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*[[Hosuk:Lab_Notes|LabNote]] ===Decoding with Agi26k0gap Padlock Probe : Masking images with MIP of all steps=== ====Masking==== *Done by Matt *MIP with images of all steps *Run PISA of the MIP **seg_data = regionprops(MaskedFile,MaskedImgFile, 'MeanIntensity', 'Area', 'Centroid'); *Matlab scripts *PISA_MaskImages_4d.m **masking, and get mean intensity, area, (x, y) position **normalize mean intensity of each dye using norm_tetra_3.m **Plotting_3.m : Plot cloud plot **norm_tetra_3.m : normalize intensity (sqrt...), **tetrahedron_4.m : Plot 3(or 4) colors on tetrahedron space **http://arep.med.harvard.edu/Polonator/2006/image_processing_pipeline/DATASETS/ **http://www.sciencemag.org/content/suppl/2005/10/12/1117389.DC1/Shendure.SOM.pdf **The server hosting the source code no longer exists. The "newer" version of the software was written in C (http://arep.med.harvard.edu/Polonator/2006/image_processing_pipeline/). Not sure whether that's something you can pick up immediately. ====Variables==== *After masking **DecodeInfoMaxNum : Total number of features by regionprops **DecodeInfo_MeanIntensity(1:MaxRolonyNum, 1:NumOfSteps) : Mean intensity of each feature **RolonyInfo_Area(1:MaxRolonyNum, 1): Area(number of pixels) of each feature **RolonyInfo_CenterY(1:MaxRolonyNum, 1) : Centroid X of each feature **RolonyInfo_CenterX(1:MaxRolonyNum, 1): Centroid Y of each feature *After normalize **Max_MeanIntensity(1:MaxRolonyNum, 1) : Max intensity of each Step1 ~ Step21 **DecodeInfo_NormMeanIntensity(1:MaxRolonyNum, 1:NumOfSteps) : Normalized by Max ***DecodeInfo_NormMeanIntensity = DecodeInfo_MeanIntensity / Max_MeanIntensity; **DecodeInfo_Sqrt(1:DecodeInfoMaxNum, 1:NumOfDecodingSlots) : sqrt of sum of sq of each intensity = sqrt[ (I_488)^2 + (I_Cy3)^2 + (I_Cy5)^2 ] **DecodeInfo_NormMeanIntensity2(1:MaxRolonyNum, 1:NumOfSteps) : norm intensity after filtered out the weak signal *** all intensity = 0 when DecodeInfo_Sqrt < thresh_val (it is 0.1 for now) **DecodeInfo_NormMeanIntensity_Result(1:MaxRolonyNum, 1:NumOfSteps) : normalized again by norm_tetra_3, plot cloud with this ***[DecodeInfo_NormMeanIntensity_Result, cms, calls, coords_3d] = norm_tetra_3(DecodeInfo_NormMeanIntensity2, NumOfSteps, thresh_subvalue); ====Normalized intensity distribution==== *all intensity = 0 when DecodeInfo_Sqrt < thresh_val *Blue : 488, Green : Cy3, Red : Cy5 [[File:DecodeResult_CloudPlots_2014-08-21.png|800px]] ====Result==== [[File:DecodeResultSummary_1_2014-08-21.png|500px]] [[File:DecodeResult_MappedfeatureTo1stROolony_2014-08-21.png|600px]] [[File:DecodeResult_MappedfeatureToBF_2014-08-21.png|600px]]
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