Poster abstracts
Poster number 24 submitted by Cho-Ying Chuang
Combined multi-color fluorescence and ultra-high resolution optical tweezers
Cho-Ying Chuang (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan), Miles L Whitmore (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan), Jess L West (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan), Matthew J Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan)
Abstract:
We present a single-molecule instrument that combines ultra-high resolution optical tweezers with multicolor confocal fluorescence microscopy. Timeshared dual optical traps were interlaced and synchronized with three fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting detectors (one for each excitation laser). Our new instrument enables the simultaneous measurement of DNA tether extension changes (e.g., from helicase or polymerase motion) and multiple fluorescently labeled observables (e.g., internal protein conformation dynamics via FRET or precise stoichiometry of complexes via multi-colored fluorophore counting). We demonstrated our instrument by measuring the binding and unbinding of fluorophore-labeled single stranded DNA oligonucleotides to a complementary tethered strand of DNA. Further, we combined multi-channel sample chambers with precise computer control of fluorescence measurement and triggered chamber motion to implement an automated ‘molecular assembly line.’ This allowed us to precisely add individual molecules of different types to a single DNA tether while conserving fluorescence photons and reducing photobleaching. In the future, these instrumentation advancements should enable the precise single-molecule assembly and measurement of complex, multi-component molecular machine systems.
References:
M.J. Comstock, Taekjip Ha, and Y.R. Chemla, Nature Methods 8, 335-340 (2011)
Keywords: optical tweezers, fluorescence microscopy, single-molecule