Development of an improved FRET imaging two-photon microscope combining lifetime and spectral imaging

FRET imaging is widely used in biology to map protein interactions in living cells. A number of recent technical developments promise to provide more accurate and quantitative measurement FRET efficiencies and protein bonding ratio. Using a reference FRET construct based on a doubly labeled DNA, one can estimate the accuracy of different techniques like spectral or lifetime imaging to resolve FRET. Further improvement have lead to the possibility of measuring in a single detector both spectral and lifetime information from the fluorescence emitted by the sample. We are currently building such a system based on an intensity modulated multi-anode PMT and have developed a global fitting algorithm to extract valuable information from combined spectral and lifetime imaging data sets.