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.
