Hosuk:LabNotes/2014-8-19: Difference between revisions
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**Since how many features would be detected or how much dense rolonies in this sample, so image in wide field of view | **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. | **eventually 63x obj. | ||
*Data is in '2014-08-13' | |||
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*Image aligning | *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 | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Cycle''' | |||
| align="center" style="background:#f0f0f0;"|'''X''' | |||
| align="center" style="background:#f0f0f0;"|'''Y''' | |||
|- | |||
| align="center" | 1 || align="center" | 0 || align="center" | 0 | |||
|- | |||
| align="center" | 2 || align="center" | -13 || align="center" | +8 | |||
|- | |||
| align="center" | 3 || align="center" | -9 || align="center" | +8 | |||
|- | |||
| align="center" | 4 || align="center" | -19 || align="center" | +4 | |||
|- | |||
| align="center" | 5 || align="center" | -16 || align="center" | +4 | |||
|- | |||
| align="center" | 5 || align="center" | -15 || align="center" | +10 | |||
|- | |||
| align="center" | 5 || align="center" | -6 || align="center" | -9 | |||
|} | |||
*#Shift, Crop | *#Shift, Crop | ||
*#Shift | *#Shift | ||
*#BF | *#BF |
Revision as of 21:20, 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
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 |
- Shift, Crop
- Shift
- BF
Result
- PGP1F_P20_S2_2014-08-09 --> , HBTissue_2014-08-12_S1