Matt:LabNotes/2015-11-15

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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