Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation

The real‐time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL‐2H3 and 5‐hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two‐photon subpicosecond 630 nm excitation. Comparison with two‐photon excitation with 590 nm photons or by three‐photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto‐fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5‐hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5‐hydroxytryptophan (3.5 ns) in solution are reduced to ∼2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL‐2H3 cells is further reduced to ∼2.0 ns when stored within secretory vesicles. Microsc. Res. Tech., 2008. © 2007 Wiley‐Liss, Inc.