What are the disadvantages of FLIM FRET?

Two limitations of FLIM is its associated high cost and limited availability of instrumentation required to measure nanosecond lifetimes. Furthermore, localized environmental influences (e.g. autofluorescence or pH alterations) can reduce the observed fluorescence lifetime, potentially resulting in inaccuracies. Additionally, FLIM tends to be a slower imaging technique, often requiring several minutes per image. Moreover, the multi-exponential lifetimes of fluorescent proteins (FPs) in live cells demand comprehensive data for FRET analysis, further slowing down FLIM-FRET measurements. Lastly, environmental factors like autofluorescence background or changes in pH can also reduce the measured fluorescence lifetime.