CN103390277B - image analysis method - Google Patents
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- CN103390277B CN103390277B CN201310310245.XA CN201310310245A CN103390277B CN 103390277 B CN103390277 B CN 103390277B CN 201310310245 A CN201310310245 A CN 201310310245A CN 103390277 B CN103390277 B CN 103390277B
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Abstract
The invention discloses a kind of image analysis method, to the digital picture obtained, adopt threshold method, high luminance pixels point according to labelling in advance is that positioning datum point positions biochip, then according to biochip attribute information set in advance, determine the effective coverage of each test point, and then, adopt threshold method, high luminance pixels point according to labelling in advance obtains the sub-positioning datum point of each effective coverage, facula position according to the sub-positioning datum point location mark test substance of effective coverage, finally detect determined facula position, signal value and the standard substance comparison of detection facula position and draw analysis result.According to the present invention can draw accurately biochip reflection result images in each detection hole/signal value.
Description
Technical field
The present invention relates to a kind of image analysis method, be specifically specifically applied to the information analysis method of biochip imaging.
Background technology
Biochip, also known as DNA chip or gene chip, they are the crystallizations that DNA hybridization probe technique combines with semi-conductor industry technology.This technology means hybridizes with being with fluorescently-labeled DNA sample molecule after being fixed on by a large amount of probe molecules on holder, by detecting the hybridization signal intensities of each probe molecule and then obtaining quantity and the sequence information of sample molecule.
Biochip technology originates from making nucleic acid molecular hybridization.So-called biochip refers generally to the microarray hybridization cake core (micro-arrays) of the biological information molecule (such as genetic fragment, DNA fragmentation or polypeptide, protein, glycan molecule, tissue etc.) that high density is fixed on mutual supporting dielectric, in array, the sequence of each molecule and position are known, and are pre-set sequence dot matrix.Micro-fluidic chip (microfluidicchips) and liquid phase biochip are ratio biochip new techniques of development after micro-array chip, and biochip technology is the substance of Systems biotechnology.
Saying more intuitively, biochip puts biological sample exactly on the materials such as one block of sheet glass, silicon chip or nylon membrane, is then collected signal by a kind of instrument, by computer analytical data result.
Although people are likely to be easy to biochip and electronic chip to connect, and it is true that both truly have a most basic common ground: have the data message of magnanimity in microsize.But they are diverse two kinds of things, on electronic chip, Boulez is semi-conductor electricity subelement one by one, and on biochip Boulez be bioprobe molecule one by one.
Last century the nineties, guide, by robot automatic printing or light, the biomolecule microarry that chemical synthesising technology manufactures on silicon chip, glass, gel or nylon membrane, it is achieved to compound, protein, nucleic acid, cell or other biological components accurately, the screening of quick, large information capacity or detection.
The intervention of biochip will necessarily amplify out the analysis of biochip, analyzes biological expression, obtains the analysis result of analyzed object.The acquisition (Imagecapture) of usual analyzing biochips result images is to be completed by charge coupled cell (chargecoupleddevice, CCD), and then obtains digital picture.General Tiff image file is the carrier that biochip experimental data is initial, by be automatically positioned and identification chip on each point, the background of quantitative each point, signal intensity, calculating point mass determinations, image is converted into initial data.
As previously mentioned, biochip finally requires over graphical analysis to obtain desired data, generally except added sample on a biochip, also can add some auxiliary information, so that the location of test point, such as fluorescent labeling is set in test point particular orientation or position, for the location of test point.And original pictorial information also can comprise other some key elements, thus generally require and first position biochip, therefore, biochip also can be arranged to the telltale mark of himself, as carried out labelling in its upper left corner or the upper right corner, according to the feature of these labellings in advance, for its fixation and recognition.Described test point is generally the detection hole according to predetermined regular array or forms sample application array by certain equipment, and detection hole is built-in with relevant biological material to be detected.Therefore, to detection hole be accurately positioned the premise being to ensure that analyzing biochips.But due to image obtain, as shooting can be subject to the impact of irradiation around so that shooting quality influenced to different extents, the coding microball fluorescence in particular for labelling is easily bleached, and then so that above-mentioned location become relatively difficulty.
In some implementations, Fluorescence Scanner, the information such as on mensuration biochip, the fluorescence intensity of each point, makes standard curve by fluorescence intensity, the content of the various material to be detected of analytical calculation are utilized.Obviously, this realization does not make full use of specifically labelled positional information, and adopts other approach, and its overall calculation amount is relatively larger, and there is no positive impact for location accurately.
Summary of the invention
For this, it is an object of the invention to provide a kind of image analysis method, it is possible to draw accurately each detection hole in biochip reflection result images/signal value.
The present invention is by the following technical solutions:
A kind of image analysis method, to the digital picture obtained, adopt threshold method, high luminance pixels point according to labelling in advance is that positioning datum point positions biochip, then according to biochip attribute information set in advance, determine the effective coverage of each test point, and then, adopt threshold method, high luminance pixels point according to labelling in advance obtains the sub-positioning datum point of each effective coverage, facula position according to the sub-positioning datum point location mark test substance of effective coverage, finally detect determined facula position, signal value and the standard substance comparison of detection facula position and draw analysis result.
Be can be seen that by such scheme, according to the present invention, depend on the production method that current biochip is general, it detection hole possessing rule or test point arrangement mode, and the feature that general phosphor dot is identified, by the optical information of optical point, utilize the predetermined positional information that threshold method combines detection hole or test point to determine facula position, and then carry out the mode detected, it is possible to relatively accurate draws the signal value of each detection hole or test point in biochip.Holistic approach is also fairly simple, it is ensured that the efficiency of process is relatively high.
Above-mentioned image analysis method, positioning datum point and sub-positioning datum point are positioned at the upper left corner of corresponding institute identified areas.
Above-mentioned image analysis method, the method finding positioning datum point is as follows:
A) to be not less than two pixels and to be not more than four pixels for the step-length described digital picture of traversal；
B) for each pixel traversed, in the neighborhood centered by this pixel, each statistical parameter that basis predetermined biochip attribute is corresponding, statistical parameter includes the average brightness value after digital picture described in the average brightness value of described digital picture, Edge Enhancement and statistical variance；
If c) certain pixel meets the following conditions, confirm as a datum mark:
C1) threshold values × 0.6, edge in the whole region of signal averaging > in the average hot spot of the Edge Enhancement image in the neighborhood centered by this pixel；
C2) gray value Edge Enhancement image with average brightness value 0.8 to 1.4 times be the pixel in the neighborhood of radius quantity and other radiuses be in a ratio of at most；
D) position weight of all datum marks of calculation procedure c) gained；
E) position weight is minimum or occurrence number is maximum datum mark is found out as positioning datum point.
Above-mentioned image analysis method, the computing formula of position weight is:
Math.Sqrt(((_topy-initY)/lgRect.RowSpan+lgRect.RowSpan)×((_topx-initX)/lgRect.ColSpan+lgRect.ColSpan))；
Wherein-topy is the Y coordinate of Current Scan pixel position
InitY is the Y coordinate of Current Scan pixel place monitoring holes upper left position
LgRect.RowSpan is the height of monitoring holes in chip attribute
_ topx is the X-coordinate of Current Scan pixel position
InitX is the X-coordinate of Current Scan pixel place monitoring holes upper left position
LgRect.ColSpan is the width of monitoring holes in chip attribute.
Above-mentioned image analysis method, biochip location is when positioning datum point is determined, is biased drawing according to the positioning datum point determined according to the position relationship of default biochip with positioning datum point.
Above-mentioned image analysis method, the defining method of effective coverage is based on biochip detecting the array way of hole or test point, and specifically, col width information high according to detection control or the ranks number of test point, row, draws graticule, form small grid, the corresponding effective coverage of each small grid.
Above-mentioned image analysis method, the method obtaining sub-positioning datum point is as follows:
1) filtration parameter that one group of signal intensity successively decreases is scanned the currently active region successively, using having of being first filled into more than setting threshold value and there is the point of circular edge as subbase on schedule；
2) obtaining the edge threshold that the currently active region is corresponding, corresponding gray scale filter value is decided to be 0.3 times of this edge threshold；
3) scan all pixels in the currently active region, find out the pixel meeting following condition:
3.1) position of this pixel is greater than the product of background value and signal intensity filtration parameter；
3.2) average signal value of this rectangular area, pixel center 3 × 3 is more than 0.8 times of background colour；
3.3) average signal value of this rectangular area, pixel center 3 × 3 is more than eight adjacent regional signal averages；
3.4) in edge enhanced images, the gray value of this pixel is maximum；
4) pixel drawn according to step 3) and the value of radius, calculate the variance of pixel in current region, average；
5) finding signal averaging to be in peak value is corresponding radius；
6) calculation procedure 3) drawn the position weight of each pixel；
7) pixel that position weight value is minimum or the weighted value that occurrence number is maximum is corresponding is found, for the sub-positioning datum point that small grid is positioned.
Above-mentioned image analysis method, if the positional information of the sub-positioning datum point drawn exceedes setting value in the condition lower deviation being benchmark with biochip attribute, then corrects this sub-positioning datum point.
Above-mentioned image analysis method, on the basis that small grid has positioned, it is determined that the position of each hot spot:
The positional information of the x1) design parameter according to biochip, and small grid, it is determined that the band of position at each hot spot place in this small grid, is designated as hot spot lattice；
X2) 0.3 times of the background value being background using hot spot lattice region is as the edge threshold values in this region；
X3) using hot spot lattice length and alleviating distention in middle-JIAO minima as the maximum of radius；
X4) all of facula information in traversal small grid；
X5) pixel with following condition is found out:
The position of this pixel is greater than 5 times of background value；
X6) for step x5) pixel that draws, goes to calculate the variance of the pixel in this region, average with the value of radius with it；
X7) spot signal meansigma methods is found to be in radius information corresponding during peak value；
X8) and using this datum mark as facula position；
X9) according to sub-positioning datum dot information, it is determined that the information of hot spot.
Detailed description of the invention
A kind of image analysis method, to the digital picture obtained, adopt threshold method, high luminance pixels point according to labelling in advance is that positioning datum point positions biochip, then according to biochip attribute information set in advance, determine the effective coverage of each test point, and then, adopt threshold method, high luminance pixels point according to labelling in advance obtains the sub-positioning datum point of each effective coverage, facula position according to the sub-positioning datum point location mark test substance of effective coverage, finally detect determined facula position, signal value and the standard substance comparison of detection facula position and draw analysis result.
Principle:
Positioning datum point and sub-positioning datum point are positioned at the upper left corner of corresponding institute identified areas, meet the general fashion of scanning, it is possible to quickly find relevant datum mark.Natural, according to different biochip attributes, such as positioning datum point can be arranged on biochip allocation really, simultaneously, positioning datum point can also show as a pair positioning datum point such as diagonal angle, it is determined that a pair positioning datum point, it is possible to biochip really allocation is directly depicted, and according to the attribute of biochip, the region residing for each detection hole or test point can be marked off accurately.
In one embodiment, the method finding positioning datum point is as follows:
A) to be not less than two pixels and to be not more than four pixels for the step-length described digital picture of traversal.
The range of choice of step-length and the relation of effect: if step-length selects relatively big, then there will be the possibility missing certain high bright spot, if selecting step-length less, the pixel quantity that can cause filtration is excessive, reduces efficiency of algorithm so that analyze image overlong time.Step-length is preferably 3 pixels, it is possible to the probability missing when ensureing better algorithm efficiency at high 2 is smaller.
B) for each pixel traversed, in the neighborhood centered by this pixel, each statistical parameter that basis predetermined biochip attribute is corresponding, statistical parameter includes the average brightness value after digital picture described in the average brightness value of described digital picture, Edge Enhancement and statistical variance.
The adquisitiones of edge threshold values is: travel through pixel from high to low according to the brightness of pixel, when pixel quantity more than current pixel point problem 10% time stop, obtaining the edge threshold values that meansigma methods is this region of front 10% pixel brightness.
The amplitude of Edge Enhancement and the impact on effect: by the use of edge threshold values, it is possible to directly filter out most background pixel, reduce the pixel quantity to calculate, greatly improve operation efficiency.
If c) certain pixel meets the following conditions, confirm as a datum mark:
C1) threshold values × 0.6, edge in the whole region of signal averaging > in the average hot spot of the Edge Enhancement image in the neighborhood centered by this pixel；
C2) gray value Edge Enhancement image with average brightness value 0.8 to 1.4 times be the pixel in the neighborhood of radius quantity and other radiuses be in a ratio of at most；
D) position weight of all datum marks of calculation procedure c) gained；
E) position weight is minimum or occurrence number is maximum datum mark is found out as positioning datum point.
In one embodiment, the computing formula of position weight is:
Math.Sqrt(((_topy-initY)/lgRect.RowSpan+lgRect.RowSpan)×((_topx-initX)/lgRect.ColSpan+lgRect.ColSpan))；
Wherein-topy is the Y coordinate of Current Scan pixel position
InitY is the Y coordinate of Current Scan pixel place monitoring holes upper left position
LgRect.RowSpan is the height of monitoring holes in chip attribute
_ topx is the X-coordinate of Current Scan pixel position
InitX is the X-coordinate of Current Scan pixel place monitoring holes upper left position
LgRect.ColSpan is the width of monitoring holes in chip attribute.
In one embodiment, biochip location is when positioning datum point is determined, it is biased drawing according to the positioning datum point determined according to the position relationship of default biochip with positioning datum point, as biased the distance transversely or longitudinally in a detection hole, or a horizontal and vertical distance in biasing one detection hole.
In one embodiment, the defining method of effective coverage is based on biochip detecting the array way of hole or test point, and specifically, col width information high according to detection control or the ranks number of test point, row, draws graticule, form small grid, the corresponding effective coverage of each small grid.
In certain embodiments, the method obtaining sub-positioning datum point is as follows:
1) filtration parameter that one group of signal intensity successively decreases being scanned the currently active region successively, using having of being first filled into more than setting threshold value and having the point of circular edge as subbase on schedule, the preferred parameter that signal successively decreases is 3.0,2.5,2.0,1.5；
2) obtaining the edge threshold that the currently active region is corresponding, corresponding gray scale filter value is decided to be 0.3 times of this edge threshold；Here consider factor for 0.3 times, if multiple is too high, have the possible datum mark of omission, if figure place is too low, can cause that qualified datum mark is too much, impact analysis efficiency)
3) scan all pixels in the currently active region, find out the pixel meeting following condition:
3.1) position of this pixel is greater than the product of background value and signal intensity filtration parameter；
3.2) average signal value of this rectangular area, pixel center 3 × 3 is more than 0.8 times of background colour；
3.3) average signal value of this rectangular area, pixel center 3 × 3 is more than eight adjacent regional signal averages；
3.4) in edge enhanced images, the gray value of this pixel is maximum；
Should be appreciated that currently used biochip, the general imaging size in a detection hole is 3 × 3 sizes exactly, if excessive, can reduce the average gray value in region, make analytical effect inaccurate.
4) pixel drawn according to step 3) and the value of radius, calculate the variance of pixel in current region, average；
5) finding signal averaging to be in peak value is corresponding radius；
6) calculation procedure 3) drawn the position weight of each pixel；
7) pixel that position weight value is minimum or the weighted value that occurrence number is maximum is corresponding is found, for the sub-positioning datum point that small grid is positioned.
In certain embodiments, if the positional information of the sub-positioning datum point drawn exceedes setting value in the condition lower deviation being benchmark with biochip attribute, then this sub-positioning datum point is corrected.
Image analysis method according to claim 7, it is characterised in that on the basis that small grid has positioned, it is determined that the position of each hot spot:
The positional information of the x1) design parameter according to biochip, and small grid, it is determined that the band of position at each hot spot place in this small grid, is designated as hot spot lattice；
X2) 0.3 times of the background value being background using hot spot lattice region is as the edge threshold values in this region；
X3) using hot spot lattice length and alleviating distention in middle-JIAO minima as the maximum of radius；
X4) all of facula information in traversal small grid；
X5) pixel with following condition is found out:
The position of this pixel is greater than 5 times of background value；
X6) for step x5) pixel that draws, goes to calculate the variance of the pixel in this region, average with the value of radius with it；
X7) spot signal meansigma methods is found to be in radius information corresponding during peak value；
X8) and using this datum mark as facula position；
X9) according to sub-positioning datum dot information, it is determined that the information of hot spot.
In such scheme, in stepb, it is determined that go out in region image data the luminance threshold of background colour in all pixels, when the brightness of pixel is lower than this threshold values, it is possible to assert that this pixel is background, when higher than this threshold values, it is believed that be high bright spot.Analyzing in image for usual one, most pixel is all background pixel.Luminance threshold obtains according to the current regional dynamics calculated, and can according to the pixel of current filter for which kind of type configures.In current method, the adquisitiones of threshold values is: travel through pixel from high to low according to the brightness of pixel, when pixel quantity more than current pixel point total amount 10% time stop, obtain the edge threshold values that meansigma methods is this region of front 10% pixel brightness, by the calculating of this threshold values, current pixel point more than 90% can be filtered out, substantially increase the efficiency of process.
In step 3.2, by the selection of step-length, can further improve efficiency, with 3 for step-length, travel through current region, can greatly reduce the pixel needing traversal, then pass through in current pixel point 3 × 3 region, background colour and believe the judgement of the information such as signal average in region, it is possible to traversed pixel will do not had to add unified checking, find the brightest core and most suitable radius so that the current pixel set selected is the brightest.
Process by above two steps, it is possible to calculative pixel is reduced very many, the computational efficiency improved greatly, make the analyzing and processing of each biochip, can complete within 2 seconds.
Claims (7)
1. the image analysis method of a biochip, it is characterized in that, to the digital picture obtained, adopt threshold method, high luminance pixels point according to labelling in advance is that positioning datum point positions biochip, then according to biochip attribute information set in advance, determine the effective coverage of each test point, and then, adopt threshold method, high luminance pixels point according to labelling in advance obtains the sub-positioning datum point of each effective coverage, facula position according to the sub-positioning datum point location mark test substance of effective coverage, finally detect determined facula position, signal value and the standard substance comparison of detection facula position and draw analysis result；
Wherein, the method finding positioning datum point is as follows:
A) to be not less than two pixels and to be not more than four pixels for the step-length described digital picture of traversal；
B) for each pixel traversed, in the neighborhood centered by this pixel, each statistical parameter that basis predetermined biochip attribute is corresponding, statistical parameter includes the average brightness value after digital picture described in the average brightness value of described digital picture, Edge Enhancement and statistical variance；
If c) certain pixel meets the following conditions, confirm as a datum mark:
C1) edge threshold × 0.6 in the whole region of signal averaging > in the average hot spot of the Edge Enhancement image in the neighborhood centered by this pixel；
C2) gray value Edge Enhancement image with average brightness value 0.8 to 1.4 times be the pixel in the neighborhood of radius quantity be in a ratio of at most with other brightness values for the pixel in the neighborhood of radius；
D) position weight of all datum marks of calculation procedure c) gained；
E) position weight is minimum or occurrence number is maximum datum mark is found out as positioning datum point；
Biochip location is when positioning datum point is determined, is biased drawing according to the positioning datum point determined according to the position relationship of default biochip with positioning datum point；
The defining method of effective coverage is based on biochip detecting the array way of hole or test point, specifically according to the ranks number of detection hole or test point, row height, col width information, draw graticule, form small grid, the corresponding effective coverage of each small grid.
2. image analysis method according to claim 1, it is characterised in that positioning datum point and sub-positioning datum point are positioned at the upper left corner of corresponding institute identified areas.
3. image analysis method according to claim 1, it is characterised in that the computing formula of position weight is:
Math.Sqrt(((_topy-initY)/lgRect.RowSpan+lgRect.RowSpan)×((_topx-initX)/lgRect.ColSpan+lgRect.ColSpan))；
Wherein _ topy is the Y coordinate of Current Scan pixel position
InitY is the Y coordinate of Current Scan pixel place monitoring holes upper left position
LgRect.RowSpan is the height of monitoring holes in chip attribute
_ topx is the X-coordinate of Current Scan pixel position
InitX is the X-coordinate of Current Scan pixel place monitoring holes upper left position
LgRect.ColSpan is the width of monitoring holes in chip attribute.
4. image analysis method according to claim 1, it is characterized in that, the defining method of effective coverage is based on biochip detecting the array way of hole or test point, specifically according to detection control or the ranks number of test point, row height, col width information, draw graticule, form small grid, the corresponding effective coverage of each small grid.
5. image analysis method according to claim 1, it is characterised in that the method obtaining sub-positioning datum point is as follows:
1) filtration parameter that one group of signal intensity successively decreases is scanned the currently active region successively, using having of being first filled into more than setting threshold value and there is the point of circular edge as subbase on schedule；
2) obtaining the edge threshold that the currently active region is corresponding, corresponding gray scale filter value is decided to be 0.3 times of this edge threshold；
3) scan all pixels in the currently active region, find out the pixel meeting following condition:
3.1) position of this pixel is greater than the product of background value and signal intensity filtration parameter；
3.2) average signal value of this rectangular area, pixel center 3 × 3 is more than 0.8 times of background colour；
3.3) average signal value of this rectangular area, pixel center 3 × 3 is more than eight adjacent regional signal averages；
3.4) in edge enhanced images, the gray value of this pixel is maximum；
4) pixel drawn according to step 3) and the value of radius, calculate the variance of pixel in current region, average；
5) signal averaging is found to be in radius corresponding during peak value；
6) calculation procedure 3) drawn the position weight of each pixel；
7) pixel that position weight value is minimum or the weighted value that occurrence number is maximum is corresponding is found, for the sub-positioning datum point that small grid is positioned.
6. image analysis method according to claim 5, it is characterised in that if the positional information of the sub-positioning datum point drawn exceedes setting value in the condition lower deviation being benchmark with biochip attribute, then this sub-positioning datum point is corrected.
7. image analysis method according to claim 5, it is characterised in that on the basis that small grid has positioned, it is determined that the position of each hot spot:
The positional information of the x1) design parameter according to biochip, and small grid, it is determined that the band of position at each hot spot place in this small grid, is designated as hot spot lattice；
X2) 0.3 times of edge threshold as this region of the background value being background using hot spot lattice region；
X3) using minima in hot spot lattice length and alleviating distention in middle-JIAO as the maximum of radius；
X4) all of facula information in traversal small grid；
X5) pixel with following condition is found out:
The brightness value of this pixel is greater than 5 times of background value；
X6) for step x5) pixel that draws, goes to calculate the variance of the pixel in this region, average with the value of radius with it；
X7) spot signal meansigma methods is found to be in radius information corresponding during peak value；
X8) and using step x7) in spot signal meansigma methods be in the pixel corresponding during peak value datum mark as facula position；
X9) according to sub-positioning datum dot information, it is determined that the information of hot spot.
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| CN110766700B (en) * | 2019-10-23 | 2022-01-28 | 吉林大学 | ICP-AES spectral image processing method based on digital micromirror |
| CN111257296B (en) * | 2020-03-20 | 2023-04-11 | 京东方科技集团股份有限公司 | Method, device and storage medium for detecting biochip sample |
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