Hosuk:LabNotes/2013-10-24
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Rolony count with PISA
1 tile, 2048 x 2048 image
- Sample : Confocal image of S2 MatTek dish at 2013-10-22
- Cells : about 70 cells, counted cells just by eye based on compact rolonies (assume nucleus)
- Size : 581.25um x 581.25um
- 1st Rolony (Cy5) parameters
- Area Upper : 100 px, Area lower : 2 px,
- Axioratio lower 0.7, Circ Upper 1.6 Circ Lower 0.4
- Perim conn 8, bkgmulti lower 2
- Count --> 5210 (about 74.4 cells per cell)
- ACTB (ATTO488) parameters
- Area Upper : 100 px, Area lower : 2 px,
- Axioratio lower 0.7, Circ Upper 1.6, Circ Lower 0.4
- Perim conn 8, bkgmulti lower 2
- Count --> 69 (about 1 cells per cell)
- Overlapped ACTB --> 40 (about 0.57 cells per cell)
- Overlapped ACTB / 1st rolony = 40/5210 = 0.77%
- Overlaped with ATTO488 on Cy5
- Blue : Cy5, Red : ATTO488 : Raw
File:ATTO488SegOverlapwithCy5 S2 20131022.png
Multiple 7 x 6 tiles, --> Cropped to 4 x 4 --> 7453 x 7453 image
- Crop this down to 4 x 4 (7453 x 7453 px)
- Cells ~ about 70 x 16 = 1120
- Size : 2.1mm x 2.1mm
- Sample : S2_20131022_20x_2kx2k_pos1_Zstack-1.2um_MaxProjection_Crop4x4_Cy5.tif
- Area Upper : 100 px, Area lower : 2 px,
- Axioratio lower 0.7, Circ Upper 1.6 Circ Lower 0.4
- Perim conn 8, bkgmulti lower 2
- Count --> 41824 (37.3 rolonies per cell)
- Sample : S2_20131022_20x_2kx2k_pos1_Zstack-1.2um_MaxProjection_Crop4x4_ATTO488.tif
- Area Upper : 100 px, Area lower : 2 px,
- Axioratio lower 0.7, Circ Upper 1.6, Circ Lower 0.4
- Perim conn 8, bkgmulti lower 2
- Count --> 363 (0.32 ACBT per cell)
- ACTB / 1st rolony = 363/41824 = 0.87%
- Overlapped ACTB --> 85 (0.076 ACBT per cell)
- Overlapped ACTB / 1st rolony = 85/41824 = 0.20%
- Overlaped with ATTO488 on Cy5
- Blue : Cy5, Red : ATTO488 : Raw
Multiple 7 x 6 tiles, --> 13077 x 11334 image
- Size : 3.7mm x 3.2mm
- processed (after rebooting PC … :( )
- 7 x 6 tiles, (13077 x 11334 px), but about 7 tiles were not images well… because of miss focusing miss
- Need to take more longer Z scanning!
- Cells ~ about 70 x 35 = 2450
- Sample : S2_20131022_20x_2kx2k_pos1_Zstack-1.2um_MaxProjection_Cy5.tif
- Area Upper : 100 px, Area lower : 2 px,
- Axioratio lower 0.7, Circ Upper 1.6 Circ Lower 0.4
- Perim conn 8, bkgmulti lower 2
- Count --> 100424 (41.0 rolonies per cell)