Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements

Abstract

For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the build-up of photo-induced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules.The concept was experimentally verified by measuring the fluorescence response of freely diffusing Cyanine fluorophores (Cy5), undergoing trans-cis isomerisation when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.

Fluorescence-based characterization of non-fluorescent transient states of tryptophan – prospects for protein conformation and interaction studies

“…Taking analyte diffusion into account for the slower transitions, all determined rates were found to be within the range of parameter values reported from FP and RTP measurements. For slower transitions already known, the influence of diffusion on the related relaxations in the TRAST curves can also provide a measure of the mobility of the analyzed molecules 59 . TRAST measurements can thus retrieve similar, and in some aspects extended, transient state information compared to RTP and FP, and can be performed under a broad range of conditions.…”

An aptamer-based single particle method for sensitive detection of thrombin using fluorescent quantum dots as labeling probes

“…Fluorescence correlation spectroscopy (FCS) is a single particle detection technique using correlation analysis of fluctuations of the fluorescence intensity due to Brownian motion of particles in a small volume (less than 1.0 fL) [33][34][35][36][37][38]. FCS has been used in study on molecular diffusion, chemical kinetics, molecular interaction in vitro and in vivo, the selection of aptamers and bioassays [39][40][41][42].…”

Transient state microscopy probes patterns of altered oxygen consumption in cancer cells

“…By TRAST, several categories of photo-induced, long-lived, dark, transient states can be monitored, including triplet [28][29][30][31][32][33], photo-ionized [32] and photoisomerized states [34]. In particular triplet states are readily quenched by oxygen, and their lifetimes depend strongly on the local oxygen concentration.…”