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Methods Enzymol. 2010;475:405-26. doi: 10.1016/S0076-6879(10)75016-3.

Force-fluorescence spectroscopy at the single-molecule level.

Author information

1
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Abstract

During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. Yet these techniques would be even more powerful if they would be combined, that is, single-molecule manipulation with an orthogonal single-molecule observation. Here, we present a recently developed approach to combine single-molecule optical tweezers with single-molecule fluorescence spectroscopy. Optical tweezers are used to manipulate and observe mechanical properties on the nanometer scale and piconewton force range. However, once the force range is in the low piconewton range or less, the spatial resolution of optical tweezers decreases significantly. In combination with fluorescence spectroscopy, like Förster resonance energy transfer (FRET), we are able to observe nanometer fluctuations and internal conformational changes in a low-force regime. The possibility to place fluorescent labels at nearly any desired position and a sophisticated design of the experiment increases the amount of information that can be extracted in contrast to pure mechanical or fluorescence experiments.

PMID:
20627166
DOI:
10.1016/S0076-6879(10)75016-3
[Indexed for MEDLINE]

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