Anna Loveland, a postdoc in the Grigorieff Lab, has a new paper, “A general approach to break the concentration barrier in single-molecule imaging” that appeared today in Nature Methods online. The paper is based on her PhD work, which was done jointly in the labs of Antoine van Oijen and Johannes Walter at Harvard.
Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule’s signal. Loveland et al. employ a new imaging approach called PhADE (photoactivation, diffusion and excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. The authors labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.
Anna B Loveland, Satoshi Habuchi, Johannes C Walte & Antoine M van Oijen (2012) A general approach to break the concentration barrier in single-molecule imaging. Nature Methods