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 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.
Brau
Ricardo R
RR
Department of Mechanical Engineering, Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Tarsa
Peter B
PB
Ferrer
Jorge M
JM
Lee
Peter
P
Lang
Matthew J
MJ
D001703:Biophysics / Q000379:methods*; D002232:Carbocyanines / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000494:pharmacology; D008027:Light; D008846:Micromanipulation; D011506:Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D013997:Time Factors
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins; C089187:cyanine dye 3; D004247:DNA