Imaging biomolecular interactions by fast three-dimensional tracking of laser-confined carrier particles.
The quantitative study of the near-equilibrium structural behavior of individual biomolecules requires high-resolution experimental approaches with longtime stability. We present a new technique to explore the dynamics of weak intramolecular interactions. It is based on the analysis of the 3D Brownian fluctuations of a laser-confined glass bead that is tethered to a flat surface by the biomolecule of interest. A continuous autofocusing mechanism allows us to maintain or adjust the height of the optical trap with nanometer accuracy over long periods of time. The resulting remarkably stable trapping potential adds a well-defined femto-to-piconewton force bias to the energy landscape of molecular configurations. A combination of optical interferometry and advanced pattern-tracking algorithms provides the 3D bead positions with nanometer spatial and >120 Hz temporal resolution. The analysis of accumulated 3D positions has allowed us not only to identify small single biomolecules but also to characterize their nanomechanical behavior, for example, the force-extension relations of short oligonucleotides and the unfolding/refolding transitions of small protein tethers.
Heinrich
Volkmar
V
Department of Biomedical Engineering and Biomedical Engineering Graduate Group, University of California at Davis, 451 East Health Sciences Drive, Davis, CA 95616, USA. vheinrich@ucdavis.edu
Wong
Wesley P
WP
Halvorsen
Ken
K
Evans
Evan
E
D000465:Algorithms; D000818:Animals; D002645:Chickens; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007834:Lasers; D008857:Microscopy, Interference / Q000295:instrumentation* / Q000379:methods*; D008863:Microspheres; D013049:Spectrin / Q000737:chemistry*; D013499:Surface Properties
D013049:Spectrin