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Record and analyze fluorescence lifetime images.
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Probing the refractive index of the microenvironment

Fluorescence lifetime is a property which is almost completely insensitive to fluorophore concentration. It provides the means of discrimination among molecules with a spectrally overlapped emission. A further important feature is the dependence of the fluorescence decay time to the microenvironment. This dependence varies between fluorophores and certain factors.

The fluorescence lifetime of e.g. GFP can be used to probe the direct local environment of the fluorophore, because the local refractive index affects fluorescence decay. The inverse GFP fluorescence lifetime scales approximately with the square of the refractive index.

Cell membranes normally have 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 2.17 ns in the cell membrane to 2.67 ns in the cytoplasm.

Reference: Klaus Suhling, Jan Siegel, David Phillips, Paul M. W. French, Sandrine Leveque-Fort, Stephen E. D. Webb, and Daniel M. Davis. “Imaging the environment of green fluorescent protein”. Biophysical Journal, 83:3589-3595 (2002).

There was, however, no correlation observed between GFP fluorescence lifetime and the viscosity of the surrounding solution. This was researched with a variety of solutes added to GFP in buffer.

Reference: Suhling, K., D. M. Davis, and D. Phillips. “The influence of solvent viscosity on the fluorescence decay and time-resolved anisotropy of green fluorescent protein”. J. Fluoresc. 12:91–95 (2002).

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