Hosuk:LabNotes/2014-8-19

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Revision as of 21:25, 22 August 2014 by >Hosuki78
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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



    1. Shift, Crop


    1. Shift
    2. BF


Result

  • PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1