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

Botchway, SW; Parker, AW; Bisby, RH; Crisostomo, AG

Abstract

The real-time uptake of serotonin, a neurotransmitter, by rat leukaemia 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 sub-picosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm (Williams et al, 1999) 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.

Citation

Botchway, S., Parker, A., Bisby, R., & Crisostomo, A. (2008). Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation. Microscopy Research and Technique, 71(4), 267-273. https://doi.org/10.1002/jemt.20548

• Journal Article Type: Article
• Publication Date: Jan 1, 2008
• Deposit Date: Jun 24, 2009
• Journal: Microscopy Research and Technique
• Print ISSN: 1059-910X
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 71
• Issue: 4
• Pages: 267-273
• DOI: https://doi.org/10.1002/jemt.20548
• Keywords: Serotonin; fluorescence; multiphoton; cellular; imaging; lifetime; microscopy; 5-hydroxytryptophan; FLIM
• Publisher URL: http://dx.doi.org/10.1002/jemt.20548