Thiophene-based dyes for probing membranes

Abstract

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.