Single-molecule fluorescence-based analysis of protein conformation, interaction, and oligomerization in cellular systems.

Single-molecule imaging (SMI) of proteins in operation has a history of intensive investigations over 20 years and is now widely used in various fields of biology and biotechnology. We review the recent advances in SMI of fluorescently-tagged proteins in structural biology, focusing on technical applicability of SMI to the measurements in living cells. Basic technologies and recent applications of SMI in structural biology are introduced. Distinct from other methods in structural biology, SMI directly observes single molecules and single-molecule events one-by-one, thus, explicitly analyzing the distribution of protein structures and the history of protein dynamics. It also allows one to detect single events of protein interaction. One unique feature of SMI is that it is applicable in complicated and heterogeneous environments, including living cells. The numbers, location, movements, interaction, oligomerization, and conformation of single-protein molecules have been determined using SMI in cellular systems.