Time-resolved single molecule FRET-
Multi-parameter single molecule fluorescence spectroscopy is an advanced technique, which provides additional and complementary information to fluorescence intensity measurements. In addition to the fluorescence intensity, the fluorescence lifetime can be resolved as a function of time for individual molecules. Fluorescence lifetime is an intrinsic quantity, and its value can be numerically compared with ensemble and time-averaged single-molecule results. Fluorescence lifetime can also be used for a better definition of FRET efficiencies at the single molecule level, and comparison between SM burst-analysis data and SM of immobilized molecules.
We have developed a system to probe the fluorescence intensity and fluorescence lifetime from individual molecules conjugated to a glass cover slip under ambient conditions. Our system employs a pulse picker to reduce the pulse rep. rate of a Ti:Sa laser, frequency doubled to yield visible pulses. The laser excitation system is coupled to a single molecule scanning confocal microscope, equipped with fast APDs. We show that using a maximum likelihood estimation (MLE) of single molecule fluorescent lifetime trajectories, the fluorescence lifetime can be determined with as little as few tens of photons. In Figure 1 a typical result of single DNA molecule labeled with TMR and Cy5 FRET pair is shown. Figure 2 displays the fluorescence lifetime of the donor dye as a function of time of that DNA molecule, after performing background correction. An accumulation of ~100 molecules produces the fluorescence lifetime histogram which displays two prominent peaks: A non- FRET peak at 2.3 ns, and a FRET peak at 1.0 ns (Figure 3). These values are with excellent agreement of bulk measurements of the same DNA, and yield an averaged FRET efficiency of E = 0.57.
- Edel, J.B., Eid, J.S. & Meller, A. (2007) Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood calculated lifetimes. J. Phys. Chem. B. 111, 2986-2990.
- Sabanayagam, C.R., Eid, J.S. & Meller, A. (2004) High-throughput scanning confocal microscope for single molecule analysis. Applied Physics Letters 84, 1216-1218.
- Sabanayagam, C.R., Eid, J.S. & Meller, A. (2005) Using fluorescence resonance energy transfer to measure distances along individual DNA molecules: Corrections due to nonideal transfer. J. Chem. Phys. 122, 061103.