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Single Wavelength Excitation Dual Color Flim for Multiplexing Genetically Encoded FRET Biosensors

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Demeautis, Claire | Sipieter, François | Roul, Julien | Chapuis, Catherine | Padilla-Parra, Sergi | Riquet, Franck | Tramier, Marc

Edité par CCSD ; Biophysical Society -

International audience. 2555-Plat. Genetically encoded Förster Resonance Energy Transfert (FRET) biosensors are powerful tools for monitoring spatiotemporal biochemical activities in living samples. By labeling a probe protein with a pair of fluorescent proteins, FRET measurement allows to follow a conformational change of the probe sensor to a specific activity. A very exciting challenge is to follow two FRET biosensors at the same time in the same sample and in the same cellular compartment. But the multiplex approach suffers from two limitations: (i) a spectral bleed-through of the first acceptor in the second donor emission band that depends on the concentration of the two biosensors and (ii) the multiple excitation wavelengths which necessitates sequential acquisition that is not adequate to follow fast signal changes in highly dynamic biochemical activities

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