Why does the phase lifetime differ from the modulation lifetime?

The lifetime from phase and modulation are the same only if all the fluorophores behave according to a single exponential decay. In this case we speak of a mono-exponential fluorescence lifetime distribution and the lifetime calculated from the phase shift and the lifetime calculated from the modulation depth are in this case identical. In case of multi-exponential lifetime distributions, the lifetimes calculated from the phase shift and from modulation depth will be different because the equations on which the lifetimes are based assume mono-exponentiality. The more exponents the more both lifetimes differ. For FRET or for tracking lifetime changes the lifetime from phase can be taken to determine the changes in lifetimes.

For multi-exponential distributions, a multi-frequency recording can be used and the phase and modulation behaviour can be fitted with a multi-exponential model, in a similar way as in time domain FLIM (TCSPC). This allows one to retrieve multiple lifetime components and their relative fractions.