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 μM, the doubly-charged
dyes showed no significant light or dark toxicity, whereas the singly-charged
dyes are phototoxic even at 0.5 μM. The doubly-charged
dyes showed
phototoxicity at concentrations greater than 10 μM, 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.