DETECTION AND QUANTIFICATION OF BIOMOLECULAR ASSOCIATION IN LIVING CELLS USING SINGLE-MOLECULE MICROSCOPY

During their random motion, biomolecules experience a manifold of interactions that transiently conjoin their paths. It is extremely difficult to measure such binding events directly in the context of a living cell: interactions may be short lived, they may affect only a minority fraction of molecules, or they may not lead to a macroscopically observable effect. We describe here a new single-molecule imaging method that allows for detecting and quantifying associations of mobile molecules. By "thinning out clusters while conserving the stoichiometry of labeling" (TOCCSL), we can virtually dilute the probe directly in the cell, without affecting the fluorescence labeling of single clusters. Essentially, an analysis region is created within the cell by photobleaching; this region is devoid of active probe. Brownian diffusion or other transport processes lead to reentry of active probe into the analysis region. At the onset of the recovery process, single spots can be resolved as well-separated, diffraction-limited signals. Standard single-molecule microscopy then allows for characterizing the spots in terms of their composition and mobility.