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 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.
Botchway
Stanley Walter
SW
Rutherford Appleton Laboratory, Central Laser Facility, HSIC, Didcot, Oxfordshire OX11 0QX, United Kingdom. s.w.botchway@rl.ac.uk
Parker
Anthony William
AW
Bisby
Roger Hugh
RH
Crisostomo
Ana Goncalves
AG
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*
D012701:Serotonin; D006916:5-Hydroxytryptophan