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=Analysis of [[Matt:LabNotes/2015-11-11 | DARTFISH Tile Image]] to compare with [[Matt:LabNotes/2015-11-6 | RNAscope]]= *Goal is to do a similar analysis to [[Matt:LabNotes/2015-11-6#Results RNAscope analysis]] and find a similar pattern *Confocal DARTFISH [[Matt:LabNotes/2015-11-11 | 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 *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 *Use CountNucleiPerYSlicesfromMIP.m which calls nucC.m **strelSize = 10 ==Number of Rolonies per Nuclei== *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 [[Matt:LabNotes/2015-9-2 | 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 *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
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