Surface-Integrated Fluorescence Correlation Spectroscopy (SI-FCS) for the quantification of transient membrane and surface binding

Das Verständnis der komplexen Netzwerke bio-molekularer Wechselwirkungen benötigt die korrekte und präzise Quantifizierung von Bindungskinetiken. Besonders von Bedeutung sind Wechselwirkungen mit Membranen oder membrangebundenen Objekten, da Membranen nicht nur Bindungskinetiken beeinflussen, sondern auch in eine große Menge entscheidender zellulärer Prozesse grundsätzlich eingebunden sind, darunter Prozesse wie Zellteilung, Signaltransduktion, Endozytose, Exozytose oder Zellmigration. Um die korrekte und präzise Quantifizierung von Bindungskinetiken zu ermöglichen, haben wir die oberflächen-integrierte Fluoreszenzkorrelationsspektroskopie (SI-FCS, engl. surface-integrated fluorescence correlation spectroscopy) entwickelt. Die Methode basiert auf der Autokorrelation von zeitlichen Fluoreszenzfluktuationen in einer Sequenz von Bildern, die mit Totalreflexions-Fluoreszenzmikroskopie (TIRF-Mikroskopie, engl. total internal reflection microscopy) aufgenommen wurden. SI-FCS kann zuverlässig die Assoziations und Dissoziationsraten von reversiblen Liganden-Rezeptor-Bindungen bestimmen. Die DNA-Hybridisierung kurzer Einzelstränge – bei denen einer auf der Oberfläche immobilisiert ist, während sich der andere, fluoreszent markierte Strang in Lösung befindet – imitierte dabei Liganden-Rezeptor-Systeme und wurde in dieser Arbeit präzise mit SI-FCS vermessen. Systematisch haben wir das Leistungsvermögen und die Limitierungen von SI-FCS untersucht und dabei die photo-induzierte Schädigung von immobilisierten Einzelsträngen als Einschränkung für lange Messzeiten oder hohe Ligandenkonzentrationen bestimmt. Die photoinduzierte Schädigung erfolgt dabei vorwiegend durch reaktive Sauerstoffspezies. In SI-FCS und in DNA-Hybrisierungs-basierter Hochauflösungsmikroskopie (DNA-PAINT) konnten wir diesen Effekt durch das biochemische Entfernen von Sauerstoff vermeiden. In einem parallelen Ansatz zeigen wir ein verbessertes Design für den bindenden DNA-Strang mit einem erhöhten Abstand zwischen dem fluoreszenten Farbstoff und der Bindungssequenz. Die Bestimmung von Bindungszeiten im Millisekunden-Bereich und kürzer mit SI-FCS ist letzlich limitiert durch exakte mathematische Modelle für die Autokorrelationsfunktion, die auch die Diffusion durch das evaneszente TIRF-Feld mit einbeziehen. Basierend auf einem Fluoropolymer mit dem Brechungsindex wässriger Proben, haben wir eine Kalibierungsprobe entwickelt, mit derer die axiale Detektionswahrscheinlichkeit für Moleküle in der TIRF-Mirkoskopie direkt bestimmt werden kann. Dabei haben wir eine tiefer in die Probe eindringende Komponente der TIRF-Anregung in der Objektiv-basierten TIRF-Mikroskopie gefunden, die bisher von der entsprechenden FCS-Theorie unberücksichtigt ist. Schließlich haben wir die praktische Anwendung von SI-FCS auf die Untersuchung von Bindungen an Membranerezeptoren und die Verteilgung von Biomolekülen zwischen Lösung und Membran optimiert. Modelle für die Liganden-Rezeptor-Wechselwirkung in Kombination mit Diffusion auf der Membran, haben wir in einer DNA-Hybridisierungs-basierten Machbarkeitsstudie verifiziert. Des Weiteren zeigen wir ein modifiziertes Modell der Autokorrelationsfunktion für die Verteilung von Biomolekülen zwischen Lösung und Membran und untersuchen die komplexe Verteilung von Peptiden in Simulationen und Experimenten mit drei unterschiedlichen Modellmembransystemen.Understanding the complex networks of biomolecular interactions requires the accurate and precise quantification of binding kinetics. Interactions with membranes or membrane-bound entities are of particular interest, as membranes do not only influence binding kinetics, but are fundamental to a large set of key cellular processes such as cytokinesis, signalling, endocytosis, exocytosis or cell migration. To enable the accurate and precise quantification of binding kinetics, we developed surface-integrated fluorescence correlation spectroscopy (SI-FCS). This method is based on the autocorrelation of temporal fluorescence fluctuations from a stream of total internal reflection fluorescence (TIRF) images. SI-FCS reliably extracts the association and dissociation rates of reversible ligand-receptor binding. The DNA hybridization of short single-strands – one immobilized on the surface, the other fluorescently labeled in solution – mimicked ligand-receptor systems and was precisely quantified with SI-FCS within this work. We systematically assessed the potential and limitations of SI-FCS and identified the photo-induced damage of immobilized single-strands as a constraint for longer measurement times and high ligand concentrations. The photo-induced damage was predominantly caused by reactive oxygen species (ROS). Not only did we show that this effect can be prevented in SI-FCS and DNA hybridization-based super-resolution microscopy (DNA-PAINT) with oxygen scavenging buffers, but we also presented an improved DNA handle design with increased distance between fluorescent dye and binding sequence. Accessing binding times with SI-FCS in the millisecond range and below is ultimately limited by accurate autocorrelation models incorporating the diffusion through the evanescent TIRF field. Based on a fluoropolymer matching the refractive index of aqueous samples, we designed a calibration slide to directly characterize the axial molecule detection function in TIRF microscopy. We found a penetrating component to the TIRF excitation in objective-type TIRF microscopy, so far unconsidered by the respective FCS theory. Finally, we optimized the implementation of SI-FCS for examining binding to membrane receptors and the partitioning of biomolecules to membranes. We verified models for ligand-receptor interaction in the presence of diffusion in the membrane using a DNA hybridization-based proof-of-concept system. Further, we presented a modified autocorrelation model for the partitioning to membranes and examined the complex partitioning of peptides in simulations and experiments on three different model-membrane systems

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site

Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments

Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy

We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates