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Biophys. J. BioFAST: First Published April 28, 2006. doi:10.1529/biophysj.106.082602
© 2006 by the Biophysical Society.

A more recent version of this article appeared on August 1, 2006.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Interlaced Optical Force-Fluorescence Measurements for Single Molecule Biophysics

Ricardo R. Brau 1, Peter B. Tarsa 1, Jorge M. Ferrer 1, Peter Lee 1 and Matthew J. Lang 1*

1 MIT

* To whom correspondence should be addressed. E-mail: mjlang{at}mit.edu.

Submitted on February 3, 2006
Revised on March 20, 2006
Accepted on 5 April 2006


  Abstract
Combining optical tweezers with single molecule fluorescence offers a powerful technique to study the biophysical properties of single proteins and molecules. However, such integration into a combined, coincident arrangement has been severely limited by the dramatic reduction in fluorescence longevity of common dyes under simultaneous exposure to trapping and fluorescence excitation beams. We present a novel approach to overcome this problem by alternately modulating the optical trap and excitation beams to prevent simultaneous exposure of the fluorescent dye. We demonstrate the dramatic reduction of trap-induced photobleaching effects on the common single molecule fluorescence dye Cy3, which is highly susceptible to this destructive pathway. The extension in characteristic fluorophore longevity, a 20-fold improvement when compared to simultaneous exposure to both beams, prolongs the fluorescence emission to several tens of seconds in a combined, coincident arrangement. Furthermore, we show that this scheme, interlaced optical force-fluorescence (IOFF), does not compromise the trap stiffness or single molecule fluorescence sensitivity at sufficiently high modulation frequencies. Such improvement permits the simultaneous measurement of the mechanical state of a system with optical tweezers and the localization of molecular changes with single molecule fluorescence, as demonstrated by mechanically unzipping a 15-base-pair DNA segment labeled with Cy3.

Key Words: Cy3, force spectroscopy, optical tweezers, photobleaching, single molecule fluorescence




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Copyright © 2006 by the Biophysical Society.