Lambert Instruments

The fluorescence lifetime is a signature almost insensitive to the fluorophore concentration that provides means of discrimination among molecules with spectrally overlapped emission. A further feature is the sensitivity of the decay time to the microenvironment. This dependence varies between different fluorophores and factors.

Refractive index
The GFP fluorescence lifetime can be directly used to probe the local environment of GFP, because this decay depends on the local refractive index. The inverse GFP fluorescence lifetime scales approximately with the square of the refractive index.
The cell membrane has a higher refractive index than the cytoplasm, namely 1,46 - 1,60 and 1,35 respectively. From fluorescence lifetime measurements of GFP in a PBS solution with increasing glycerol concentrations, the expected lifetime of GFP differs from 2,17ns in the cell membrane to 2,67ns in the cytoplasm.

Some environmental factors do not have an influence on the fluorescence lifetime:

Viscosity
No correlation between the lifetime of GFP and the viscosity of the solution surrounding the GFP was observed with a variety of solutes added to GFP in buffer.