Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood calculated lifetimes

PHYS 3

Amit Meller, ameller@bu.edu1, Evrim Atas, eatas@bu.edu1, Jason Sutin, jason@sutin.net1, and Joshua Edel2. (1) Department of Biomedical Engineering, Boston University, 44 Cummington St, Boston, MA 02215, (2) Institute of Biomedical Engineering, Imperial College, South Kensington Campus, London, SW7 2AZ, United Kingdom
Time-resolved detection of single fluorophores using the principles of time-correlated single-photon counting (TCSPC) has become popular in the past few years. The fluorescence lifetime of individual dyes is an intrinsic property of the molecule, affected only by its chemical environment. Thus SM lifetime data can be directly compared with bulk (or ensemble averaged) measurements. In contrast, the detected photon intensity is an extrinsic quantity, which depends on experimental factors, such as excitation intensity, collection efficiency, and the position of the dye; complicating our ability to compare SM intensity data with bulk measurements. We present a maximum likelihood estimator routine to compensate for localized background fluorescence and instrument response in SM TCSPC. With this algorithm we robustly extract fluorescent lifetimes decays with as few as 20 photons, and measure fluorescence lifetime trajectories of surface-immobilized DNA coupled with donor-acceptor FRET pairs. Our method is used for accurate determination of the FRET efficiencies.
 

Single Molecule Spectroscopy, Imaging and Manipulation of Biomolecular Systems
8:20 AM-12:00 PM, Sunday, August 19, 2007 BCEC -- 157A, Oral

Division of Physical Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007