Single-Molecule Light Microscopy

The complexity of biological processes requires experimental techniques which are able to resolve events on appropriate temporal and spatial scales. As all biological processes are ultimately driven by the dynamics and interactions of individual molecules, studies on the single‐molecule level provide important insights about a large variety of parameters at thermodynamic equilibrium and without ensemble averaging. In the life sciences, single‐molecule experiments are preferentially performed using fluorescence light microscopy owing to its high sensitivity, its temporal resolution and its ability to address live and thus dynamic specimen. By today, a range of single‐molecule techniques such as single‐pair Förster resonance energy transfer (FRET), single‐molecule tracking and different counting techniques are readily available to characterise molecular interactions, conformational dynamics, complex stoichiometries and translational mobilities in biological systems both in vitro and in situ.