Hosuk:LabNotes/2013-10-24

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Revision as of 21:21, 24 October 2013 by >Hosuki78
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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)