Measuring FRET in living cells with FLIM

Interactions between proteins can be demonstrated by fluorescence resonance energy transfer (FRET) [1]. The proteins under test must be fused with donor and acceptor molecules. At FRET energy is transferred non-radiatively from donor to acceptor. FRET can be detected in two ways: the change in emission-ratio between donor and acceptor can be measured, or the decrease in lifetime of the fluorescence emitted by the donor molecule is measured. This change in lifetime is detected by fluorescence lifetime imaging (FLIM) [2]. The advantage of FLIM is that it is independent of intensity variations over the sample, and it does not suffer from “bleed-through” of excitation or emission light to the acceptor as in ratio measurements. Fluorescence lifetime imaging can now be done on a wide field fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an accousto-optical modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. The LIFA is used at The Netherlands Cancer Institute to measure FRET between CFP and YFP (the Cyan and Yellow mutants of Green Fluorescent Protein) labeled vesicles in living HeLa cells. Results of this are shown.