FRETSCAL

For the FRET analysis of images of yeast cells labeled with YFP and CFP.

FRETSCAL screens images of cells that contain CFP and YFP tagged proteins in order to determine the extent of energy transfer(FRET)between the two fluorophores. The graphical user interface (GUI) of FRETSCAL allows the user to specify criteria that are used to identify areas of interest (AOIs) in the images. The criteria include the signal to background ratios, the fit of the signal intensity to a Gaussian distribution, the minimal distance between AOIs, the size of the AOI, intensity limits, and the position of the surrounding background area. FRETSCAL generates cropped TIFS of the AOIs, including a user specified area surrounding the AOI for context. The user can screen through the AOIs, choosing to keep or discard AOIs based on both visual inspection and image statistics. The final output is a text file that includes the intensity data and a calculation of the FRET index FretR. FretR can be used as an estimate for the distance between the tagged proteins. FRETSCAL comes with a user manual and a collection of sample images.