Since the 1990s, advances in single-molecule imaging and manipulation have led to the emergence of single-molecule biology (ie, the probing and understanding of biological behaviors on a single-molecule basis). Due to contributions from many laboratories around the world, this has changed the way many biological problems are addressed and generated much new knowledge.
One early example of single-molecule biology is the study of turnovers of the enzyme cholesterol oxidase.1 The enzyme contains a flavin moiety that is naturally fluorescent in its oxidized form, but not in its reduced form. Each on/off cycle of fluorescence emission corresponds to an enzymatic cycle, enabling the observation of enzymatic reactions in real time. On a single-molecule basis, a chemical reaction occurs in a stochastic way (ie, waiting time for a chemical reaction to occur is random) rather than deterministic as is the case of a large number of molecules. The ability to observe a single-molecule chemical reaction in real time is important because many biomolecules, such as DNA, exist as single molecules in live cells.