Personal notebook entry for FRET -- last updated 20150615
You need a control where there is FRET and one where there is no FRET. For instance, a short linker between the donor-acceptor and a long rigid linker between donor-----acceptor. The below method from Day R 2015 uses an additional control, a mutated acceptor with a short linker. (More details about this RET control at ref 12 below http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1366774/.)
For FLIM measurements, this paper also uses cumarin as a standard. I found that eosin works well because the fluorescence is strong over a wide spectrum and the lifetime changes dramatically and predictively when it is dehydrated vs mixed with water.
Example no-frills ratio FRET macros for ImageJ for opening & measuring all files in a directory. User first has to figure our background values and enter them into macro. Then user traces cells (or gets the computer to do this) and saves in ROI file with the same name except RoiSet.zip at the end. Macro then lists ratio for each cell on whole area measurement. Macro here.