Christian G. Hübner

Simple scheme for rapid three-dimensional orientation determination of the emission dipole of single molecules

One of the unique features of single molecule absorption and emission is their anisotropy due to the well-defined transition dipole(s) for both processes allowing the determination of the molecule’s orientation. While polarization-resolved techniques are usually capable of detecting only a projection of the transition dipole, several methods have been proposed in order to determine the full three-dimensional orientation. Here, we report on a detection scheme that allows for a shot-noise limited determination of the emission dipole orientation utilizing an annular mirror, a polarizing beam splitter in conjunction with three detectors in a scanning confocal optical microscope.

Three-dimensional orientational colocalization of individual donor-acceptor pairs

We report on the determination of the three-dimensional orientation of the donor and acceptor transition dipoles in individual fluorescence resonance energy transfer (FRET) pairs by means of scanning optical microscopy with annular illumination. Knowledge of the mutual orientation of the donor and acceptor dipole is mandatory for reliable distance determination based on FRET efficiency measurements. In our model system perylenediimide as the donor and terryelenediimide as the acceptor are coupled via a stiff p-terphenyl linker. The absorption dipoles of the donor and acceptor are selectively addressed by the 488 nm and 647 line of an Ar/Kr mixed gas laser, respectively. A clear deviation from collinearity is observed with a distribution of misalignment angles peaked around 22 degrees.

Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics.

We report on the simultaneous detection of the fluorescence lifetime, spectrum, and three-dimensional dipole orientation determination of single perylene diimide molecules deposited on a silica surface as a model system for studying fluorophore internal and orientational dynamics. We employ a multi-parameter detection scheme to demonstrate how jumps in the orientation of the molecule can be disentangled from spectral jumps, both leading to changes of the detected total fluorescence intensity. The fluorescence lifetime determined simultaneously from the same photons is also sensitive to the orientation of the dipole with respect to the interface between media with different refractive indices. The correlated changes of the lifetime and orientation we observe are in good agreement with theory.

Three-dimensional orientation determination of the emission dipoles of single molecules: The shot-noise limit

The power of three-dimensional orientation detection of single emitting dipoles using a sophisticated scheme with three detectors in a confocal microscope is quantitatively explored by means of Monte Carlo simulations. We show that several hundreds of photons are sufficient for a reliable orientation determination. In typical single-molecule experiments, time resolutions in the submillisecond range for orientation trajectories become accessible. Experimental data on fluorescent latex beads and single perylene monoimide molecules show that a properly aligned setup can perfectly reproduce the simulated data. The simulations and experimental data highlight the potential of our method and give practical guidelines for its application.