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.

Thermally Reduced Graphene Oxide Nanohybrids of Chiral Functional Naphthalenediimides for Prostate Cancer Cells Bioimaging

“…Electrochemical Cyclic Voltammetry analysis was carried out using a standard 3 electrode system comprising a platinum working electrode, an Ag/AgCl (in solution of KCl) reference electrode and platinum wire counter electrode and the ferrocene/ferrocenium (Fc/Fc + ) redox couple as the internal standard was used. Two-photon excitation experiments were performed at the Rutherford Appleton Laboratory following the methodology described by Botchway et al [ 73 ] A mode locked Mira titanium sapphire laser (Coherent Lasers Ltd, USA), generating 180 fs pulses at 75 MHz and emitting light at a wavelength of 710-970 nm was used for the 2-photon excitation. The laser was pumped by a solid state continuous wave 532 nm laser (Verdi V18, Coherent Laser Ltd), with the oscillator fundamental output of 915 ± 2 nm or 810 ± 2 nm.…”

Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes

“…In contrast, lifetime-based approaches remain severely underexplored. Yet lifetime-based sensing and imaging is a powerful complement to the usual methods (5,6). A major benefit is that emission lifetimes are normally independent of concentration and can be calibrated absolutely, unlike intensity measurements, which are subject to error from fluctuations in the efficiency of delivery and detection of light.…”

Fluorescence Lifetime Imaging of Propranolol Uptake in Living Glial C6 Cells

“…However, excitation on the red edge at 310-320 nm allows selective stimulation of propranolol fluorescence in the presence of tryptophancontaining proteins in a manner analogous to that previously described for observation of serotonin and 5-hydroxytryptophan fluorescence [18,23]. For propranolol, the long wavelength absorption peak is at 289 nm (ε = 5,900 M −1 cm −1 ) whilst the extinction coefficient at 315 nm is 1,730 dm 3 mol −1 cm −1 .…”

“…The microscope system for fluorescence lifetime imaging has been previously described [18]. Briefly, it is based on a Ti: sapphire laser (Coherent Mira) pumping an optical parametric oscillator (APE) coupled to an inverted microscope (Nikon TE2000U) with a water-immersion ultraviolet corrected objective (Nikon VC x60, NA 1.2).…”

“…In contrast, two-photon microscopy using red and near infrared wavelengths with the benefit of confining the region of excitation to the focal volume of a femtosecond laser beam focussed to the diffraction limit, which further allows pseudoconfocal imaging [14,15] with reduced overall phototoxicity. Multiphoton microscopy has applications in pharmaceutical sciences [16] effectively permitting imaging of UV intrinsic fluorescence from biochemical chromophores such as serotonin [17,18] and dehydroergosterol [19].…”