Fluorescence lifetime mapping as an alternative way to follow intracellular processes

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate many biochemical processes. In addition to emission intensity detection, it measures the fluorescence lifetime (FL) of the fluorophore on a scanned sample area. Since FL can be modulated by processes like energy transfer or be sensitive to physical/chemical variations, its measurement is exploited to monitor variation in cellular environment. For example, fluorescence resonant energy transfer (FRET) between suitable donor-acceptor groups is widely used to recognize specific intracellular interactions. In this field, FLIM-FRET approach offers some advantages in comparison to steady-state emission: the almost non dependence of FL on the label concentration and the ability to distinguish interacting and non interacting donor fractions. In this work, FLIM-FRET experiments are performed in HeLa and SH-SY5Y cells, allowing the analysis of Ca2+ dynamics in different cell compartments (mitochondria, cytosol or Golgi apparatus), by means of Cameleon sensors. These sensors are genetically-encoded indicators for Ca2+, in which Ca2+-responsive elements alter the FRET efficiency between two fluorescent proteins as a function of intracellular Ca2+ concentration. In some sensors, the Cyan Fluorescent Protein donor unit has been replaced by mCerulean3, characterized by single FL decay and higher brightness. FLIM analysis has permitted a better determination of FRET efficiency, avoiding the most common artifacts of intensity-based approaches, such as intermolecular FRET induced by high sensor concentrations. FLIM is also exploited for the intracellular localization of 30 nm sized gold nanoparticles in Human Umbilical Vein Endothelial Cells. Since gold nanoparticles are fluorescent under two-photon excitation with a short FL (about 200 ps), it has been possible to distinguish and to map their contribution from the residual cell autofluorescence (FL of few ns), within the same spectral range.