Matt:LabNotes/2015-11-15
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Analysis of DARTFISH Tile Image to compare with RNAscope
- Goal is to do a similar analysis to Matt:LabNotes/2015-11-6#Results RNAscope analysis and find a similar pattern
- Confocal DARTFISH images should match Tile 2 position of RNAscope sample and totals 4,584um long (equivalent to Y-slices 1-21)
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