Interlaced Optical Force-Fluorescence Measurements for Single Molecule Biophysics

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 longevi...

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Bibliographic Details
Published in:Biophysical journal 2006-08, Vol.91 (3), p.1069-1077
Main Authors: Brau, Ricardo R., Tarsa, Peter B., Ferrer, Jorge M., Lee, Peter, Lang, Matthew J.
Format: Article
Language:English
Subjects:
Biochemistry
Biophysics - methods
Carbocyanines - pharmacology
DNA - chemistry
Equipment Design
Fluorescence
Fluorescent Dyes - pharmacology
Light
Micromanipulation
Molecules
Proteins
Proteins - chemistry
Spectrometry, Fluorescence - instrumentation
Spectrometry, Fluorescence - methods
Spectroscopy, Imaging, Other Techniques
Spectrum analysis
Time Factors
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Summary: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.
ISSN:0006-3495
1542-0086
DOI:10.1529/biophysj.106.082602