FLIM-FRET  One of the most difficult aspects of quantifying FRET is the problem of spectral bleed through, and interpreting what is bleeding light signal, and what is caused by real FRET. This problem is greatly enhanced when looking at two proteins, which are likely expressed at different levels. One way around this is FRET-AB, which only looks at gain of fluorescence by the donor once the acceptor has been bleached. However, this is not accurate for quantification, as bleaching the donor can result in bleaching of the acceptor, often underestimating FRET efficiency. FLIM, or fluorescence lifetime measure, is a method that measures how long it takes for any fluorophore to go from it’s excited state to its ground state, while releasing a photon. This time is affected by FRET, and results in decreased time as the energy resonates to the acceptor fluorophore. By using picosecond pulsed lasers and gated photon counters, the precise lifetime of a fluorophore  can be measured, and this relies on nano-environmental conditions such as hydrophobicity, pH, and FRET. What is critical, is that FLIM can detect FRET quantitatively independent of fluorophore concentration. Thus, FLIM- FRET is perfect for two molecule interaction studies in vivo. For a thorough explanation of FLIM, see the Becker-Hickl website. FLIM is an example of using fluorescence beyond just that of intensity and location of a protein, and allows us to understand the environment of a protein at atomic resolution, down to 8nm, far greater than what is possible with even electron microscopy.