Hosuk:LabNotes/2013-12-11
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NCIH1975 Rolony : 2nd sample
- Sample : Glass-bottom MatTek dish – 1 dishes
- NCIH1975 P27 (from Illumina)
- Process 12/09 ~ 12/11
- Image Cy5-1st Rolony detection probe (1uM in 30% Formamide + 2xSSC)
Image condition
- Laser = 2%
- PMT Gain = 550
- Z stack = 0.25um step, 199 images
- allows higher resolution along Z axis, and better 3D view
Result(Preliminary)
- This cells were more denser than previous sample.
- The number of rolonies were many.
- PISA counting = 14557
- PISA Param
- Gaussian std = 3, upper = -3e-4
- area upper = 100, lower = 2, axratio lower = 0.7
- circ upper = 1.6, lower = 0.8, perim conn = 8
- bkgmult lower = 7
- PISA Param
3D view with ImageJ
- Procedure : Stack all images --> Crop (because ImageJ can’t handle a big size) --> Run 3D plugin
- Max Projection image (2048x2048 size, Cropped, 444x444 size)
File:MAX Fullsize Cropped Stack NCIH1975-20131211 Step3 Pos1.png
- 3D view (360 degree Rotation)
File:3DView MAX Cropped Stack NCIH1975-20131211 Step3 Pos1.png