When motion slows: intrinsic photophysics of thioflavin T and X cations at cryogenic temperatures

Gas-phase spectroscopy reveals the intrinsic photophysics of dyes in the absence of a microenvironment, such as a solvent or protein matrix. Here we report fluorescence-excitation and emission spectra of two thioflavin cations, thioflavin T (ThT⁺) and thioflavin X (ThX⁺), isolated in vacuo and at cryogenic temperatures using a custom-built setup named LUNA2 (Luminescence iNstrument in Aarhus) where ions are mass-selected and trapped in a cylindrical Paul trap in connection with a liquid nitrogen reservoir. We determine absorption band maxima at 422 ± 2 nm and 434 ± 1 nm for ThT⁺ and ThX⁺, respectively. Time-dependent density functional theory calculations also predict the absorption by ThX⁺ to be redshifted compared to ThT⁺, by 59 meV in agreement with the experimental shift of 81 meV. Despite the low temperature, the emission spectra are broad and featureless, resulting in large Stokes shifts (few tenths of an eV). Based on the time-response in a photomultiplier detector, we measure excited-state lifetimes that are significantly shorter than those for other fluorescent ions previously studied by our group, e.g. rhodamines, oxazines, and protein biochromophores. Our findings suggest that there is no local S₁ minimum with similar geometry as in the ground state, and that the ions undergo barrierless twist motion in the electronically excited state. This conclusion is supported by previous theoretical calculations for ThT⁺.