Single molecule detection on surfaces

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

Detection efficiency in total internal reflection fluorescence microscopy

We present a rapid and flexible framework for the accurate calculation of the detection efficiency of fluorescence emission in isotropic media as well as in the vicinity of dielectric or metallic interfaces. The framework accounts for the dipole characteristics of the emitted fluorescence and yields the absolute detection efficiency by taking into account the total power radiated by the fluorophore. This analysis proved to be useful for quantitative measurements, i.e. the fluorescence detection at a glass–water interface for total internal reflection fluorescence microscopy in an epi- and a trans-illumination configuration

Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as the fluorophore density and the photo-activation and photo-switching rates

Spectral Cross-Cumulants for Multicolor Super-resolved SOFI Imaging

Super-resolution optical fluctuation imaging (SOFI) provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution as well as the sampling can be increased up to n-fold in all three spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a novel multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increase the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and multiple experiments with different cellular structures, fluorophores and filter sets. Based on an eigenvalue/ vector analysis we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a novel route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. This makes simultaneous multiplexed super-resolution fluorescence imaging widely accessible to the life science community interested to probe colocalization between two or more molecular species.Comment: main: 21 pages & 4 figures, supplementary 20 pages & 16 figure

Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels

The use of photoactivatable dyes in STED microscopy has so far been limited by two—photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Here we report ONB‐2SiR, a fluorophore that can be both photoactivated in the UV and specifically de‐excited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water‐soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general

Three-dimensional Super-resolution Optical Fluctuation Imaging

Super-resolution optical fluctuation imaging (SOFI) achieves three-dimensional super-resolution by computing higher-order spatio-temporal cross-cumulants of stochastically blink-ing fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wider range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders. We present a balanced SOFI algorithm for mapping molecular parameters and for linearizing the brightness response and we outline a MATLAB toolbox for two- and three-dimensional SOFI analysis. We show super-resolved three-dimensional cell structures imaged with a multi-plane wide-field microscope. The simultaneous acqui-sition of several focal planes significantly reduces the acquisition time and helps limiting the photo-bleaching of the marker fluorophores

Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI)

Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wide range of blinking conditions. The main drawback of SOFI is the nonlinear response to brightness and blinking heterogeneities in the sample, which limits the use of higher cumulant orders for improving the resolution. Balanced super-resolution optical fluctuation imaging (bSOFI) analyses several cumulant orders for extracting molecular parameter maps, such as molecular state lifetimes, concentration and brightness distributions of fluorophores within biological samples. Moreover, the estimated blinking statistics are used to balance the image contrast, i.e. linearize the brightness and blinking response and to obtain a resolution improving linearly with the cumulant order. Using a widefield total-internal-reflection (TIR) fluorescence microscope, we acquired image sequences of fluorescently labelled microtubules in fixed HeLa cells. We demonstrate an up to five-fold resolution improvement as compared to the diffraction-limited image, despite low single-frame signal-to-noise ratios. Due to the TIR illumination, the intensity profile in the sample decreases exponentially along the optical axis, which is reported by the estimated spatial distributions of the molecular brightness as well as the blinking on-ratio. Therefore, TIR-bSOFI also encodes depth information through these parameter maps. bSOFI is an extended version of SOFI that cancels the nonlinear response to brightness and blinking heterogeneities. The obtained balanced image contrast significantly enhances the visual perception of super-resolution based on higher-order cumulants and thereby facilitates the access to higher resolutions. Furthermore, bSOFI provides microenvironment-related molecular parameter maps and paves the way for functional super-resolution microscopy based on stochastic switching

Optical Coherence Correlation Spectroscopy (OCCS)

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