Single molecule detection and fluorescence correlation spectroscopy on surfaces

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

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

Single molecule detection on surfaces

Marcel Leutenegger

Monitoring biological relevant reactions on the single molecule level based on fluorescence spectroscopy techniques has become one of the most promising approaches for understanding a variety of phenomena in biophysics, biochemistry and life science. By applying techniques of fluorescence spectroscopy to labeled biomolecules a manifold of important parameters becomes accessible. For example, molecular dynamics, energy transfer, and ligand-receptor reactions can be monitored at the molecular level. This huge application field was and still is a major drive for innovative optical methods as it opens the door for new quantitative insights of molecular interactions on a truly micro- and nano-scopic scale. This thesis contributes new single molecule detection (SMD) concepts, correlation analysis and optical correlation spectroscopy to study fluorophores or labeled biomolecules close to a surface. The search beyond the classical confocal volume towards improved confinement was a key objective. In a first approach, fluorescence correlation spectroscopy (FCS) using near field light sources to achieve highly confined observation volumes for detecting and measuring fluorophores up to micromolar concentration was investigated. In a second approach, FCS and fluorescence intensity distribution analysis (FIDA) based on dual-color total internal reflection fluorescence (TIRF) microscopy was conceived to achieve a common observation volume for dual-color fluorescence measurements. This resulted in two novel fluorescence fluctuation spectroscopy instruments providing observation volumes of less than 100al. The first instrument generates a near field observation volume around and inside nano-apertures in an opaque metal film. Back-illumination of such an aperture results in a highly confined excitation field at the distal aperture exit. This instrument was characterized with FCS and observation volumes as small as 30al were measured. The second instrument confines the observation volume with total internal reflection (TIR) at a glass-water interface. Today, the last-generation instrument provides a dual-color ps pulsed excitation and time-resolved detection for coincidence analysis and time-correlated single photon counting. It was characterized with FCS and FIDA and observation volumes of 70al to 100al were achieved. Moreover, the presence of the interface favors emission into the optically denser medium, such that nearly 60% of the emitted fluorescence can be collected. This very efficient light collection resulted in a two- to three-fold stronger fluorescence signal and led to a high signal to background ratio, which makes this instrument particularly suitable for SMD studies on surfaces. In parallel to these experimental investigations, a theoretical analysis of the total SMD process including an analysis of optical focus fields, molecule-interface interactions, as well as the collection and detection efficiency was performed. This analysis was used as a guideline for steady instrument improvements and for the understanding of the SMD process. Finally, SMD concepts were applied for a first investigation of in vitro expression of an odorant receptor and for monitoring the vectorial insertion into a solid-supported lipid membrane. These receptors were incorporated and immobilized in the lipid membrane. With increasing expression time, an increasing amount of receptors as well as an increasing aggregation was observed. The incorporation density and the receptor aggregation were investigated with TIRF microscopy and image correlation spectroscopy.

EPFL2007

Plasmon enhanced luminescence from fullerene molecules excited by local electron tunneling

Frédéric Rossel

This thesis reports on the optical properties of fullerene molecules forming nanoscale crystals grown on ultrathin NaCl films on Au(111) investigated by Scanning Tunneling Microscope (STM)-induced light emission spectroscopy. In this regard, an existing optical setup has been improved. The new instrument facilitates the alignment of the detection lens with the tunnel junction. STM images of the investigated samples show that, upon adsorption on a NaCl covered Au(111) surface, C60 and C70 crystallize into multilayer islands with a hexagonally arranged close-packed top layer in which electron-poor regions on one molecule face electron-rich regions on the adjacent molecule. Luminescence spectra from these nanocrystals induced by electrons tunneling through a double barrier STM junction are presented. Their bias dependence as well as their similarity with laser-induced photoluminescence spectra indicate a hot electron injection mechanism followed by a radiative decay associated with the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO–LUMO) gap of the molecules. Local fluorescence and phosphorescence of C70 molecules are identified, leading to unambiguous chemical recognition on the nanoscale. Moreover, the molecular luminescence is demonstrated to be selectively enhanced by localized surface plasmons in the STM tip-sample gap, the tunnel junction acting as an optical antenna. Finally, the results of a scanning tunneling microscopy and scanning tunneling spectroscopy investigations of self-assembled islands of Azure A molecules on a Au(111) substrate are presented. Although no final conclusion can be drawn on the molecular arrangement owing to the impossibility to recognize specific parts of the molecule, the presence of these structures is found to modify the Au(111) herringbone reconstruction.