Abstract:Fluorophores that are fixed during image acquisition produce a diffraction pattern that is characteristic of the orientation of the fluorophore's underlying dipole. Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by applying Gaussian-based fitting algorithms to in-focus images of individual fluorophores; when applied to fixed dipoles, this can lead to a bias in the range of 5-20 nm. We introduce a method for the joint estimation of position and orientation of dipoles, based on the representation of a physically realistic image formation model as a 3-D steerable filter. Our approach relies on a single, defocused acquisition. We establish theoretical, localization-based resolution limits on estimation accuracy using Cramér-Rao bounds, and experimentally show that estimation accuracies of at least 5 nm for position and of at least 2 degrees for orientation can be achieved. Patterns generated by applying the image formation model to estimated position/orientation pairs closely match experimental observations.

We present a novel concept for optical spectroscopy called nonlinear correlation spectroscopy (NLCS). NLCS analyses coherent field fluctuations of the second and third harmonic light generated by diffusing nanoparticles. Particles based on noncentrosymmetric nonlinear materials such as KNbO(3) show a strong second as well as third harmonic response. The method and the theory are introduced and experimental NLCS results in fetal calf serum are presented showing the promising selectivity of this technique for measurement in complex biological environments.

The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.

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