Iwan Märki

Super-resolution orientation estimation and localization of fluorescent dipoles using 3-D steerable filters

Fluorophores that are fixed during image acquisition produce a diffraction pattern that is characteristic of the orientation of the fluorophores 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.

Triplet Imaging of Oxygen Consumption During the Contraction of a Single Smooth Muscle Cell (A7r5)

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.

Direct Observation of Transitions between Surface-Dominated and Bulk Diffusion Regimes in Nanochannels

The diffusion of charged proteins in liquid-filled nanometer-sized apertures with charged surfaces has been investigated with fluorescence correlation spectroscopy (FCS). Based on a two-dimensional (2D) multicomponent diffusion model, key parameters such as the number of molecules diffusing freely inside the nanochannel or interacting with the surfaces, together with the specific diffusion parameters, could be extracted. Different regimes of diffusion have been observed and described by a model, which takes into account the steric exclusion, the reversible surface adsorption of the biomolecules, and the exclusion-enrichment effect that is due to the charge of the proteins and the ionic strength of the solution. Conditions where the diffusion of proteins through nanoconfined spaces can be of the same magnitude as in the bulk were both predicted and experimentally verified.

High volume confinement in two-photon total-internal- reflection fluorescence correlation spectroscopy

We report results on two-photon total-internal-reflection fluorescence correlation spectroscopy with radially polarized light. The combination of liquid crystal spatial light modulator, providing radial polarization, with ultrafast laser (picosecond Nd:GdVO4 laser) allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

Three-dimensional nano-localization of single fluorescent emitters

We present a combination of self-interference microscopy with lateral super-resolution microscopy and introduce a novel approach for localizing a single nano-emitter to within a few nanometers in all three dimensions over a large axial range. We demonstrate nanometer displacements of quantum dots placed on top of polymer bilayers that undergo swelling when changing from an air to a water environment, achieving standard deviations below 10 nm for axial and lateral localization.

Imaging of G protein-coupled receptors in solid-supported planar membranes at the single molecule level

Odorant receptors are an excellent example of natural superiority in specifically binding specific, small and hydrophobic molecules. They are of particular interest in the development of a sensor platform for G protein-coupled receptors (GPCRs). Odorant receptors (OR5) of Rattus norvegicus were incorporated into model membranes by in vitro synthesis and vectorial incorporation for achieving natural receptor function. The vectorial insertion of OR5 into the planar membrane and their lateral distribution, their interactions and their mobility within the membrane are of great importance for ligand-receptor interaction. We applied total internal reflection fluorescence (TIRF) microscopy and image analysis to assess the insertion and the OR5 distribution as well as the lateral mobility of these receptors at the single molecule level. The vectorial incorporation of OR5 into planar lipid membranes was investigated with TIRF microscopy and image segmentation. With increasing expression time, the OR5 incorporation density and aggregation increased linearly by about 0.02μm-2min-1. The expression and incorporations of single OR5s were completed within about 8 minutes. The mobility of the incorporated receptors was measured with fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photo-bleaching (FRAP). These measurements revealed that the incorporated receptors were immobilized with this class of lipid membranes.

High volume confinement in two-photon fluorescence correlation spectroscopy with radially polarized light

We present the results on two-photon total-internal-reflection fluorescence correlation spectroscopy. The combination of liquid crystal spatial light modulator, providing radial polarization, with ultrafast laser (picosecond Nd:GdVO4 laser) allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

Assessing the Cellular Uptake Pathway for Poly-Lysine Analogues using Triplet Lifetime Imaging

Research on synthetic delivery vectors is of major interest for cell imaging and manipulation, as they allow an efficient transfer of nucleic acids, therapeutic proteins or small drugs into the cells. We have developed a library of L-lysine analogues that allow for highly efficient gene delivery with low cytotoxicity. However little is known on the exact mechanism of uptake and the final intracellular destination of the synthetic carriers. Therefore we have developed a novel optical technique based on a modulated excitation allowing for intracellular imaging of the triplet-lifetime and -yield of fluorophores attached to the delivery vector. Both these parameters are highly dependant on the intracellular environment thus provide insight into the subcellular localization of the labelled carrier. The method combines high temporal and spatial resolution and is compatible with a multiplicity of fluorophores. We performed series of model experiments to compare the triplet lifetime and triplet yield behaviour during the natural uptake mechanism to a series of controlled conditions. The latter include microinjection of fluorescently labelled carriers directly into the cytoplasm and cell nucleus as well as in vitro measurements under conditions mimicking physiological, acidic, or DNA rich environments. To validate our technique the results from the triplet imaging were compared with two complementary methods: carrier localization by subcellular fractionation and confocal laser scanning microscopy. --- Reference --- Geissbuehler et al. Triplet imaging of Oxygen consumption during the contraction of a single smooth muscle cell (A7r5). Biophysical Journal (2010) vol. 98 (2) pp. 339-349

Steerable filters for orientation estimation and localization of fluorescent dipoles

Fluorescence localization microscopy (i.e., PALM, STORM) has enabled optical imaging at nanometer-scale resolutions. The localization algorithms used in these techniques rely on fitting a 2-D Gaussian to the in-focus image of individual fluorophores. For fixed fluorophores, however, the observed diffraction pattern depends on the orientation of the underlying molecular dipole and does not necessarily correspond to a section of the systems point spread function. By using a physically realistic image formation model for dipoles to perform the fit, both the position and orientation of the dipole can be estimated with high accuracy, improving upon Gaussian localization. In this paper, we present an algorithm for joint position and orientation estimation based on a 3-D steerable filter, and show that the results are near-optimal with respect to the Cramér-Rao bounds. We show that patterns generated using estimated positions and orientations closely fit experimental measurements.

Three-Dimensional Localization of Nano-Emitters with Nanometer-Level Precision

We show nanometer-level localization accuracy of a single quantum-dot in three dimensions by self-interference and diffraction-pattern analysis. We believe that this approach has the capacity to push optical microscopy to the molecular level.