A Membrane-Bound FRET-Based Caspase Sensor for Detection of Apoptosis Using Fluorescence Lifetime and Total Internal Reflection Microscopy

摘要

A caspase sensor based on Forster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane-bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal-to-background ratio is increased considerably. In comparison with conventional or laser scanning microscopy, this permits long-term observation of apoptosis in live cell cultures using very low absorption and avoiding light-induced damages of the samples.