Hosuk:LabNotes/2014-8-19: Difference between revisions

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====Analysis====
====Analysis====
=====Arrange data=====
*1. Arrange data
*File name (decided at imaging) : [Sample]_[Resolution]_[Step#]_[Pos#]_z##_ch##.tif
*#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) 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
*#*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
*Step# after confocal imaging
*#*Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF
**Step0 : 1st Rolony --> ch00 : Cy3, ch01 : BF
*#Maximum Intensity Projection(MIP) at each channel
**Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF
*#*Re-format the Step1~Step7 --> Step1~21
 
*#*Step1 --> Step1:488, Step2:Cy3, Step3:Cy5
 
*#*Step2 --> Step4:488, Step5:Cy3, Step6:Cy5
*Maximum Intensity Projection(MIP) at each channel
*#*...Step7 --> Step19:488, Step20:Cy3, Step21:Cy5
**Re-format the Step1~Step7 --> Step1~21
*#Image aligning
**Step1 --> Step1:488, Step2:Cy3, Step3:Cy5
*#*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]
**Step2 --> Step4:488, Step5:Cy3, Step6:Cy5
*#*Get offset using Bright field images (ch03) of each Cycle : Step1, Step4, Step7, ... Step19
**...Step7 --> Step19:488, Step20:Cy3, Step21:Cy5
*#**I've done by manually, but eventually need automation
 
*#*ex)Offset
 
*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 (ch03) of each Cycle : Step1, Step4, Step7, ... Step19
***I've done by manually, but eventually need automation
 
*ex)Offset
{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Cycle'''
| align="center" style="background:#f0f0f0;"|'''Cycle'''
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| align="center" | Crop Size || align="center" | 12  || align="center" | 12
| align="center" | Crop Size || align="center" | 12  || align="center" | 12
|}
|}


*#Shift, Crop of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
*#Shift, Crop of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
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*




====Result====
====Result====
*PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1
*PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1

Revision as of 21:32, 22 August 2014


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

  • 1. Arrange data
    1. 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
    2. Step# after confocal imaging
      • Step0 : 1st Rolony --> ch00 : Cy3, ch01 : BF
      • Step1~7 : ch00 : 488, ch01 : Cy3, ch02 : Cy5, ch03 : BF
    3. 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
    4. 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 (ch03) 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 of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
      • Name : Crop_[file name]



Result

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