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*[[Hosuk:Lab_Notes|LabNote]] ===Rolony count with PISA=== ====1 tile, 2048 x 2048 image ==== *Sample : Confocal image of S2 MatTek dish at [[Hosuk:LabNotes/2013-10-22|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 : [[Media:ATTO488SegOverlapwithCy5_S2_20131022.tif|Raw]] [[File:ATTO488SegOverlapwithCy5_S2_20131022.png|600px]] ====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 : [[Media:ATTO488SegOverlapwithCy5_S2_Tile_4x4_20131022.tif|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) *Sample : S2_20131022_20x_2kx2k_pos1_Zstack-1.2um_MaxProjection_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 --> 892''' (0. 32 ACBT per cell) *ACTB / 1st rolony = 892/100424 = 0.89% **'''Overlapped ACTB --> 213''' (0.087 ACBT per cell) **Overlapped ACTB / 1st rolony = 213/100424 = 0.21% *Overlaped with ATTO488 on Cy5 **Blue : Cy5, Red : ATTO488 : [[Media:ATTO488SegOverlapwithCy5_S2_Tile_7x6_20131022.tif|Raw]]
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