Real-time Cellular Uptake of Serotonin Using Fluorescence Lifetime Imaging with 2-photon Excitation – a Unique Biological Window

Serotonin (5-hydroxytryptamine) is an endogenous compound located mainly in the enterochromaffin cells of the gastrointestinal tract, serotonergic neurons, and platelets. Physiologically 5-HT acts as a neurotransmitter, neuromodulator and vasoconstrictor [1]. Imaging studies related to the processes of synthesis and serotonin sequestering/release from secretory vesicles are essential for the understanding of these mechanisms. The real-time uptake of serotonin by rat basophilic leukaemia mast cell line RBL2H3 and 5-hydroxytryptophan (5-HTP) by Chinese hamster V79 cells (Figure 1) has been studied by monitoring ultraviolet (340 nm) fluorescence induced by twophoton sub-picosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm [2] shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from autofluorescence of other cellular components (e.g. flavins, NADH). Using fluorescence lifetime imaging (FLIM) of cells, we observe 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 [3]. The use of this technique has shown the potential for evaluation of serotonin distribution with high dimensional resolution within a cell as well as to determine its intracellular concentration. It is equally applicable to the study of other biomolecules with biophysical characteristics similar to tryptophan derivatives. We also imaged the uptake of serotonin in the presence of drugs such as propranolol (beta-blocker) and fluoxetine (selective serotonin reuptake inhibitor) using both RBL and C6 glial cells. Results obtained reflect the pharmacological actions of these agents.