Optical probing of single fluorescent molecules and proteins

Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis on the underlying principles methodo logy and the application of single-molecule analysis at room temperature. T his Minireview is mainly focused on the application of confocal and near-fi eld optical microscopy to investigate the photodynamics of individual molec ules embedded in ultrathin polymer layers. We discuss rotational mobility o f individual probe molecules in polystyrene and poly(methylmethacrylate) th in films, fluorescence lifetime trajectories and their spatial distribution , and real-time singlet- triplet dynamics. As a whole, the single-molecule photodynamics observed is due to the dynamic nature of both polymers at roo m temperature, where local polymer conformational dynamics modulates the ox ygen concentration and diffusion on a molecular scale, influencing the fluo rescence lifetime and intersystem crossing parameters. We also discuss the photodynamics of individual autofluorescent proteins, in particular the on/ off blinking and the apparent stability of the protein against bleaching. T hese studies illustrate the unique information obtainable with the single-m olecule approach, information that is otherwise hidden in ensemble-averaged measurements.