FRET - Image analysis - Spillover factors (Page 1)

Just as for image acquisition, the details for image analysis will depend on the image analysis software. We demonstrate our analysis using the Softworx program that is part of our DeltaVision system. The first step is to determine the Spillover factors described in the Introduction. This is accomplished by imaging two strains in which one of your proteins of interest is tagged with either YFP or CFP alone. For example we demonstrate how we determined the YFP Spillover factor for a spindle pole body (SPB) component. In the image below, Spc110 is tagged with YFP. No other fluorescent protein is labeled. We had run the FRET acquisition series and acquired 4 bundled images, just as if we were examining FRET between a pair of proteins. In the left strip the depressed and highlighted "545" button displays the YFP channel. The Data Inspector panel allows us to create a 5 x 5 pixel box that encompasses the image of the SPB. The pixel intensity data is saved to a space delimited text output file.