RESUMEN
Single dye molecules are used as local probes to map the spatial distribution of the squared electric field components in the focus of a high numerical aperture lens. Simulated field distributions are quantitatively verified by experimentally obtained fluorescence excitation maps. We show that annular illumination can be used to engineer the field distribution in the focus at a dielectric/air interface such that electric field components in all directions acquire comparable magnitudes. The 3D orientation of molecular absorption dipoles can be determined by comparing measured to simulated image patterns. The presence of longitudinal electric field components in a focus is of particular interest in tip-enhanced scanning near-field optical microscopy.
RESUMEN
The absorption dipole orientation of single fluorescent molecules is determined by mapping the spatial distribution of the squared electric field components in a high-numerical-aperture laser focus. Annular illumination geometry and the vicinity of a plane dielectric/air interface strongly enhance the longitudinal field component and the transverse fields perpendicular to the polarization direction. As a result, all three excitation field components in the focus are of comparable magnitude. The scheme holds promise to monitor rotational diffusion of single molecules in complex environments.
RESUMEN
A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.
RESUMEN
One limitation of the conventional Shack-Hartmann sensor is that the spots of each microlens have to remain in their respective subapertures. We present an algorithm that assigns the spots to their reference points unequivocally even if they are situated far outside their subaperture. For this assignment a spline function is extrapolated in successive steps of the iterative algorithm. The proposed method works in a single-shot technique and does not need any aid from mechanical devices. The reconstruction of a simulated steep aspherical wave front (approximately 100 lambda/mm slope) is described as well as experimental results of the measurement of a spherical wave front with a huge peak-to-valley value (approximately 400 lambda). The performance of the method is compared with the unwrapping method, which has been published before.
