• Acceptor photobleaching;
  • anisotropy;
  • fluorescence lifetime imaging microscopy (FLIM);
  • fluorescence resonance energy transfer (FRET);
  • homo-FRET


The phenomenon of resonance energy transfer first described by Theodor Förster presents the opportunity of retrieving information on molecular proximity, orientation and conformation on the nanometre scale from (living) samples with conventional fluorescence microscopes (or even macroscopic devices). During the past 10 years Förster (or fluorescence) resonance energy transfer (FRET) microscopy has been revolutionized by the vast progress in fluorescent protein and in situ fluorescent labelling technology as well as by the commercial availability of advanced quantitative microscopy instrumentation. FRET microscopy is now routinely used in modern cell biology research. This short review will guide the reader through the most established FRET microscopy techniques, their inherent strengths and limitations, potential pitfalls, and assist the reader in making an educated choice on the FRET microscopy method most suited for their specific application.