In addition to intensity and wavelength, fluorescence lifetime imaging can be utilized to visualize the factors that affect the fluorescence lifetime properties of the probe molecule i.e. the state of environment in the immediate vicinity of the probe molecule.
The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state. For a single exponential decay, the fluorescence intensity as a function of time after a brief pulse of excitation light is described as
where I0 is the initial intensity immediately after the excitation pulse.
Fluorescence lifetime is independent of dye concentration, photobleaching, light scattering and excitation light intensity.
The fluorescence lifetime is extermely sensitive to the changes in immediated molecular environment. The factors affecting the fluorescence lifetime include pH, ion concentration, viscosity, hydrophobic properties, oxygen concentration, molecular binding, molecular interactions by radiationless energy transfer when two proteins approach each other or electron transfer when two moleucles are almost in contact.
Therefore, fluorescence lifetime imaging allows us to perform e.g. accurate ion concentration measurement and Fluorescence Resonance Energy Transfer (FRET) analysis.
There are two methods of fluorescence lifetime imaging: the time-domain method and the frequency-domain method.
Time-domain FLIM - The sample is excited using a pulsed light source and the fluorescence is detected at varoious delay values using a time-gated imaging detector. The fluorescence lifetime image is obtained by processing the time-gated images.
Frequency-domain FLIM - Here the sample is excited using an intensity modulated light source and the fluorescence is detected using a gain modulated imaging detector. Fluorescence lifetime is calculated by measuring the phase shift of fluorescence and the reduction in its amplitude.
Fluorescence Lifetime Imaging Microscopy: Spatial resolution of biochemical processes in the cell Philippe I. H. Bastiaens and Anthony Squire Trends in Cell Biology 1999, 9, 48-51