Matt:LabNotes/2015-11-15
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Analysis of DARTFISH Tile Image to compare with RNAscope[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- Counted number of in bins of 752 pixels
Counts of Nuclei in 21 Y-slices[edit]
- 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
- Use CountNucleiPerYSlicesfromMIP.m which calls nucC.m
- strelSize = 10
Number of Rolonies per Nuclei[edit]
- Results are in C:\Users\Matt\Documents\RolonyImages\11-11-2015\20151029_BA8_DARTFISHsuppv2_20XDecoding\Aligned\BA8_suppv2_2015-10-29\Excels
- Update wiki later with graphs
- Note: 20X Decoding (with 0.75 NA) is not good enough
- In 9 positions equaling 2.677mm^2
- Decoded 6,908 rolonies (and this includes double counting rolonies of overlapping regions)
- 225 genes
- 178 genes > 3 counts
- In 4 positions of 63X decoding equaling 0.1354mm^2
- Decoded 1,641 rolonies using 3D Decoding (~10% more than 2D Decoding)
- That would be >30,000 rolonies in 2.677mm^2
- In 9 positions equaling 2.677mm^2
- Maybe try a 40X 1.3NA objective?
- Especially for mouse embryo project
- 292um FOV should be enough to fit each embryo section
- 1.3NA should be high enough resolution for 3D Decoding
- Especially for mouse embryo project