Matt:LabNotes/2015-11-15: Difference between revisions

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>Mzcai
(Created page with "=Analysis of DARTFISH Tile Image to compare with RNAscope= *Goal is to do a similar analysis to [[Matt:LabNotes/20...")
 
>Mzcai
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**Pos8: 2046
**Pos8: 2046
**Pos9: 2046
**Pos9: 2046
*Finding size of overlap between each position
**Based on stage coordinates expect overlap size to be 295px = 84um
**To find actual overlap compare positions of decoded rolonies
***Did this manually so only looked at most abundant gene 'SNAP25'
***In the same region, more rolonies decoded at the bottom of previous image then top of latter image
****E.g. More SNAP25 in bottom 279px of Pos1 than in top 279px of Pos2
****Could be due to photobleaching? "uneven illumination" or equivalent phenomena for confocal?
*Offsets (calculated average Y-offset of SNAP25 rolonies that overlapped)
**Pos1->2: 279
**Pos2->3: 289
**Pos3->4: 298
**Pos4->5: 278
**Pos5->6: 278
**Pos6->7: 305
**Pos7->8: 280
**Pos8->9: 299
*Stitched Tile A:
**Use count of previous position for overlapping region
*Stitched Tile B:
**Use count of latter position for overlapping region
==Counts of Genes in 21 Y-slices==
*Counted number of in bins of 752 pixels
==Counts of Nuclei in 21 Y-slices==
*Since Positions 1-4 of 9 during imaging had weird nuclei morphology I took altPositions directly adjacent (no shift in y direction) and treat those as if
*Due to these altPositions I can't make one long tile image so will just count nuclei in the appropriate regions
**eg Y-slice 1 = 1-752 of Pos 1
**eg Y-slice 3 = 1505-2048 of altPos1 + 280-489 of altPos2
==Number of Rolonies per Nuclei==

Revision as of 06:53, 18 November 2015

Analysis of DARTFISH Tile Image to compare with RNAscope

Running Matlab 2D Decoding

  • Used 2D Decoding
    • Program folder: C:\Users\Matt\Dropbox\GradZhangLab\Matlab\DARTFISH_PxPx_Organized\
    • Data folder: C:\Users\Matt\Documents\RolonyImages\11-11-2015\20151029_BA8_DARTFISHsuppv2_20XDecoding\
  • First time trying on images taken with 20X objective (normally 63X) but kept the 2048x2048 pixel resolution
    • Each image went from 184um to 584um width
    • Pixel size went from 0.09um to 0.3um
    • Axial resolution went from ~0.5um to ~2um
    • Z step size went from 0.3um to 1.04um
  • DecodePxPx.m paramaters set in Info.m file
    • Area Lower = 2 pixels
    • Area Upper = 15 pixels
    • TreshDistance = 0.05 (normalized intensity below which to call '0')
  • Alignment Offset (iCycle 2 as reference) Results:
    • Offset, Pos 1, iCycle 2 & 1, [x, y] = [1, 1]
    • Offset, Pos 1, iCycle 2 & 3, [x, y] = [0, -1]
    • Offset, Pos 1, iCycle 2 & 4, [x, y] = [1, -2]
    • Offset, Pos 1, iCycle 2 & 5, [x, y] = [0, -2]
    • Offset, Pos 2, iCycle 2 & 1, [x, y] = [1, 0]
    • Offset, Pos 2, iCycle 2 & 3, [x, y] = [1, 0]
    • Offset, Pos 2, iCycle 2 & 4, [x, y] = [1, -2]
    • Offset, Pos 2, iCycle 2 & 5, [x, y] = [0, -2]
    • Offset, Pos 3, iCycle 2 & 1, [x, y] = [1, 0]
    • Offset, Pos 3, iCycle 2 & 3, [x, y] = [1, -1]
    • Offset, Pos 3, iCycle 2 & 4, [x, y] = [1, -2]
    • Offset, Pos 3, iCycle 2 & 5, [x, y] = [0, -2]
    • Offset, Pos 4, iCycle 2 & 1, [x, y] = [1, 1]
    • Offset, Pos 4, iCycle 2 & 3, [x, y] = [0, -1]
    • Offset, Pos 4, iCycle 2 & 4, [x, y] = [0, -2]
    • Offset, Pos 4, iCycle 2 & 5, [x, y] = [0, -1]
    • Offset, Pos 5, iCycle 2 & 1, [x, y] = [1, 1]
    • Offset, Pos 5, iCycle 2 & 3, [x, y] = [0, -1]
    • Offset, Pos 5, iCycle 2 & 4, [x, y] = [0, -2]
    • Offset, Pos 5, iCycle 2 & 5, [x, y] = [0, -1]
    • Offset, Pos 6, iCycle 2 & 1, [x, y] = [0, 1]
    • Offset, Pos 6, iCycle 2 & 3, [x, y] = [0, -1]
    • Offset, Pos 6, iCycle 2 & 4, [x, y] = [0, -1]
    • Offset, Pos 6, iCycle 2 & 5, [x, y] = [0, -1]
    • Offset, Pos 7, iCycle 2 & 1, [x, y] = [0, 1]
    • Offset, Pos 7, iCycle 2 & 3, [x, y] = [0, 0]
    • Offset, Pos 7, iCycle 2 & 4, [x, y] = [0, -1]
    • Offset, Pos 7, iCycle 2 & 5, [x, y] = [0, -1]
    • Offset, Pos 8, iCycle 2 & 1, [x, y] = [0, 0]
    • Offset, Pos 8, iCycle 2 & 3, [x, y] = [0, -1]
    • Offset, Pos 8, iCycle 2 & 4, [x, y] = [0, -1]
    • Offset, Pos 8, iCycle 2 & 5, [x, y] = [0, -1]
    • Offset, Pos 9, iCycle 2 & 1, [x, y] = [0, 1]
    • Offset, Pos 9, iCycle 2 & 3, [x, y] = [0, 0]
    • Offset, Pos 9, iCycle 2 & 4, [x, y] = [0, -1]
    • Offset, Pos 9, iCycle 2 & 5, [x, y] = [0, 0]

Stitching Results Into Tile

  • DecodePxPx.m results containing coordinate info is stored in StatTable matrix saved in 'Analysis_BA8_suppv2_2015-10-29_PosX_Try_1.mat'
    • Column order: Index, Area, Avg Intensity, X Coordinate, Y Coordinate, Total Count
  • Output following info into one consolidated excel file:
    • Column order: GeneName, GeneBarcode, Area, X Coordinate, Y Coordinate
    • Use script RolonyPositionExcels.m
  • Aligned/Cropped image sizes
    • Pos1: 2044
    • Pos2: 2046
    • Pos3: 2046
    • Pos4: 2044
    • Pos5: 2044
    • Pos6: 2046
    • Pos7: 2046
    • Pos8: 2046
    • Pos9: 2046
  • Finding size of overlap between each position
    • Based on stage coordinates expect overlap size to be 295px = 84um
    • To find actual overlap compare positions of decoded rolonies
      • Did this manually so only looked at most abundant gene 'SNAP25'
      • In the same region, more rolonies decoded at the bottom of previous image then top of latter image
        • E.g. More SNAP25 in bottom 279px of Pos1 than in top 279px of Pos2
        • Could be due to photobleaching? "uneven illumination" or equivalent phenomena for confocal?
  • Offsets (calculated average Y-offset of SNAP25 rolonies that overlapped)
    • Pos1->2: 279
    • Pos2->3: 289
    • Pos3->4: 298
    • Pos4->5: 278
    • Pos5->6: 278
    • Pos6->7: 305
    • Pos7->8: 280
    • Pos8->9: 299
  • Stitched Tile A:
    • Use count of previous position for overlapping region
  • Stitched Tile B:
    • Use count of latter position for overlapping region

Counts of Genes in 21 Y-slices

  • Counted number of in bins of 752 pixels

Counts of Nuclei in 21 Y-slices

  • Since Positions 1-4 of 9 during imaging had weird nuclei morphology I took altPositions directly adjacent (no shift in y direction) and treat those as if
  • Due to these altPositions I can't make one long tile image so will just count nuclei in the appropriate regions
    • eg Y-slice 1 = 1-752 of Pos 1
    • eg Y-slice 3 = 1505-2048 of altPos1 + 280-489 of altPos2

Number of Rolonies per Nuclei