Hosuk:LabNotes/2014-8-19: Difference between revisions
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====Analysis==== | ====Analysis==== | ||
*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''' | ||
Line 75: | Line 68: | ||
| 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
- 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 (ch03) of each Cycle : Step1, Step4, Step7, ... Step19
- I've done by manually, but eventually need automation
- ex)Offset
- File name (decided at imaging) : [Sample]_[Resolution]_[Step#]_[Pos#]_z##_ch##.tif
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 of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
- Name : Crop_[file name]
- Shift, Crop of ch00, ch01, ch02 images(Image_Aligning_Crop_v5.m)
Result
- PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1