Confocal microscopy of single molecules of the green fluorescent protein

Single molecule detection has been extended into life sciences by use of strongly fluorescent labels. The green fluorescent protein (GFP) as a self-fluorescent biomolecule has attracted considerable attention. Here, single molecules of the GFP-mutant Glu222Gln are immobilized in a polyvinylalcohol matrix and detected by confocal fluorescence microscopy. Although this mutant stabilizes one of both conformers of the wild-type GFP, the investigation of its fluorescence dynamics reveals strong signal fluctuations. This fluorescence behaviour is—at least partly—caused by reversible photochemical changes of the protein framework, that can relax into the fluorescent state on different timescales. Thus, this protein appears particularly appropriate for studying the microheterogeneity of the macromolecule GFP on a single molecule level.