J. Hohlbein, and C.G. Hübner, Journal of Chemical Physics, 129, 094703, 2008, [link]
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.
two-dimensional geometrical confinement for either single diffusing molecules or fluorescent polymerized nanowires. The membranes for measurements of single molecule diffusion featured a pore diameter of 35-40nm, a porosity (volume fraction of the pores) of 20-25%, and a thickness of 35 µm. Thus, the aspect ratio of the pores is ≃1000. In comparison, the size of the diffraction limited laser focus is roughly 2µm in height (long axis) and 0.5µm in diameter (short axes), resulting in an aspect ratio of 4. Therefore, if the long axis of the pores is aligned with the long axis of the confocal microscope, the probe molecules are forced to diffuse parallel to the long axis of the laser focus. Apparent one-dimensional diffusion within nanoporous alumina was shown for different probes such as Alexa Fluor 488 and the enhanced green fluorescent protein (eGFP). As compared to three-dimensional diffusion in free solution, the mean diffusion time through the focus increases within the pores. The factor of increase was theoretically derived as the squared aspect ratio of the laser focus resulting in a value of 16. Indeed, for Alexa Fluor 488 an increase in the mean diffusion time by a factor of 19 was found. In the case of eGFP a factor of 14 was obtained.
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.
We report on fluorescence fluctuations of nanoparticles diffusing through a laser focus. Subject to an intensity threshold the fluorescence signal is transformed into time traces of on and off periods. The distribution functions of the experimental on and off times follow power laws t^-alpha over several orders of magnitude with exponents alpha equal 1.5-2. At long times the distribution functions cross over to exponential decays. For the interpretation of the experimental data a diffusion-reaction equation is proposed which covers both, the diffusion controlled recurrence and the photon statistics as the relevant processes.
Hohlbein lab - Wageningen 