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*[[Hosuk:Lab_Notes|LabNote]] ===Multiple scanning with different PMT gain to avoid saturation of rolonies=== *Data : NCIH1975 20gap test sample (..\2014-11-04\Arrange\1.MIP\0.1stRolony) *File : NCIH1975_20gapTest_S3_TruncatedT4_Cycle0_63x_2k_Pos1.tif (PMT 520) *NCIH1975_20gapTest_S3_TruncatedT4_Cycle0_63x_2k_Pos1_PMT660.tif (PMT 660) *Low PMT (PMT 520) has nice rolonies inside nucleus *High PMT (PMT 660) has nice rolonies outside nucleus, there are a lot of rolonies which are not seen in low PMT gain image. ====Procedure==== *#Run PISA at each image *#Make mask images by immultiply(data, BW) *#Sum mask images *#Run PISA again with the summed image ====Result==== *worked well! *Rolony count **Low PMT image : 4701 **High PMT image : 7944 **Low PMT image : 10520 *Dr. Zhang's comment : **Weak intensity (~a few thousand)could be recognized as a real signal, because of lower backgrond noise, and that's why we use PMT. **Thus a single scanning with a low PMT gain would be enough if we separate higher signal region (such as nucleus). **So I need try to separate nucleus and other part with image processing, instead of scanning multiple times. **Check FISSEQ data of Jayβs paper and find X-Y coordinate of genes, and compare the location of the genes to my result [[File:MultiScanTest_2014-11-04_NCIH1975.png|1000px]]
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