FLIM-FRET analysis using Ca2+ sensors in HeLa cells

In this work, FLIM-FRET experiments are performed in HeLa cells expressing a genetically encoded Ca²⁺ indicator (GECIs) based on FRET, called Cameleon probes. These sensors allow the analysis of Ca²⁺ dynamics in different cell compartments, such as mitochondria and cytosol. Cameleons are composed by two fluorescent proteins (FPs, donor and acceptor moieties) and two Ca²⁺ responsive elements that alter the FRET efficiency between the FPs, as a function of intracellular Ca²⁺ concentration. In particular, we focus on new generated Cameleon sensors where the Cyan Fluorescent Protein donor moiety has been replaced by mCerulean3. Since it is characterized by single fluorescence lifetime decay and higher brightness, mCerulean3 based sensors has permitted the application of FLIM approach for the determination of FRET efficiency. FLIM measurements, compared to intensity-based FRET analysis, have the advantages of being independent of fluorophore concentration, excitation intensity fluctuation, sample thickness, or photobleaching.