title: Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics.
abstract: We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.
authors.forename: Joachim
authors.initials: J
authors.lastname: Ross
authors.forename: Peter
authors.initials: P
authors.lastname: Buschkamp
authors.forename: Daniel
authors.initials: D
authors.lastname: Fetting
authors.forename: Achim
authors.initials: A
authors.lastname: Donnermeyer
authors.forename: Christian M
authors.initials: CM
authors.lastname: Roth
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
mesh_terms: D003116:Color; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods*; D055096:Optical Devices; D012680:Sensitivity and Specificity; D013816:Thermodynamics
chemical_list: D004247:DNA