1. Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics
- Author
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Daniel Fetting, Achim Donnermeyer, Joachim Ross, Philip Tinnefeld, Christian Roth, and P. Buschkamp
- Subjects
Confocal ,Excitation filter ,Color ,Nanotechnology ,Sensitivity and Specificity ,law.invention ,law ,Microscopy ,Materials Chemistry ,Fluorescence Resonance Energy Transfer ,Physical and Theoretical Chemistry ,Spectroscopy ,Microscopy, Confocal ,Chemistry ,business.industry ,Lasers ,Optical Devices ,DNA ,Laser ,Surfaces, Coatings and Films ,Förster resonance energy transfer ,Biomolecular complex ,Optoelectronics ,Thermodynamics ,business ,Excitation - 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.
- Published
- 2007