN201910403659 A CN 201910403659A CN 110136118 A CN110136118 A CN 110136118A
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- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T7/00—Image analysis
- G06T7/0002—Inspection of images, e.g. flaw detection
- G06T7/0012—Biomedical image inspection
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- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
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- G06T7/10—Segmentation; Edge detection
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- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
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- G06T7/136—Segmentation; Edge detection involving thresholding
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- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
- G06T2207/10—Image acquisition modality
- G06T2207/10056—Microscopic image
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- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
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- G06T2207/20—Special algorithmic details
- G06T2207/20092—Interactive image processing based on input by user
- G06T2207/20104—Interactive definition of region of interest [ROI]
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- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T2207/00—Indexing scheme for image analysis or image enhancement
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- G06T2207/30242—Counting objects in image
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Abstract
The present invention provides a kind of method for cell count based on contours extract, is related to a kind of method for cell count.To solve the problems, such as that existing cytometry is cumbersome, it is not high to count accuracy rate, the present invention carries out OTSU binaryzation to grayscale image and negates；Tally straight line is detected according to reversed binary picture and calculates straight line angle；It is deleted according to angle and selects straight line；Grayscale image and straight line binary image are corrected, and projection histogram is made according to straight line binary picture, determine counting region boundary and intercepts counting region；Top cap transformation is carried out to counting region；Image is handled using gamma transformation, OTSU binaryzation and opening operation；It searches profile and determines number of cells.The present invention can be used for the accurate counting of cell.
Description
Technical field
The present invention relates to a kind of method for cell count, and in particular to a kind of method for cell count based on contours extract.
Background technique
With the development of society, pharmaceutically Bioexperiment is widely used, cell experiment is indispensable one
Point, the cell of culture requires the density ability well-grown for having certain under general condition, so to carry out cell count.It counts
As a result it is indicated with every milliliter of cell number, the principle and method of cell count are identical as blood count.
Existing counting means mainly include the artificial counting method using cell counting board, based on image analysis technology
Automated enumeration instrument, and the automated enumeration instrument using electric-resistivity method (Coulter principle).Wherein, artificial counting method is most
To be universal, by suspension cell sample injection cell counting board, (cell counting board is a kind of common cell count work to experimenter
Tool, is medically commonly used to counting red corpuscles, leucocyte etc. and gains the name, be also commonly used for calculating micro- life such as some bacteriums, fungi, yeast
The quantity of object is a kind of common biology tool) counting chamber, under the microscope to visually observe and be carried out based on manually by rule
Number.The major defect of the method is: (1) since the depth of counting chamber itself is several times as much as cell dimensions, will result in cell in this way
Differential suspension wherein after sample injection, thus the cellular morphology observed can difference, cause the inaccuracy of count results
It judges incorrectly with cell activity；It (2) is usually 10 μ L by the sample of rule injection cell counting board, but in microscope observation area
Sample size in domain is only sub-fraction, and often less than 1 μ L, such cell sample is distributed whether uniformly will in counting chamber
Very big influence is caused to result；(3) be to carry out artificial counting according to certain rule when counting, the difference of operator's level with
And fatigue strength caused by visually observing just introduces very big human error；(4) process is relatively complicated when cell counting board uses, and needs
Periodically to be calibrated the precision to guarantee measurement；And a kind of measurement of sample can only be once carried out in cell counting board,
It is more inconvenient；A general cell count averagely needs to spend 20 minutes, and the efficiency of cell count is lower.Based on image analysis skill
Though the self-reacting device of art avoids macroscopic difficulty, still has following deficiency: (1) introducing disposable counting
The use of piece consumptive material increases user's testing cost；(2) count slice is similar with cell counting board in structure, so there is also
Cell differential suspension leads to the problem of result inaccuracy and activity erroneous judgement on tally；(3) the same with artificial counting, most of base
The result error caused by the instrument of image method has that test sample amount is few is big.Traditional Coulter counter device is whole
Integrated level it is not high, operate not simple and direct enough, be in addition exactly that traditional Coulter counter does not have cell sample motility rate and comments
The function of sentencing, therefore counting can be not accurate enough；Always there are some dead cells due to a variety of causes, total cell in cell colony
Percentage shared by middle living cells is called cell viability, vigor is generally also checked by separation cell in tissue, to understand separation
Process whether cell is had damage.Cell after recovery will also check vigor, understand the effect for freezing and recovering；So
These traditional Coulter counter devices cannot all be completed.
Summary of the invention
The present invention is to solve the problems, such as that existing cytometry is cumbersome, it is not high to count accuracy rate, provides one kind
Method for cell count based on contours extract.
A kind of method for cell count based on contours extract of the present invention, is achieved through the following technical solutions:
Step 1: greyscale transformation is carried out to cellscan image under microscope, then to the gray scale obtained after greyscale transformation
Figure carries out binary conversion treatment, obtains binary picture, is negated to obtain reversed binary picture to the binary picture；
Step 2: detecting the straight line in reversed binary picture and calculating straight line angle；
Step 3: counting the frequency that the straight line angle occurs, determine straight corresponding to the highest angle, θ 0 of the frequency of occurrences
Line, and retain the straight line and straight line normal thereto as grid lines；
Result figure is selected Step 4: obtaining grid lines binaryzation and deleting: one Zhang great little of creation, type and the reversed binary picture
It is identical, and pixel value is set as 0 image, and step 3 is deleted the grid lines that choosing obtains and is plotted on the figure, pixel value is set as
255；
Step 5: cellscan image, grayscale image, grid lines binaryzation delete and select result under microscope described in rotation correction
Figure；
Step 6: determining cell count region: from vertical direction with the rotation correction that obtains step 5 in horizontal direction
Grid lines binary picture afterwards is projected, the length threshold of straight line after setting projection, in throwing vertically and horizontally
The straight line peak value for being greater than length threshold is found in shadow histogram, and then determines grid line position, according to obtained grid line position
Cell count region is intercepted in cellscan image and grayscale image under microscope after rotation correction；
Step 7: carrying out top cap transformation to the counting region grayscale image that step 6 obtains: according to the operator radiuscope of setting
The mask core size for calculating top cap transformation, using top cap conversion process counting region gray level image, prominent cell compartment；
Step 8: carrying out gamma transformation to the top cap transformation results figure obtained by step 7；
Step 9: carrying out binaryzation and opening operation to the gamma transformation results figure obtained by step 8: using at OTSU
The result figure that reason step 8 obtains, and use size for ew1、eh1Mask verification binary picture carry out opening operation, disconnect cell
Between connection；
Step 10: searching profile to the result figure that step 9 obtains, profile number is number of cells.
Present invention feature the most prominent and significant beneficial effect are:
A kind of method for cell count based on contours extract according to the present invention, deletes choosing and rotation using grid lines binaryzation
Turn to correct and determine cell count region, top cap transformation, gamma transformation, OTSU binaryzation, opening operation then are carried out to counting region
Equal image procossings determine number of cells to search profile；Using top cap transformation, gamma transformation removal background area to count results
Influence, accuracy rate can be obviously improved.The method of the present invention is easy to operate, efficiency of the practice is higher, and counts accuracy rate height, compares
The prior art, cell count accuracy rate improve about 8%.
Detailed description of the invention
Fig. 1 is flow chart of the present invention；ROI indicates area-of-interest (Region Of Interest)；
Fig. 2 is heretofore described reversed binary picture；
Fig. 3 is deleted for heretofore described grid lines binaryzation and is selected result figure；
Fig. 4 is the projection histogram of heretofore described horizontal direction；
Fig. 5 is the projection histogram of heretofore described vertical direction；
Fig. 6 is in the present invention according to intercepting cell count area in original image of the grid line position after rotation correction and grayscale image
Domain schematic diagram；
Fig. 7 is heretofore described top cap transformation results figure；
Fig. 8 is heretofore described gamma transformation results figure；
Fig. 9 is the result figure obtained after binaryzation in step 9 and opening operation in the present invention；
Figure 10 is in the present invention by searching contour detecting cell compartment effect picture.
Specific embodiment
Specific embodiment 1: being illustrated in conjunction with Fig. 1~Figure 10 to present embodiment, one kind that present embodiment provides
Method for cell count based on contours extract, specifically includes the following steps:
Step 1: greyscale transformation is carried out to cellscan image under microscope, then to the gray scale obtained after greyscale transformation
Figure carries out binary conversion treatment, obtains binary picture, is negated to obtain reversed binary picture (such as Fig. 2 institute to the binary picture
Show)；
Step 2: detecting the straight line in reversed binary picture and calculating straight line angle；
Step 3: counting the frequency that the straight line angle occurs, the highest angle, θ of the frequency of occurrences is determined0Corresponding is straight
Line, and retain the straight line and straight line normal thereto as grid lines, that is, removing remaining angle is not θ0、θ1Miscellaneous straight line,
Selected result figure (as shown in Figure 3) Step 4: obtaining grid lines binaryzation and deleting: creation one Zhang great little, type with it is described
Reversed binary picture is identical, and wherein the pixel value of pixel is set as 0 image, and step 3 is deleted the grid lines that choosing obtains and is drawn
It makes on the figure, pixel value is set as 255；
Step 5: cellscan image, grayscale image, grid lines binaryzation delete and select result under microscope described in rotation correction
Figure；
Step 6: determining cell count region: from vertical direction with the rotation correction that obtains step 5 in horizontal direction
Grid lines binary picture afterwards is projected, obtain vertically and horizontally projection histogram (Fig. 4 be the present invention in institute
State the projection histogram of horizontal direction；Fig. 5 is the projection histogram of heretofore described vertical direction)；Straight line after setting projection
Length threshold, in projection histogram vertically and horizontally find be greater than length threshold straight line peak value, in turn
Grid line position is determined, according to cellscan image and grayscale image under microscope of the obtained grid line position after rotation correction
Middle interception cell count region (as shown in Figure 6)；
Step 7: carrying out top cap transformation to the counting region grayscale image that step 6 obtains: according to the operator radiuscope of setting
The mask core size for calculating top cap transformation, using top cap conversion process counting region gray level image, prominent cell compartment；Such as Fig. 7 institute
Show；
Step 8: carrying out gamma transformation (gamma transformation) to the top cap transformation results figure obtained by step 7；Such as Fig. 8
It is shown；
Step 9: carrying out binaryzation and opening operation to the gamma transformation results figure obtained by step 8: using at OTSU
The result figure that reason step 8 obtains, and use size for ew1、eh1Mask verification binary picture carry out opening operation, disconnect cell
Between connection；As shown in Figure 9；
It is as shown in Figure 10 contours extract effect picture, profile Step 10: searching profile to the result figure that step 9 obtains
Number is number of cells.
Specific embodiment 2: the present embodiment is different from the first embodiment in that, it is (big using OTSU in step 1
Saliva algorithm, a kind of pair of image that Japanese scholars OTSU was proposed in 1979 carry out the highly effective algorithm of binaryzation) grayscale image is carried out
Binary conversion treatment.
Other steps and parameter are same as the specific embodiment one.
Specific embodiment 3: present embodiment is unlike specific embodiment two, the step 2 specifically:
The straight line in binary picture obtained using accumulated probability Hough transformation to step 1 is detected, according on straight line
Any two points coordinate (xi,yi),(xj,yj), straight line angle, θ, calculation formula is calculated are as follows:
θ=arctan ((xi-xj)/(yi-yj))
Other steps and parameter are the same as one or two specific embodiments.
Specific embodiment 4: present embodiment, unlike specific embodiment one, two or three, the step 5 has
Body are as follows:
It is deleted according to the grid lines binaryzation in step 4 and result figure is selected to determine that it rotates angle, θ1, rotated image width1、
Rotated image height1Are as follows:
w1=| h0·sinθ1+w0·cosθ1|
h1=| w0·sinθ1+h0·cosθ1|
Wherein, w0、h0The respectively described grid lines binaryzation deletes the original width for selecting result figure, original height, rotation center
ForUtilize θ1、w1、h1Image rotation matrix is calculated, then cell under the microscope is swept according to image rotation matrix
Tracing picture, grayscale image, grid lines binaryzation, which are deleted, selects result figure to carry out rotation correction.
Other steps and parameter are identical as specific embodiment one, two or three.
Specific embodiment 5: present embodiment is unlike specific embodiment four, the step 8 specifically:
A (m, n)=a (m, n)/255 is normalized to the pixel value of top cap transformation results figure, wherein convert for top cap
The pixel coordinate value of result figure, a (m, n) are pixel value, and selection is greater than 1 gamma value, to the pixel value fetching after normalization
Number a (m, n)=a (m, n)gamma, so that high gray value regional dynamics range expands.
Other steps and parameter are identical as specific embodiment four.
Specific embodiment 6: present embodiment, unlike specific embodiment five, operator described in step 7 is partly
Diameter is 2.
Other steps and parameter are identical as specific embodiment five.
The present invention can also have other various embodiments, without deviating from the spirit and substance of the present invention, this field
Technical staff makes various corresponding changes and modifications in accordance with the present invention, but these corresponding changes and modifications all should belong to
The protection scope of the appended claims of the present invention.
Claims (6)
1. a kind of method for cell count based on contours extract, which is characterized in that specifically includes the following steps:
Step 1: under microscope cellscan image carry out greyscale transformation, then to the grayscale image obtained after greyscale transformation into
Row binary conversion treatment, obtains binary picture, is negated to obtain reversed binary picture to the binary picture；
Step 2: detecting the straight line in reversed binary picture and calculating straight line angle；
Step 3: counting the frequency that the straight line angle occurs, the highest angle, θ of the frequency of occurrences is determined0Corresponding straight line, and
Retain the straight line and straight line normal thereto as grid lines；
Result figure is selected Step 4: obtaining grid lines binaryzation and deleting: one Zhang great little of creation, type and the reversed binary picture phase
Together, and pixel value be set as 0 image, step 3 is deleted into the obtained grid lines of choosing and is plotted on the figure, pixel value is set as 255；
Step 5: cellscan image, grayscale image, grid lines binaryzation delete and select result figure under microscope described in rotation correction；
Step 6: determining cell count region: from vertical direction and after the rotation correction that obtains step 5 in horizontal direction
Grid lines binary picture is projected, and the length threshold of straight line, straight in projection vertically and horizontally after setting projection
The straight line peak value for being greater than length threshold is found in square figure, and then determines grid line position, is being revolved according to obtained grid line position
Cell count region is intercepted in cellscan image and grayscale image under microscope after transferring to another school just；
Step 7: carrying out top cap transformation to the counting region grayscale image that step 6 obtains: being calculated and pushed up according to the operator radius of setting
The mask core size of cap transformation, using top cap conversion process counting region gray level image, prominent cell compartment；
Step 8: carrying out gamma transformation to the top cap transformation results figure obtained by step 7；
Step 9: carrying out binaryzation and opening operation to the gamma transformation results figure obtained by step 8: being walked using OTSU processing
Rapid eight obtained result figures, and use size for ew1、eh1Mask verification binary picture carry out opening operation, disconnect cell between
Connection；
Step 10: searching profile to the result figure that step 9 obtains, profile number is number of cells.
2. a kind of method for cell count based on contours extract according to claim 1, which is characterized in that used in step 1
OTSU carries out binary conversion treatment to grayscale image.
3. a kind of method for cell count based on contours extract according to claim 2, which is characterized in that the step 2 tool
Body are as follows:
The straight line in binary picture obtained using accumulated probability Hough transformation to step 1 is detected, according to any on straight line
Two o'clock coordinate (xi,yi),(xj,yj), straight line angle, θ, calculation formula is calculated are as follows:
θ=arctan ((xi-xj)/(yi-yj))。
4. a kind of according to claim 1,2 or 3 method for cell count based on contours extract, which is characterized in that the step
Rapid five specifically:
It is deleted according to the grid lines binaryzation in step 4 and result figure is selected to determine that it rotates angle, θ1, rotated image width w1, rotation
Picture altitude h afterwards1Are as follows:
w1=| h0·sinθ1+w0·cosθ1|
h1=| w0·sinθ1+h0·cosθ1|
Wherein, w0、h0The respectively described grid lines binaryzation deletes the original width for selecting result figure, original height, and rotation center isUtilize θ1、w1、h1Image rotation matrix is calculated, then cell under the microscope is swept according to image rotation matrix
Tracing picture, grayscale image, grid lines binaryzation, which are deleted, selects result figure to carry out rotation correction.
5. a kind of method for cell count based on contours extract according to claim 4, which is characterized in that the step 8 tool
Body are as follows:
A (m, n)=a (m, n)/255 is normalized to the pixel value of top cap transformation results figure, wherein m, n is top cap transformation knot
The pixel coordinate value of fruit figure, a (m, n) are pixel value, and selection is greater than 1 gamma value, to the pixel value fetching number after normalization
A (m, n)=a (m, n)gamma。
6. a kind of method for cell count based on contours extract according to claim 5, which is characterized in that described in step 7
Operator radius is 2.
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| CN112507991A (en) * | 2021-02-04 | 2021-03-16 | 季华实验室 | Method and system for setting gate of flow cytometer data, storage medium and electronic equipment |
| CN114511851A (en) * | 2022-01-30 | 2022-05-17 | 南水北调中线干线工程建设管理局 | Hairspring algae cell statistical method based on microscope image |
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