Hosuk:LabNotes/2014-8-19
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Decoding with Agi26k0gap Padlock Probe
- Image resolution : 20x obj, 4096 x 4086, z step = 1.04um
- Since how many features would be detected or how much dense rolonies in this sample, so image in wide field of view
- eventually 63x obj.
- Data is in '2014-08-13'
Analysis
Arrange data
- File name (decided at imaging) : [Sample]_[Resolution]_[Step#]_[Pos#]_z##_ch##.tif
- ex) PGP1F : PGP1F_Agi26k0gap_Decode_P20_S2_2014-08-09_20x_4k_Step1_Pos2_z01_ch01.tif
- ex) Tissue section : HBTissue_2014-08-12_RCAagain_20x_4k_Pos1_z00_ch00.tif
- Step# after confocal imaging
- Step0 : 1st Rolony --> ch00 : Cy3, ch01 : BF
- Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF
- Maximum Intensity Projection(MIP) at each channel
- Re-format the Step1~Step7 --> Step1~21
- Step1 --> Step1:488, Step2:Cy3, Step3:Cy5
- Step2 --> Step4:488, Step5:Cy3, Step6:Cy5
- ...Step7 --> Step19:488, Step20:Cy3, Step21:Cy5
- Image aligning
- Step# in one cycle is an exact position, no shift happened --> Cycle N = Step[3(N-1)+1], Step[3(N-1)+2], Step[3(N-1)+3]
- Get offset using Bright field images of each Cycle : Step1, Step4, Step7, ... Step19
- I've done by manually, but eventually need automation
- ex)Offset
Cycle | X | Y |
1 | 0 | 0 |
2 | -13 | +8 |
3 | -9 | +8 |
4 | -19 | +4 |
5 | -16 | +4 |
5 | -15 | +10 |
5 | -6 | -9 |
Average Offset | -11.14 | +0.71 |
Round | -11 | +1 |
New Offset | ||
1 | -11 | +1 |
2 | -2 | +7 |
3 | +2 | +7 |
4 | -8 | +3 |
5 | -5 | +3 |
5 | -4 | -11 |
5 | +5 | -10 |
Crop Size | 12 | 12 |
- Shift, Crop
- Shift
- BF
Result
- PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1