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Interlaced Optical Force-Fluorescence Measurements for Single Molecule Biophysics

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

^* Biological Engineering Division, and Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts

Correspondence: Address reprint requests to Matthew J. Lang, Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139. Tel.: 617-452-2631; E-mail: mjlang{at}mit.edu.

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, 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-basepair DNA segment labeled with Cy3.

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