Optical Coherence Correlation Spectroscopy (OCCS)

We present optical coherence correlation spectroscopy (OCCS), a novel method to study the motion dynamics of nanoparticles within many observation volumes simultaneously. For this purpose, OCCS combines the key features of dark-field optical coherence microscopy (dfOCM) and fluorescence correlation spectroscopy (FCS). dfOCM is a Fourier domain optical coherence microscopy technique offering micrometer three-dimensional spatial resolution and microseconds temporal sampling. The rapid temporal sampling is achieved by detecting the weakly back-scattered light by nanoparticles that is optically amplified upon interference with the strong reference light. For instance, our near-infrared dfOCM system is able to detect diffusing gold nanoparticles as small as 30 nm in diameter every 50 us, which is sufficiently fast to observe the motion dynamics of these particles. On the other hand, FCS is a classical method to investigate the motion dynamics of diffusing fluorescent markers and their photo-physical and/or photo-chemical processes at the single-molecule level. Whereas FCS suffers from photo-bleaching, triplet blinking and saturation of the fluorescence emission of the marker fluorophores, OCCS overcomes these limitations by detecting the back-scattered light from nanoparticles. Using a mode-locked Ti:Sapphire laser (780 nm central wavelength), we performed experiments with nanoparticles ranging from 30 nm to 100 nm in diameter. We present these initial results and an associated theoretical fit model for the extraction of the particles’ concentrations and diffusion parameters. The experimental determination of the diffusion time and concentration of gold nanoparticles based on this method is presented as a proof of principle and shows the potential of this technique. Moreover, the interferometric detection allows observing the back-scattered light from many sample volumes along the optical axis. This simultaneous detection enables assessing the axial flow of nanoparticles, which is similar to a lateral flow measurement in dual-focus FCS.

Arno Pino Bouwens, Stéphane Broillet, Stefan Geissbühler, Theo Lasser, Marcel Leutenegger, Christophe Pache, Akihiro Sato, Daniel Pawel Szlag

A classical technique to monitor dynamical processes at the single-molecule level is fluorescence correlation spectroscopy (FCS). However, FCS requires fluorescent labels that are typically limited by photobleaching and saturation. We present a new method, optical coherence correlation spectroscopy (OCCS), based on noble-metal nanoparticles that overcome those photobleaching and saturation limitations. OCCS is a correlation spectroscopy technique based on dark-field optical coherence microscopy (dfOCM), a Fourier domain optical coherence microscopy technique. OCCS is based on the amplified backscattered light caused by diffusing nanoparticles. Due to the interferometric principle of OCCS, several sampling volumes along the optical axis are measured simultaneously with high detection sensitivity. This adds the possibility to assess axial flow, which is similar to a lateral flow measurement in dual-focus fluorescence correlation. Using a mode-locked Ti:Sapphire laser (780nm central wavelength) we performed experiments with nanoparticles down to 30nm in diameter. We present these first experimental results and an associated theoretical fit model allowing the extraction of the particles’ concentrations and diffusion parameters. The experimental determination of the diffusion time and concentration of gold nanoparticles based on this method is presented as a proof of principle and shows the potential of this technique. In the near future, we aim at investigating smaller gold nanoparticles assessing biological phenomena. As a first application we apply this method to membrane receptor interaction using functionalized nanoparticles.

2013

Optical Coherence Correlation Spectroscopy (OCCS)

Arno Pino Bouwens, Stéphane Broillet, Stefan Geissbühler, Theo Lasser, Marcel Leutenegger, Christophe Pache, Akihiro Sato

A classical technique to monitor dynamical processes at the molecular level is fluorescence correlation spectroscopy (FCS). FCS requires fluorescent labels that are typically limited by photobleaching and saturation. We present a new method that uses noble-metal nanoparticles instead of fluorophores: optical coherence correlation spectroscopy (OCCS). OCCS is a correlation spectroscopy technique based on dark-field optical coherence microscopy, a Fourier domain optical coherence tomography technique. In OCCS, several sampling volumes are measured simultaneously with high detection sensitivity. OCCS measures the time correlation function of the light back-scattered by the nanoparticles. Using a mode-locked Ti:Sapphire laser (780nm central wavelength) we performed first experiments with different nanoparticles down to 30nm in diameter. We present experimental results and a preliminary model to fit the correlation curves and extract the particles’ concentrations and diffusion coefficients. The experimental determination of the diffusion times of gold nanoparticles using this model is presented, showing the potential of our method. In the near future, we aim at investigating smaller gold nanoparticles that interfere less with the biological phenomena under study.

2012

Structure and function of the nicotinic acetylcholine receptor

Jörg Grandl

The subject of this thesis is the study of the structure and function of the human muscle nicotinic acetylcholine receptor (nAChR), which is a typical member of the large class of ligand-gated ion channels. Appropriate techniques, like site-directed mutagenesis, patch-clamp and fluorescence microscopy, and combinations thereof, were applied to gain insight into selected aspects of the structure-function relationship of this receptor. Special emphasis was put on the investigation of single receptors, wherever this promised scientific benefit. Experiments with the aim to study structural rearrangements of nAChR upon ligand binding by fluorescence resonance energy transfer (FRET) were undertaken. Such intramolecular motions are expected to link ligand binding to channel gating and are therefore fundamental to understand the function of nAChR. Double-cysteine mutants were produced and receptors were labelled on living cells in order to measure changes in FRET efficiency due to ligand binding. A novel class of functionally modified ligands for the nAChR was characterized by patch-clamp technique. Derivatives that carry a thiol-reactive group were shown to be full and very potent agonists. On the bases of a structural model of the binding site, residues were selected, that are likely to be in contact with the bound ligand. Equivalent single-cysteine mutants were produced to covalently bind these agonists and thereby to gain information about the mechanisms of ligand binding and receptor activation. Ligands that were coupled to fluorophores turned out to be either partial or full agonists with nanomolar efficacies for the nAChR. Their use to study the diffusion of single nAChR on living HEK293-cells for different functional states is described in detail. The dependency of the lateral diffusion on the activation state (closed/opened/desensitized) was studied. Additionally these ligands served to demonstrate the feasibility to simultaneously combine patch-clamp and fluorescent binding experiments on a single-molecule level. Acetylcholine receptors carrying a single point-mutation, related to the congenital myasthenic syndrome, were found to stabilize an intermediate conductance state and were studied in detail by single-channel electrophysiology. The dependency of the gating behavior on different ligands, ligand concentration and transmembrane voltage was investigated. Mutants, carrying equivalent mutations on different subunits were produced to reveal the origin of this effect. Quantitative characterization was obtained by dwell-time and spectral noise-analysis, in order to gain insight on sub-millisecond fluctuations of the channel lining transmembrane regions of the nAChR and the gating mechanism in general.

EPFL2005

Structure and function of the nicotinic acetylcholine receptor

Jörg Grandl

EPFL2005