ABSTRACT
Förster resonance energy transfer (FRET) is an effective and high-resolution method to monitor protein-protein interactions in live or fixed specimens. FRET imaging can be implemented with either an upright or inverted microscope depending on the particular application. Some wild-type proteins such as EosFP proved to be useful as marker proteins in several imaging application without further optimization. Suitable fluorophore FRET partners are the key to the success of a FRET application. Many visible fluorescent proteins have been employed in combination with FRET microscopy to visualize dynamic protein interactions under physiological conditions. Confocal imaging is a point-detection-based approach in which a tightly focused spot is scanned across the sample and the excited fluorescence is collected through the objective, spatially filtered with a pinhole to remove the out-of-focus signals, and detected by a photo multiplier tube detector. Major new FRET developments in the area of fluorescence lifetime determinations, either in the time or frequency domain will exploit techniques like Fluorescence Lifetime Imaging.
