CN107723344A - New automatic fluorescence signal acquisition analysis method based on dPCR - Google Patents
New automatic fluorescence signal acquisition analysis method based on dPCR Download PDFInfo
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Abstract
The invention belongs to image processing field, particularly a kind of new automatic fluorescence signal acquisition analysis method for being based on dPCR (digital Polymerase Chain Reaction digital polymerase chain reactions).This method extracts simultaneously analysis of fluorescence signal using the thought of image-region division --- image mosaic --- signal collection and analysis.Fluorescence signal acquisition analysis method is applied to the result in dPCR later stages and detected by this method in a manner of engineering first, whole system is set to realize full-automation by reasonably designing, avoid artificial interference such as hand labeled, measurement error caused by progressive scan etc., accuracy is good, convenient operation and control, effectively increase the accuracy of detection and detection efficiency of system, original image size when the scattered image perfection gathered can be spliced into design by splicing step, highly reduce artwork, facilitate analysis and detection of the later stage to fluorescence signal, improve the accuracy of detection.
Description
Technical field
The invention belongs to image processing field, particularly one kind to be based on dPCR (digital Polymerase Chain
Reaction digital polymerase chain reactions) new automatic fluorescence signal acquisition analysis method.
Background technology
The concept of dPCR technologies from propose till now, it is short less than the vicennial time in have development at full speed.This
Item technology expands outside organisms to gene, is the Protocols in Molecular Biology that specific DNA fragmentation is expanded for amplifying.
At present, dPCR technologies are as the Absolute quantification detection of target gene, detection GMOs, gene defect screening, cancer prevention, gene
The powerful of the applications such as abrupt climatic change, have in terms of biomedical and accurate medical treatment and be extremely widely applied.It is but existing
Concentrated in most of researchs and optimize and improve in itself for dPCR chips, and it is glimmering for the whole dPCR systems middle and later periods
The problem of optical signal detecting, but turns into " gray area " of this great research.
Found by the retrieval to existing literature data, document [1] (Qiangyuan Zhu, Lin Qin and et al,
Digital PCR on an integrated self-priming compartmentalization chip, Lab
Chip,Lab Chip,2014,1176:1185) Qiangyuan Zhu of Zhejiang University et al. were once mentioned and devises a kind of use
PDMS is made, the valveless of Vacuum Package, the microchip formula dPCR of no pump, and experimental verification is carried out to it, but article is in the positive
Reaction counts, and is not further discussed in the uniformity and repeatability of sample.
Document [2] (Heyries K.A.et al., Megapixel digital PCR, Nature Methods, 2011,
8(8):649:651) explanation of the collection and analysis that terminate rear fluorescence signal to thermal cycle reaction was once mentioned.The document pair
DPCR chips carry out region division, manually mark to carry out region recognition, then with 0.5pixels/ μm of precision to institute
Zoning is progressively scanned, and counts fluorescence intensity.Although having obtained final result, hand labeled adds operation
Complexity, be unfavorable for the integrated development of system, and progressive scan reduces detection efficiency.
Later stage fluorescence signal acquisition and the process of analysis decide the accuracy of detection of system, in addition whole course of reaction into
Lose, because the fluorescence signal acquisition and parser that are directed to dPCR systems in the prior art are few, and Laser Scanning Confocal Microscope
Object lens scope can not cover whole chip, so which limits the efficient and integrated development of whole dPCR systems, because
This collection to fluorescence signal and analysis after PCR reactions terminate accomplish that automation with rapidly and efficiently changing is one urgently to be resolved hurrily
Problem.
The content of the invention
The defects of present invention is in the presence of the research of existing dPCR technologies, propose it is a kind of based on dPCR it is new it is complete from
Dynamic fluorescence signal acquisition analysis method, divided using image-region the thought of --- image mosaic --- signal collection and analysis come
Simultaneously analysis of fluorescence signal is extracted, full-automation is realized, avoids the interference of spuious optical signal, not only increase the accurate of testing result
Degree and detection efficiency, and provide thinking for the high efficiency and integrated development of dPCR technologies.
The present invention realizes that the technical scheme of above-mentioned purpose is, a kind of new automatic fluorescence signal acquisition based on dPCR point
Analysis method, its innovative point are：The step of this method, is as follows：
Step 1：The making of dPCR chips.DPCR chips for we using micro-nano process technology produce with special
The high density pore type dPCR chips of mark, reference picture 2, its overall dimensions are N*N ㎜, and region A is the part of reaction microchamber, side
Edge size is M*M ㎜, has six special cross in the annular region that dPCR chip edges and reaction microchamber A edges are formed
Note, P1, P2, P3 in reference picture 2, P4, P5, P6, it is specified that be the origin of coordinates with the immediate summits of P1 on dPCR chips, P1, P2
The central point line and direction for pointing to P2 is x positive directions, P1, P5 central point line and direction for pointing to P5 is y positive directions, if setting
P1 centre coordinate is P1 (a, b) (0<a,b<(N-M)/2), then other 5 points centre coordinates are respectively P2 (N-a, b), P3
(a, N/2), P4 (N-a, N/2), P5 (a, N-b), P6 (N-a, N-b)；The rectangle region that the center of six cross marks is formed
The size in domain is (N-2a) * (N-2a).
Step 2：Create cross mark identification database.To in step 1 dPCR chips shoot, obtain it is of all kinds (times
Rate size, definition, cross mark integrity degree are incomplete same) positive picture and negative picture, wherein, positive picture refers to
Picture containing cross mark, negative picture refer to the picture for not containing cross mark.By setting all positive pictures
ROIs (Region Of Interest abbreviation, refer to the rectangular area containing cross mark), create the identification number of cross mark
According to storehouse.
Step 3：Initialization, all directions and each summit are set, gather image.The step is completed by following sub-step.
Sub-step 1：Reference picture 2, using the direction of P1, P2 central point line as horizontal direction, with P1, P5 center
The direction of point line is as vertical direction；Matrix is converted image information into, it is big according to the pixel of the next pictures of CCD camera
The small e*f and size d of corresponding dPCR chips reaction microchamber, finds the conversion relation between pixel and unit micron；Utilize CCD
Conversion relation between the size N*N and pixel and micron of pixel size e*f, dPCR chip of the next pictures of camera
Be calculated division pickup area total number sNr (1) * sNr (2) (represent respectively in the horizontal direction with vertical direction needed for adopt
Collect photo number) and the stepping-in amount stepSize (1) and stepSize (2) of programmable fine motion control platform (represent respectively
Stepping-in amount in horizontal direction and vertical direction)；Adjacent two pictures are obtained by e-stepSize (1) and f-stepSize (2)
Between overlap distance overlap (1) and overlap (2) (represent respectively in the horizontal direction with vertical direction it is adjacent two figure
The overlap distance of piece), judge overlap (1)>E/4~e/3 and overlap (2)>Whether f/4~f/3 sets up, if into
It is vertical, carry out sub-step 2, if invalid, increase sNr and according to the methods described of sub-step 1 recalculate stepSize and
Overlap, untill condition is met, carry out sub-step 2；
Sub-step 2：Optional one is used as initial vertax from four summits of chip, with the initial vertax most similar one
Individual cross mark is designated as Pz (z value is 1,2,5,6), will contain the three parts such as initial vertax, Pz and part reaction microchamber
Picture is as first shooting picture, and the position of the picture is current location, and current position coordinates are by initial vertax coordinate come table
Show；
Sub-step 3：Initialization current location is initial position, and initial position co-ordinates are represented by current position coordinates, from first
Beginning, position began stepping through all areas of current dPCR chips, it then follows the principle progressive scan of vertical direction after first horizontal direction,
In scanning process, the theory after jth moved further is determined according to the stepping-in amount of initial position co-ordinates and programmable fine motion control platform
Position coordinates (Xj0,Yj0), while programmable fine motion control platform completes jth step according to the stepping-in amount stepSize of above-mentioned calculating
Mobile, the position coordinates after movement is designated as (Xj0’,Yj0'), judge (Xj0,Yj0) and (Xj0’,Yj0') whether identical, if identical,
Carry out the next moved further of waiting of taking pictures；If it is different, programmable continuous five trials of fine motion control platform are moved to (Xj0,Yj0) position
Put, in five moving process, the position coordinates after movement is designated as (X every timeji,Yji) (i=1,2,3,4,5), i-th movement
After the completion of, judge (Xji,Yji) and (Xj0,Yj0) whether identical, and obtain physical location (Xji,Yji) and theoretical position (Xj0,
Yj0) deviation er, if (Xji,Yji) and (Xj0,Yj0) identical, then taken pictures and carry out the moved further of jth+1, if five times have been moved
Into rear (Xji,Yji) and (Xj0,Yj0) still different, then theoretical position (X is automatically adjusted to according to deviation erj0,Yj0), then taken pictures
And carry out the moved further of jth+1；
Sub-step 4：Scanning is fully completed, and obtains picture O11、O12、……、OsNr(2)sNr(1), carry out step 4.
Step 4：Splice picture with vertical direction in the horizontal direction, the position difference of two pictures to be spliced on each direction
There are two kinds of situations, reference picture 3 (a) (b) and Fig. 4 (a) (b), completed by following sub-step：
Sub-step 1：Take the preceding two pictures O of t (t=1,2 ..., sNr (2)) rowst1, Oti (i=2,3 ... ...,
SNr (1)), the comparison block with 1/30~1/50*overlap pixel width is set, in picture Ot1, carry out phase between Oti
Close property compare, by the correlation in k row q row comparison domains be designated as R1, R2 ..., Rn, find maximum correlation Rmax=
Max { R1, R2 ..., Rn } and lap position kth ' row q ' row (reference picture 3a1, Ot1Size be e ' * f '), then take Ot1
(k '+1：F ') OK, (1, q ') row, take (the 1 of Oti：F-k ') OK, (1, e) row, it is spliced into a new picture (reference picture
3a2) and delete original picture Ot1, using new picture as the pictures of t rows first, it is designated as Ot1, then by Ot1With next figure
Piece Oti is spliced according to above-mentioned steps, the like, until the row all sNr (1) pictures all complete by splicing；Obtain
The newest picture O that often row splicing is completed11, O21... ..., Ot1, then rotor step 2.
Sub-step 2：Remember O11, O21... ..., Ot1Respectively O1, O2... ..., Ot, take O1, Oj (j=2,3 ... ..., t) two
Pictures, the comparison block with 1/30~1/50*overlap pixel width is set, in picture O1, carry out phase between Oj
Close property compare, by the correlation in kk row qq row comparison domains be designated as R11, R22 ..., Rnn, find maximum correlation
Rmmax=max { R11, R22 ..., Rnn } and lap position kth ' ' row q ' ' row (reference picture 4a1), then take (the 1 of O1：
K ' ') OK, (1, q ' ') row, take (the 1 of Oj：F2) OK, (q ' '+1, e2) is arranged, and is spliced into a new picture (reference picture 4a2) simultaneously
Delete original picture O1, using new picture as current first pictures, O is designated as again1, then by O1With next pictures Oj
Spliced according to sub-step 2, until all sNr (2) pictures all complete by splicing, obtain newest full picture O1, ginseng
According to Fig. 5, its size is designated as N ' * M '.
Step 5：Reference picture 5, by picture O1Six regions are divided into, are designated as M1, M2, M3, M4, M5, M6 respectively, each
The size in region is N '/2*M '/3, and a cross mark is respectively contained in six regions；Mi (i=1,2,3,4,5,6) is taken,
Cross mark in the cross mark identification database identification Mi created according to step 2, and find cross using canny operators
The edge of note, by Hough transition detection and associated straight lines are drawn, so as to obtain the four edges edge straight line of cross mark in Mi, note
For li1, li2, li3, li4 (reference picture 6)；Four crosspoints of four straight lines are finally calculated according to the algorithm of analytic geometry,
Vi1, vi2, vi3 are designated as, vi4 (reference picture 6), so as to obtain the centre coordinate of six marks, is designated as c1 (Xc1, Yc1), c2
(Xc2, Yc2), c3 (Xc3, Yc3), c4 (Xc4, Yc4), c5 (Xc5, Yc5), c6 (Xc6, Yc6) (reference picture 7).
Step 6：By tan-1((Ycj-Yci)/(Xcj-Xci)) (j=2,4,6；I=1,3,5) on calculated level direction c1 and
C2, c3 and c4, c5 and c6 relative deflection angle and be designated as α 1, α 2, α 3, by tan-1((Xcj- Xci)/(Ycj-Yci)) (j=3,
4,5,6；I=1,2,3,4) c1 and c3 on vertical direction, c1 and c5, c3 and c5, c2 and c4, c2 and c6, c4 and c6 phase are calculated
To deflection angle and α 4, α 5, α 6, α 7, α 8, α 9 are designated as, takes its average value, i.e. α respectivelyx=(α 1+ α 2+ α 3)/3, αy=(α 4+ α 5
+ α 6+ α 7+ α 8+ α 9)/6, αxAnd αyAs picture O1In the horizontal direction with the deflection angle on vertical direction, α=(α is takenx+αy)/
2, then α is O1Deflection angle, according to Yc1-Yc2Relation with zero determines O1Yawing moment, so as to by O1By clockwise (or
Direction anglec of rotation α counterclockwise), new picture (reference picture 8) is obtained, is designated as O again1, its size is designated as N " * M ".
Step 7：Repeat step 5, obtain new picture O1The centre coordinate of upper six marks, is designated as c1 (X againc1, Yc1),
c2(Xc2, Yc2), c3 (Xc3, Yc3), c4 (Xc4, Yc4), c5 (Xc5, Yc5), c6 (Xc6, Yc6), and go to step 8；
Step 8：Reference picture 8, crops O1On portion beyond the rectangular area G that is made up of ci (i=1,2 ... ..., 6)
Point, and (or reducing) is amplified into the part within G, obtain new picture and be designated as OO, make described in OO size and step 1
DPCR chips are identical with the size of a part, are (N-2a) * (N-2a).
Step 9：Reference picture 9, using the centre coordinate of cross mark P1 on dPCR chips as basic point coordinate, (0,0) is designated as,
Coordinate centered on the center of circle of i-th (i=1,2 ... ..., n-1, n, n are the total number of reaction microchamber) individual reaction microchamber, makees a diameter of
D circle, it is designated as justifying CEi, the position for justifying CEi is the position of upper i-th of the reaction microchamber of picture OO, will justify in CEi (including side
Edge) value a little be arranged to i, its residual value is disposed as 0, (N-2a) * (N-2a) matrix B is established, if data i in matrix B
Position be b1 rows b2 row, then extract the data f that b1 rows b2 is arranged in picture OOy, take fyAverage value and be designated as Fi, Fi is
The fluorescence intensity of i-th of reaction microchamber on dPCR chips, count identical Fi number and be designated as Numi, so as to obtain experiment knot
Fruit, experimental result are represented by fluorescence intensity Fi and its number Numi.
The beneficial effects of the invention are as follows：(1) fluorescence signal acquisition analysis method is applied in a manner of engineering first
The result detection in dPCR later stages, makes a complete engineering detecting system, proposes a kind of new full-automatic fluorescence letter
Number capturing analysis method, at present, both at home and abroad there is not yet document report similar to the present invention.(2) made by reasonably designing
Whole system realizes full-automation, avoids to measure caused by artificial interference such as hand labeled, progressive scan etc. and misses
Difference, accuracy is good, convenient operation and control, effectively increases the accuracy of detection and detection efficiency of system.(3) walked by above-mentioned splicing
The scattered image perfection gathered can be spliced into original image size during design suddenly, height reduces artwork, facilitates the later stage pair
The analysis and detection of fluorescence signal, improve the accuracy of detection.
Brief description of the drawings
Fig. 1 is the flow chart of the present invention.
Fig. 2 is the structure chart of dPCR chips.
The relative position situation map of two pictures when Fig. 3 (a), (b) are row splicing；(a1) compare when, (b1) splices for row
The lap position schematic diagram that block is searched out；(a2), (b2) is the schematic diagram being stitched together with the pictures of a line two.
The relative position situation map of two pictures when Fig. 4 (a), (b) are row splicing；(a1) compare when, (b1) splices for row
The lap position schematic diagram that block is searched out；(a2), (b2) is the schematic diagram that the pictures of same row two are stitched together.
Fig. 5 is the picture and six division schematic diagrames containing cross mark region after the completion of all splicings.
Fig. 6 is four straight lines of cross mark and four intersection points of four straight line intersections detected in picture Mi.
Fig. 7 is the Centered Graphs of six cross marks detected.
Fig. 8 is the figure after the picture rotation that splicing is completed.
Fig. 9 is the schematic diagram that reaction microchamber location matrix is created according to dPCR chips designed size.
Figure 10 is the structure chart for the dPCR chips that reaction microchamber is 50 μm.
Figure 11 is the splicing result of the pictures of the first row first and the second pictures in experiment.
Figure 12 (a) is the picture for identifying the cross mark in Mi in experiment using cross mark identification database；(b) it is
The part of cross mark is extracted in experiment from Mi.
Figure 13 is to be detected in experiment and mark edge line and the cross mark figure in crosspoint in M1.
Figure 14 is the centre coordinate figure of six marks on the dPCR chips detected in experiment.
The part picture that Figure 15 is coincide by the location matrix that reaction microchamber is built in experiment with physical location.
Figure 16 is laboratory test results.
Embodiment
The present invention is elaborated further below in conjunction with accompanying drawing.
A kind of the step of new automatic fluorescence signal acquisition analysis method based on dPCR, is followed successively by：
Step 1：The making of dPCR chips.We have been produced highly dense with special marking using micro-nano process technology
Spend pore type dPCR chips, such as Figure 10：Its overall dimensions is 9.1*9.1 ㎜, and the marginal dimension of the part of middle reaction microchamber is
8380*8380 μm, there are six special cross in the annular region that dPCR chip edges and reaction microchamber A edges are formed
Note, is designated as P1, P2, P3, P4, P5, and P6 is, it is specified that P1, P2 central point line and direction for pointing to P2 is x positive directions, in P1, P5
The heart point line and direction for pointing to P5 is y positive directions, if setting P1 centre coordinate as P1 (200,200) (unit for μm), then its
His 5 points of centre coordinate is respectively P2 (8900,200), P3 (200,4550), P4 (8900,4550), P5 (200,8900),
P6 (8900,8900) (unit is μm)；Reference picture 10, the size of each reaction microchamber is 50 μm, two neighboring reaction microchamber
The distance between (EF) be 55 μm, in adjacent rows adjacent three reaction microchambers the center of circle (3 points of A, B, C) form equilateral triangle
Shape, to wait six pieces of formal distribution to have 76 reaction microchambers on dPCR chips per a line in each piece, there is 29*2 rows, then should
26448 (76*29*2*6) individual reaction microchambers are shared on chip.
Step 2：Create cross mark identification database.DPCR chips are shot, obtain 218 it is of all kinds (times
Rate size, definition, cross mark integrity degree are incomplete same) positive picture and negative picture, wherein, positive picture refers to
Picture containing cross mark, there are 109, negative picture refers to the picture for not containing cross mark, there are 109.Pass through setting
The ROIs of 109 positive pictures, create the identification database of cross mark.
Step 3：Initialization, all directions and each summit are set, gather image.The step is completed by following sub-step.
Sub-step 1：Reference picture 2, using the direction of P1, P2 central point line as horizontal direction, with P1, P5 center
The direction of point line is as vertical direction；Matrix is converted image information into, it is big according to the pixel of the next pictures of CCD camera
Small 1938*1460 and the size 50 of corresponding dPCR chips reaction microchamber, find the conversion relation between pixel and unit micron；
Utilize the conversion relation between the pixel size of the next pictures of CCD camera, the size of dPCR chips and pixel and micron
The total number sNr that division pickup area is calculated is that 5*7 (is respectively the number of collection photo needed for horizontal direction and vertical direction
Mesh) and the stepping-in amount stepSize of programmable fine motion control platform be respectively 1820 and 1300 (represent respectively in the horizontal direction and
Stepping-in amount on vertical direction)；Obtained by e-stepSize (1) and f-stepSize (2) overlapping between adjacent two pictures
Distance overlap is 118 and 160 (overlap distances for representing adjacent two pictures with vertical direction in the horizontal direction respectively),
Judge overlap (1)>300 and overlap (2)>Whether 500 set up, if set up, carries out sub-step 2, if invalid,
Increase sNr simultaneously recalculates stepSize and overlap according to the methods described of sub-step 1, untill condition is met, obtains
SNr is respectively 7 and 9, carries out sub-step 2；
Sub-step 2：P1 is chosen from four summits of chip as initial vertax, it is most similar one with the initial vertax
Cross mark is designated as P1, initial vertax, P will be contained1Picture with the three parts such as part reaction microchamber is as first shooting figure
Piece, the position of the picture is current location, and current position coordinates are represented by initial vertax coordinate；
Sub-step 3：Initialization current location is initial position, and initial position co-ordinates are represented by current position coordinates, from first
Beginning, position began stepping through all areas of current dPCR chips, it then follows the principle progressive scan of vertical direction after first horizontal direction,
In scanning process, the theory after jth moved further is determined according to the stepping-in amount of initial position co-ordinates and programmable fine motion control platform
Position coordinates (Xj0,Yj0), while programmable fine motion control platform completes jth step according to the stepping-in amount stepSize of above-mentioned calculating
Mobile, the position coordinates after movement is designated as (Xj0’,Yj0'), judge (Xj0,Yj0) and (Xj0’,Yj0') whether identical, if identical,
Carry out the next moved further of waiting of taking pictures；If it is different, programmable continuous five trials of fine motion control platform are moved to (Xj0,Yj0) position
Put, in five moving process, the position coordinates after movement is designated as (X every timeji,Yji) (i=1,2,3,4,5), i-th movement
After the completion of, judge (Xji,Yji) and ((Xj0,Yj0) whether identical, and the deviation er of physical location and theoretical position is obtained, if
(Xji,Yji) and (Xj0,Yj0) identical, then taken pictures and carry out the moved further of jth+1, if (X after the completion of five movementsji,Yji) with
(Xj0,Yj0) still different, then theoretical position (X is automatically adjusted to according to deviation erj0,Yj0), then taken pictures and carry out the step of jth+1
It is mobile；
Sub-step 4：Scanning is fully completed, and obtains picture O11、O12、……、O97, carry out step 4.
Step 4：Splice picture with vertical direction in the horizontal direction, the position difference of two pictures to be spliced on each direction
There are two kinds of situations, reference picture 3 and Fig. 4, completed by following sub-step：
Sub-step 1：Take the preceding two pictures Ot of t (t=1,2 ..., 9) rows1, Oti (i=2,3 ... ..., 7), if
The comparison block with 10 pixel width is put in picture Ot1, correlation comparison is carried out between Oi, by k row q row comparison domains
Correlation be designated as R1, R2 ..., Rn, find correlation Rmax=max { R1, R2 ..., Rn } and the lap position of maximum
Kth ' row q ' row (reference picture 3a1, Ot1Size be e ' * f '), then take Ot1(k '+1：F ') OK, (1, q ') row, take Oti's
(1：F-k ') OK, (1, e) row, it is spliced into a new picture and deletes original picture Ot1, using new picture as current
First pictures, are designated as Ot1(reference picture 11, the splicing result of two pictures before the first row), then by Ot1With next pictures Oti
Spliced according to above-mentioned steps, the like, completed until all 7 pictures of t rows all splice；Obtain current newest
Picture O11, O21 ... ..., Ot1, then rotor step 2.
Sub-step 2：Remember O11, O21... ..., Ot1Respectively O1, O2... ..., Ot, take O1、Oj(j=2,3 ... ..., t) two
Picture, the comparison block with 10 pixel width is set in picture O1, correlation comparison is carried out between Oj, by kk row qq row ratios
Compared with the correlation in region be designated as R11, R22 ..., Rnn, find maximum correlation Rmmax=max R11, R22 ...,
Rnn } and lap position kth " row q " row, then take (the 1 of O1：K ") OK, (1, q ") row, take (the 1 of Oj：F2) OK, (q "+1,
E2) arrange, be spliced into a new picture and delete original picture O1, using new picture as current first pictures, again
It is designated as O1, then by O1Spliced with next pictures Oj according to sub-step 2, until 9 pictures all complete by splicing, obtained most
New full picture O1, its size is designated as N ' * M '.
Step 5：Reference picture 5, by picture O1Six regions are divided into, are designated as M1, M2, M3, M4, M5, M6 respectively, each
The size in region is N '/2*M '/3, and a cross mark is respectively contained in six regions；Mi (i=1,2,3,4,5,6) is taken,
Cross mark (reference picture 12) in the cross mark identification database identification Mi created according to step 2, and utilize canny operators
The edge of cross mark is found, by Hough transition detection and associated straight lines are drawn, so as to obtain four of cross mark in Mi
Edge line, it is designated as li1, li2, li3, li4 (reference pictures 13, for the straight line detected in first cross mark M1 and its friendship
Point)；Four crosspoints of four straight lines are finally calculated according to the algorithm of analytic geometry, are designated as vi1, vi2, vi3, vi4 (references
Figure 13), so as to obtain the centre coordinate of six marks, c1 (X are designated asc1, Yc1), c2 (Xc2, Yc2), c3 (Xc3, Yc3), c4 (Xc4,
Yc4), c5 (Xc5, Yc5), c6 (Xc6, Yc6) (reference picture 14).
Step 6：By tan-1((Ycj-Yci)/(Xcj-Xci)) on (j=2,4,6, i=1,3,5) calculated level direction c1 and
C2, c3 and c4, c5 and c6 relative deflection angle and be designated as α 1, α 2, α 3, by tan-1((Xcj- Xci)/(Ycj-Yci)) (j=3,
4,5,6, i=1,2,3,4) c1 and c3 in vertical direction, c1 and c5, c3 and c5, c2 and c4, c2 and c6, c4 and c6 phase are calculated
To deflection angle and α 4, α 5, α 6, α 7, α 8, α 9 are designated as, takes its average value, i.e. α respectivelyx=(α 1+ α 2+ α 3)/3, αy=(α 4+ α 5
+ α 6+ α 7+ α 8+ α 9)/6, αxAnd αyAs picture O1In the horizontal direction with the deflection angle on vertical direction, α=(α is takenx+αy)/
2, then α is O1Deflection angle, according to Yc1-Yc2Relation with zero determines O1Yawing moment, so as to by O1By side counterclockwise
To anglec of rotation α, new picture is obtained, is designated as O again1, its size is designated as N " * M ".
Step 7：Repeat step 5, obtain new picture O1The centre coordinate of upper six marks, is designated as c1 (X againc1, Yc1),
c2(Xc2, Yc2), c3 (Xc3, Yc3), c4 (Xc4, Yc4), c5 (Xc5, Yc5), c6 (Xc6, Yc6), and go to step 9；
Step 8：Reference picture 8, crops O1Upper ciPart beyond the rectangular area G that (i=1,2 ... ..., 6) is formed,
And (or reducing) is amplified into the part within G, obtain new picture and be designated as OO, make OO size and dPCR cores described in step 1
Piece is identical with the size of a part, is 8700*8700.
Step 9：Reference picture 9, using the centre coordinate of cross mark P1 on dPCR chips as basic point coordinate, (0,0) is designated as,
Coordinate centered on the center of circle of i-th (i=1,2 ... ..., 26448) individual reaction microchamber, makees a diameter of d circle, is designated as justifying CEi, circle
CEi position is the position (reference picture 15) of upper i-th of the reaction microchamber of picture OO, by (including edge) institute in circle CEi a little
Value be arranged to i, its residual value is disposed as 0, establishes 8700*8700 matrix B, if data i position is b1 rows in matrix B
B2 is arranged, then extracts the data f that b1 rows b2 is arranged in picture OOy, take fyAverage value and be designated as Fi, Fi is i-th on dPCR chips
The fluorescence intensity of reaction microchamber, count identical Fi number and be designated as Numi, so as to obtain experimental result, experimental result is by fluorescence
Intensity Fi and its number Numi expressions, reference picture 16.
Claims (1)
1. a kind of new automatic fluorescence signal acquisition analysis method based on dPCR, it is characterised in that comprise the following steps：
Step 1：Make dPCR chips, dPCR chips for we using micro-nano process technology produce with special marking
High density pore type dPCR chips, its overall dimensions are N*N ㎜, and region A is the part of reaction microchamber, and marginal dimension is M*M ㎜,
There are P1, P2, P3 in six special cross marks in the annular region that dPCR chip edges and reaction microchamber A edges are formed,
P4, P5, P6 are, it is specified that with the immediate summits of P1 be the origin of coordinates, P1, P2 central point line and the side for pointing to P2 on dPCR chips
To for x positive directions, P1, P5 central point line and direction for pointing to P5 is y positive directions, if setting P1 centre coordinate as P1 (a, b)
(0<a,b<(N-M)/2), then other 5 points centre coordinates are respectively P2 (N-a, b), P3 (a, N/2), P4 (N-a, N/2), P5
(a, N-b), P6 (N-a, N-b)；The size for the rectangular area that the center of six cross marks is formed is (N-2a) * (N-
2a)；
Step 2：Create cross mark identification database；To in step 1 dPCR chips shoot, obtain multiplying power size, definition,
The incomplete same positive picture of cross mark integrity degree and negative picture, wherein, positive picture refers to containing cross mark
Picture, negative picture refer to the picture for not containing cross mark；By the ROIs for setting all positive pictures；, create cross
The identification database of note；
Step 3：Initialization, all directions and each summit are set, gather image；The step is completed by following sub-step：
Sub-step 1：Using the direction of P1, P2 central point line as horizontal direction, made with the direction of P1, P5 central point line
For vertical direction；Convert image information into matrix, according to the pixel size e*f of the next pictures of CCD camera with it is corresponding
The size d of dPCR chip reaction microchambers, finds the conversion relation between pixel and unit micron；Utilize next of CCD camera
Conversion relation between the size N*N and pixel and micron of pixel size e*f, dPCR chip of picture is calculated division and adopted
Collect region total number sNr (1) * sNr (2) (represent respectively in the horizontal direction with vertical direction needed for gather photo number) and
The stepping-in amount stepSize (1) and stepSize (2) of programmable fine motion control platform (are represented in the horizontal direction and vertical square respectively
Upward stepping-in amount)；Overlap distance between adjacent two pictures is obtained by e-stepSize (1) and f-stepSize (2)
Overlap (1) and overlap (2), overlap (1) and overlap (2) represent respectively in the horizontal direction with phase on vertical direction
The overlap distance of adjacent two pictures, judges overlap (1)>E/4~e/3 and overlap (2)>Whether f/4~f/3 sets up,
If set up, sub-step 2 is carried out, if invalid, increase sNr and recalculates stepSize according to the methods described of sub-step 1
And overlap, untill condition is met, carry out sub-step 2；
Sub-step 2：Optional one is used as initial vertax from four summits of chip, most similar one ten with the initial vertax
Word mark is designated as Pz, and z value is 1,2,5,6, and the picture containing three parts such as initial vertax, Pz and part reaction microchambers is made
For first shooting picture, the position of the picture is current location, and current position coordinates are represented by initial vertax coordinate；
Sub-step 3：Initialization current location is initial position, and initial position co-ordinates are represented by current position coordinates, from initial bit
Put all areas for beginning stepping through current dPCR chips, it then follows the principle progressive scan of vertical direction, scanning after first horizontal direction
During, the theoretical position after jth moved further is determined according to the stepping-in amount of initial position co-ordinates and programmable fine motion control platform
Coordinate (Xj0,Yj0), while programmable fine motion control platform completes jth moved further according to the stepping-in amount stepSize of above-mentioned calculating,
Position coordinates after movement is designated as (Xj0’,Yj0'), judge (Xj0,Yj0) and (Xj0’,Yj0') whether identical, if identical, clapped
According to the next moved further of waiting；If it is different, programmable continuous five trials of fine motion control platform are moved to (Xj0,Yj0) position, five
In secondary moving process, the position coordinates after movement is designated as (X every timeji,Yji) (i=1,2,3,4,5), after the completion of ith movement,
Judge (Xji,Yji) and (Xj0,Yj0) whether identical, and obtain physical location (Xji,Yji) and theoretical position (Xj0,Yj0) deviation
Er, if (Xji,Yji) and (Xj0,Yj0) identical, then taken pictures and carry out the moved further of jth+1, if (X after the completion of five movementsji,
Yji) and (Xj0,Yj0) still different, then theoretical position (X is automatically adjusted to according to deviation erj0,Yj0), then taken pictures and carry out
J+1 moved furthers；
Sub-step 4：Scanning is fully completed, and obtains picture O11、O12、……、OsNr(2)sNr(1), carry out step 4；
Step 4：Splice picture with vertical direction in the horizontal direction, the position of two pictures to be spliced has two respectively on each direction
Kind situation, completed by following sub-step：
Sub-step 1：Take the preceding two pictures O of t (t=1,2 ..., sNr (2)) rowst1, Oti (i=2,3 ... ..., sNr
(1)), the comparison block with 1/30~1/50*overlap pixel width is set, in picture Ot1, carry out between Oti it is related
Property compare, by the correlation in k row q row comparison domains be designated as R1, R2 ..., Rn, find maximum correlation Rmax=max
{ R1, R2 ..., Rn } and lap position kth ' row q ' row, then take Ot1(k '+1：F ') OK, (1, q ') row, take (the 1 of Oti：
F-k ') OK, (1, e) row, it is spliced into a new picture and deletes original picture Ot1, using new picture as t rows first
Pictures, it is designated as Ot1, then by Ot1Spliced with next pictures Oti according to above-mentioned steps, the like, until the row institute
Having sNr (1) pictures, all splicing is completed；Obtain the newest picture O that every row splicing is completed11, O21... ..., Ot1, then rotor
Step 2；
Sub-step 2：Remember O11, O21... ..., Ot1Respectively O1, O2... ..., Ot, take O1, Oj (j=2,3 ... ..., t) two pictures,
Comparison block with 1/30~1/50*overlap pixel width is set, in picture O1, correlation comparison is carried out between Oj,
By the correlation in kk row qq row comparison domains be designated as R11, R22 ..., Rnn, find maximum correlation Rmmax=max
{ R11, R22 ..., Rnn } and lap position kth " row q " row, then take (the 1 of O1：K ") OK, (1, q ") row, take (the 1 of Oj：
F2) OK, (q "+1, e2) is arranged, and is spliced into a new picture and is deleted original picture O1, using new picture as current first
Pictures, O is designated as again1, then by O1Spliced with next pictures Oj according to sub-step 2, until all sNr (2) pictures
All splicing is completed, and obtains newest full picture O1, its size is designated as N ' * M '；
Step 5：By picture O1Six regions are divided into, are designated as M1, M2, M3, M4, M5, M6 respectively, the size in each region is
N '/2*M '/3, respectively contain a cross mark in six regions；Mi (i=1,2,3,4,5,6) is taken, is created according to step 2
Cross mark in cross mark identification database identification Mi, and using the edge of canny operators searching cross mark, by suddenly
Husband's transition detection simultaneously draws associated straight lines, so as to obtain the four edges edge straight line of cross mark in Mi, is designated as li1, li2, li3,
li4；Four crosspoints of four straight lines are finally calculated according to the algorithm of analytic geometry, are designated as vi1, vi2, vi3, vi4, so as to
The centre coordinate of six marks is obtained, is designated as c1 (Xc1, Yc1), c2 (Xc2, Yc2), c3 (Xc3, Yc3), c4 (Xc4, Yc4), c5 (Xc5,
Yc5), c6 (Xc6, Yc6)；
Step 6：By tan-1((Ycj-Yci)/(Xcj-Xci)) (j=2,4,6；I=1,3,5) c1 and c2, c3 on calculated level direction
Relative deflection angle and α 1, α 2, α 3 are designated as with c4, c5 and c6's, by tan-1((Xcj-Xci)/(Ycj-Yci)) (j=3,4,5,6；
I=1,2,3,4) c1 and c3 on vertical direction, c1 and c5, c3 and c5, c2 and c4, c2 and c6, c4 and c6 relative deflection are calculated
Angle is simultaneously designated as α 4, α 5, α 6, α 7, α 8, α 9, takes its average value, i.e. α respectivelyx=(α 1+ α 2+ α 3)/3, αy=(α 4+ α 5+ α 6+ α 7
+ α 8+ α 9)/6, αxAnd αyAs picture O1In the horizontal direction with the deflection angle on vertical direction, α=(α is takenx+αy)/2, then α be
For O1Deflection angle, according to Yc1-Yc2Relation with zero determines O1Yawing moment, so as to by O1By (or counterclockwise) clockwise
Direction anglec of rotation α, obtains new picture, is designated as O again1, its size is designated as N " * M "；
Step 7：Repeat step 5, obtain new picture O1The centre coordinate of upper six marks, is designated as c1 (X againc1, Yc1), c2 (Xc2,
Yc2), c3 (Xc3, Yc3), c4 (Xc4, Yc4), c5 (Xc5, Yc5), c6 (Xc6, Yc6), and go to step 8；
Step 8：Crop O1On part beyond the rectangular area G that is made up of ci (i=1,2 ... ..., 6), and by within G
Part zooms in or out, and obtains new picture and is designated as OO, makes chi of the OO size with dPCR chips described in step 1 with a part
It is very little identical, it is (N-2a) * (N-2a)；
Step 9：Using the centre coordinate of cross mark P1 on dPCR chips as basic point coordinate, it is designated as (0,0), i-th (i=1,
2 ... ..., n-1, n, n are the total number of reaction microchamber) coordinate centered on the center of circle of individual reaction microchamber, make a diameter of d circle, note
For circle CEi, circle CEi position is the position of upper i-th of the reaction microchamber of picture OO, by (including edge) institute in circle CEi a little
Value be arranged to i, its residual value is disposed as 0, (N-2a) * (N-2a) matrix B is established, if data i position is b1 in matrix B
Row b2 is arranged, then extracts the data f that b1 rows b2 is arranged in picture OOy, take fyAverage value and be designated as Fi, Fi is i-th on dPCR chips
The fluorescence intensity of individual reaction microchamber, count identical Fi number and be designated as Numi, so as to obtain experimental result, experimental result is by glimmering
Luminous intensity Fi and its number Numi is represented.
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