We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localisation of protein-protein interactions in situ. An Example of a new fluorescent protein variant with enhanced properties are given and the development of a FRET biosensor for simultaneous measurement of multiple intra- and inter-molecular interactions is illustrated by experimental evidence of an energy transfer cascade via multiple acceptors. The juxtaposition of interacting population and FRET efficiency is elucidated, with a priori knowledge, by multi-exponential analysis.

© 2003 SPIE

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