FRET FRET is an acronym for Forster Resonant Energy Transfer. This is an exciting, yet often poorly understood method of detection protein-protein interactions in vivo.  FRET relies on the simple physical property that one fluorophore can resonate energy with another, if there is a spectral overlap between the donor fluorophore’s emission wavelengths, and the acceptor fluorophore’s excitation wavelengths, and the two fluorphore’s dipole moments are aligned, and the two fluorophores are very close together in space. For fluorescent proteins, this distance is roughly 10nm, which is considered a direct interaction biochemically. When two compatible fluorophores come together in space, energy can resonate  between the two fluorophores, causing the donor to produce less photons of light, and the acceptor to produce more photons of light. See an excellent FRET animation HERE. What does lack of FRET between two proteins imply? Nothing. Given the variables explained above, two molecules can directly interact, but not FRET. How is FRET best detected between two proteins? Simple, fast Method: FRET-Acceptor Bleaching (AB). See FRET-AB video HERE. This method is not prone to artifacts, but often underestimates FRET efficiency. Complex, by highly quantifiable method: FLIM-FRET. See this site for an explanation of FLIM- FRET. This method is very accurate, but requires very specialized equipment. What About Stimulated Emission FRET? SE-FRET is ONLY A VALID METHOD WHEN TWO FLUOROPHORES ARE KNOWN TO HAVE EQUAL CONCENTRATIONS.  Thus, SE-FRET is excellent for sensors, in where the two fluorophores are fused to one molecule, hence 1:1 in concentration. However SE-FRET for two proteins is highly complex and often prone to artifacts. Want to Learn more?  Molecular Imaging: FRET Microscopy and Spectroscopy. by Ammasi Periasamy (Editor), Richard Day (Editor) ISBN- 10: 0195177207