Single-molecule spectroscopy of protein folding dynamics-expanding scope and timescales

► Single-molecule spectroscopy can now be used to probe timescales from nanoseconds to weeks. ► Nanosecond-FCS allows the investigation of the submicrosecond dynamics of unfolded and intrinsically disordered proteins. ► Microfluidic mixing methods provide nonequilibrium single-molecule information from submillisecond to second timescales. ► The rigorous analysis of photon statistics has allowed the average transition path time of protein folding to be measured. ► Single-molecule spectroscopy starts to bridge the gap between folding in vitro and in vivo.