Hosuk:LabNotes/2013-10-24: Difference between revisions

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===Rolony count with PISA===
===Rolony count with PISA===
====1 tile, 2048 x 2048 image ====
====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


*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)


*1st Rolony (Cy5) parameters
*1st Rolony (Cy5) parameters
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[[File:ATTO488SegOverlapwithCy5_S2_20131022.png|600px]]
[[File:ATTO488SegOverlapwithCy5_S2_20131022.png|600px]]


====Multiple 7 x 6 tiles, --> 11334 x 13077 image ====
*too big to process...




Line 39: Line 36:
*Crop this down to 4 x 4 (7453 x 7453 px)
*Crop this down to 4 x 4 (7453 x 7453 px)
**Cells ~ about 70 x 16 = 1120
**Cells ~ about 70 x 16 = 1120
*Size : 2.1mm x 2.1mm




Line 56: Line 54:


*ACTB / 1st rolony = 363/41824 = 0.87%
*ACTB / 1st rolony = 363/41824 = 0.87%
**Overlapped ACTB --> 85 (0.076 ACBT per cell)
**'''Overlapped ACTB --> 85''' (0.076 ACBT per cell)
**Overlapped ACTB / 1st rolony = 85/41824 = 0.20%
**Overlapped ACTB / 1st rolony = 85/41824 = 0.20%


Line 62: Line 60:
*Overlaped with ATTO488 on Cy5
*Overlaped with ATTO488 on Cy5
**Blue : Cy5, Red : ATTO488 : [[Media:ATTO488SegOverlapwithCy5_S2_Tile_4x4_20131022.tif|Raw]]
**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]]

Latest revision as of 21:44, 24 October 2013


Rolony count with PISA[edit]

1 tile, 2048 x 2048 image[edit]

  • 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[edit]

  • 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[edit]

  • 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 : Raw