%0 Journal Article
%A López Duarte, Ismael
%A Chairatana, Phoom
%A Wu, Yilei
%A Pérez-Moreno, Javier
%A Bennett, Philip M.
%A Reeve, James E.
%A Boczarow, Igor
%A Kaluza, Wojciech
%A Hosny, Neveen A.
%A Stranks, Samuel D.
%A Nicholas, Robin J.
%A Clays, Koen
%A Kuimova, Marina K.
%A Anderson, Harry L.
%T Thiophene-based dyes for probing membranes
%D 2015
%@ 1477-0520
%@ 1477-0539
%U https://hdl.handle.net/20.500.14352/114887
%X We report the synthesis of four new cationic dipolar push-pull dyes, together with an evaluation of their photophysical and photobiological characteristics pertinent to imaging membranes by fluorescence and second harmonic generation (SHG). All four dyes consist of an N, N-diethylaniline electron-donor conjugated to a pyridinium electron-acceptor via a thiophene bridge, with either vinylene (-CH=CH-) or ethynylene (-C=C-) linking groups, and with either singly-charged or doubly-charged pyridinium terminals. The absorption and fluorescence behavior of these dyes were compared to a commercially available fluorescent membrane stain, the styryl dye FM4-64. The hyperpolarizabilities of all dyes were compared using hyper-Rayleigh scattering at 800 nm. Cellular uptake, localization, toxicity and phototoxicity were evaluated using tissue cell cultures (HeLa, SK-OV-3 and MDA-231). Replacing the central alkene bridge of FM4-64 with a thiophene does not substantially change the absorption, fluorescence or hyperpolarizability, whereas changing the vinylene-links to ethynylenes shifts the absorption and fluorescence to shorter wavelengths, and reduces the hyperpolarizability by about a factor of two. SHG and fluorescence imaging experiments in live cells showed that the doubly-charged thiophene dyes localize in plasma membranes, and exhibit lower internalization rates compared to FM4-64, resulting in less signal from the cell cytosol. At a typical imaging concentration of 1 mu M, the doubly-charged dyes showed no significant light or dark toxicity, whereas the singly-charged dyes are phototoxic even at 0.5 mu M. The doubly-charged dyes showed phototoxicity at concentrations greater than 10 mu M, although they do not generate singlet oxygen, indicating that the phototoxicity is type I rather than type II. The doubly-charged thiophene dyes are more effective than FM4-64 as SHG dyes for live cells.
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