Lifetime and spectrally resolved characterization of the photodynamics of single fluorophores in solution using the anti-Brownian electrokinetic trap.

We report simultaneous determination of fluorescence intensity, lifetime, and emission spectrum over time scales on the order of seconds for single molecules in solution, using the anti-Brownian electrokinetic trap. We demonstrate the technique with trapped single fluorophores of Atto647N and Alexa647. Three emission states with distinct intensities, lifetimes, and emission peaks are found in the case of Atto647N. Transitions between states happen occasionally. We characterize the three states and quantify the transition probabilities between states using concurrent intensity, lifetime, and spectrum data. Alexa647, on the other hand, showed little dynamics. These results represent a significant advance in the ability to identify and characterize different dynamical states of single molecules in aqueous solution with high precision and millisecond time resolution.