Fluorescence Lifetime Imaging
By measuring the time a fluorophore takes to decay from its excited state to when it emits a photon, fluorescence lifetime imaging microscopy (FLIM) reveals both the behavior and the location of that molecule. Alterations in the label's immediate environment, such as changes in temperature, pH, hydrophobicity, or the binding of a labeled protein to another, provide alternative decay paths for the excited fluorophore, changing its lifetime. Lifetime measurements can therefore reveal cellular changes that result from drug treatment, development, or even malignant transformation and are unaffected by the excitation laser's intensity or the fluorophore's concentration.
LOCI uses FLIM to study the binding of NADH to proteins in developing cells and cancerous cells. NADH is naturally fluorescent, but the fluorescence lifetime doubles from 0.5 to 1 nanoseconds when the coenzyme binds to protein. The excited-state lifetime of a fluorescent molecule is intensity independent and sensitive to changes in the local microenvironment of the fluorophore.
At LOCI, the Spectral Lifetime Multiphoton Microscope (SLIM) and Optical Workstation (OWS) microscopes use time domain frequency to measure fluorescence lifetimes: a short pulse of light excites the sample, and fluorescence emission is recorded as a function of time in nanoseconds.