title: Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation.
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.
authors: Rutherford Appleton Laboratory, Central Laser Facility, HSIC, Didcot, Oxfordshire OX11 0QX, United Kingdom. s.w.botchway@rl.ac.uk
authors: Stanley Walter
authors: SW
authors: Botchway
authors: Anthony William
authors: AW
authors: Parker
authors: Roger Hugh
authors: RH
authors: Bisby
authors: Ana Goncalves
authors: AG
authors: Crisostomo
mesh_terms: D006916:5-Hydroxytryptophan / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D005347:Fibroblasts / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008168:Lung / Q000166:cytology; D008407:Mast Cells / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton; D017785:Photons; D051381:Rats; D012701:Serotonin / Q000378:metabolism*
chemical_list: D012701:Serotonin; D006916:5-Hydroxytryptophan