Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation |
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Authors: | Botchway Stanley Walter Parker Anthony William Bisby Roger Hugh Crisostomo Ana Goncalves |
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Affiliation: | Rutherford Appleton Laboratory, Central Laser Facility, HSIC, Didcot, Oxfordshire OX11 0QX, United Kingdom. s.w.botchway@rl.ac.uk |
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Abstract: | 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 approximately 2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to approximately 2.0 ns when stored within secretory vesicles. |
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Keywords: | serotonin fluorescence multiphoton cellular imaging lifetime microscopy 5‐hydroxytryptophan FLIM |
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