Simultaneous Multicolor Fluorescence Cross-Correlation Spectroscopy to Detect Higher Order Molecular Interactions Using Single Wavelength Laser Excitation

Fluorescence cross-correlation spectroscopy is a powerful method for the study of molecular interactions and dynamics in solution and even in living cells. Usually, in the optical setup, either two laser beams have to be superimposed in their respective confocal volumes or two- photon excitation is used for a dual-color detection system. It has been shown recently that fluorescence cross correlation can be achieved with spectrally similar fluorophores using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this study, we show that SW-FCCS allows the simultaneous excitation of up to three fluorophores in which the cross correlation of their fluctuation signals is detected separately in three detection channels. The experimental and theoretical model to describe triple pairwise cross correlations incorporating cross talk and possible changes in emission characteristics such as quenching upon binding are outlined. The effectiveness of SW-FCCS to detect binding of three interacting partners is experimentally verified with a standard ligand-receptor model, biotin- streptavidin, where differently labeled biotin ligands and their binding to a third-color labeled streptavidin are studied. The cross-correlation amplitudes and their changes with stoichiometric binding are analyzed and the upper limits of dissociation constants are determined. Performed with appropriate negative controls, SW-FCCS can determine interaction patterns between ligands and receptors.

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

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.

EPFL2006

Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: Evidence that the homopentameric 5-hydroxytryptamine type 3(As) receptor binds only one ligand

Kirstin Friedrich, Rudolf Hovius, Horst Vogel

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)-4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligand-receptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864].

1999

Ligand-Protein Affinity Studies Using Long-Lived States of Fluorine-19 Nuclei

Geoffrey Bodenhausen, Roberto Buratto, Estel Canet Martinez, Daniele Mammoli

The lifetimes T-LLS of long-lived states or T-LLC of long-lived coherences can be used for the accurate determination of dissociation constants of weak protein ligand complexes. The remarkable contrast between signals derived from LLS or LLC in free and bound ligands can be exploited to search for weak binders with large dissociation constants K-D > 1 mM that are important for fragment-based drug discovery but may escape detection by other screening techniques. Alternatively, the high sensitivity of the proposed method can be exploited to work with large ligand-to-protein ratios, with an evident advantage of reduced consumption of precious proteins. The detection of F-19-F-19 long-lived states in suitably designed fluorinated spy molecules allows one to perform competition binding experiments with high sensitivity while avoiding signal overlap that tends to hamper the interpretation of proton spectra of mixtures.

American Chemical Society2016

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

EPFL2006