Development and application of super-resolution fluorescence microscopy

Für eine drastische Erweiterung des Anwendungsfeldes der optischen Mikroskopie sorgte die in den letzten Jahren aufgekommene sogenannte Superauflösungsmikroskopie. Das klassische Modellsystem, um das Auflösungsvermögen derartiger Techniken zu vergleichen und zu quantifizieren, stellen Komponenten des eukaryontischen Zytoskeletts dar. Auch im Rahmen dieser Arbeit wurden derartige Zellstrukturen verwendet und anhand dessen ein quantitativer Wert für die Auflösung der Blink-Mikroskopie erhalten. Das System der Wahl waren dabei Aktinfilamente in vitro, in fixierten und in lebenden Zellen. Darüberhinaus wurde auch ein deutlich systematischerer Ansatz verfolgt, indem eine Matrix aus gezielt angeordneten Farbstoffmolekülen als Vergleichssystem verwendet wurde. Dabei wurde die gezielte Platzierung sowohl virtuell in Monte-Carlo-Simulationen, als auch real mithilfe eines Rasterkraftmikroskops durchgeführt. Die Platzierung von Farbstoffmolekülen durch die Verwendung von Rasterkraftmikroskopie wies allerdings einige Nachteile auf, wie etwa eine sehr aufwändige Durchführung und das Fehlen einer Möglichkeit zur Parallelisierung. Aus diesem Grund wurde im Folgenden auf die sogenannte DNA-Origami-Technik zurück gegriffen, um gezielte Farbstoffanordnungen zu generieren. Diese dienten nun als Testprobe sowohl für die Blink-Mikroskopie, als auch für die superauflösenden Techniken PAINT und SHRImP. Schließlich wurden DNA-Origamis als generelle Auflösungsstandards für die lokalisierungsbasierte Superauflösungsmikroskopie etabliert, wobei zur automatisierten Auswertung ein spezieller Algorithmus entwickelt wurde. Die Erweiterung auf den dreidimensionalen Fall gelang durch die Nutzung von Astigmatismus-basierter 3D-Lokalisierungsmikroskopie und die Verwendung einer speziell dafür designten dreidimensionalen DNA-Origami-Struktur. Die Möglichkeit einer unkomplizierten Auswertung wurde auch in diesem Fall durch die Entwicklung eines automatisierten Detektionsalgorithmus sichergestellt.In recent years the so called super-resolution microscopy yielded to a dramatic increase of applications in optical microscopy. The common used model system to compare and quantify the resolution values of those techniques is given by components of the eukaryotic cytoskeleton. Also within this work those cell structures were used for gaining a quantitative value of the resolution of Blink Microscopy. Thereby the systems of choice were actin filaments in vitro, in stained cells and in living cells. Furthermore a much more systematic approach was carried out by using a matrix of regular arranged dye molecules as a comparison system. Thereby the accurate placement was done virtually in Monte-Carlo-simulations as well as in reality by using an atomic force microscope. However, the arrangement of fluorescent molecules by using an atomic force microscope contains a number of disadvantages, e.g. the quite high effort of the process and the lack of possibilities for parallelization. For this reason in the following the so called DNA origami technique was used for generating specific arrangements of dye molecules. These structures now functioned as calibration probe for Blink Microscopy as well as for the super-resolution techniques PAINT and SHRImP. Finally DNA origamis were established as general resolution standards for localization based super-resolution microscopy. Thereby for automated data analysis a specific algorithm was developed. The expansion to the three-dimensional case succeeded by applying astigmatism-based 3D-localization-microscopy to a specific for this case designed three-dimensional DNA-origami-structure. The option for uncomplicated data analysis was also ensured by developing of an automatic detection algorithm

A New Perspective on the Multidimensionality of Divergent Thinking Tasks

In the presented work, a shift of perspective with respect to the dimensionality of divergent thinking (DT) tasks is introduced moving from the question of multidimensionality across DT scores (i.e., fluency, flexibility, or originality) to the question of multidimensionality within one holistic score of DT performance (i.e., snapshot ratings of creative quality). We apply IRTree models to test whether unidimensionality assumptions hold in different task instructions for snapshot scoring of DT tests across Likert-scale points and varying levels of fluency. It was found that evidence for unidimensionality across scale points was stronger with be-creative instructions as compared to be-fluent instructions which suggests better psychometric quality of ratings when be-creative instructions are used. In addition, creative quality latent variables pertaining to low-fluency and high-fluency ideational pools shared around 50% of variance which suggests both strong overlap, and evidence for differentiation. The presented approach allows to further examine the psychometric quality of subjective ratings and to examine new questions with respect to within-item multidimensionality in DT

Super-Resolution Imaging of C-Type Lectin and Influenza Hemagglutinin Nanodomains on Plasma Membranes Using Blink Microscopy

AbstractDendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts

Self-Regeneration and Self-Healing in DNA Origami Nanostructures

DNA nanotechnology and advances in the DNA origami technique have enabled facile design and synthesis of complex and functional nanostructures. Molecular devices are, however, prone to rapid functional and structural degradation due to the high proportion of surface atoms at the nanoscale and due to complex working environments. Besides stabilizing mechanisms, approach for the self‐repair of functional molecular devices are desirable. Here we exploit the self‐assembly and reconfigurability of DNA origami nanostructures to induce the self‐repair of defects of photoinduced and enzymatic damage. With different examples of repair in DNA nanostructures, we distinguish between unspecific self‐regeneration and damage specific self‐healing mechanisms. Using DNA origami nanorulers studied by atomic force and superresolution DNA PAINT microscopy, quantitative preservation of fluorescence properties is demonstrated with direct potential for improving nanoscale calibration samples

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and non-fluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by ‘‘blinking’’ with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This ‘‘continuous switching mode’’ is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.

Intrinsically Resolution Enhancing Probes for Confocal Microscopy

In recent years different implementations of superresolution microscopy based on targeted switching (STED, GSD, and SSIM) have been demonstrated. The key elements to break the diffraction barrier are two distinct molecular states that generate a saturable nonlinear fluorescence response with respect to the excitation intensity. In this paper, we demonstrate that a nonlinearity can even be encoded in fluorescent probes, which then increase the resolution of a standard confocal microscope. This nonlinearity is achieved by an intensity dependent blocking of the resonance energy transfer between a donor and one or more acceptor fluorophores, utilizing radical anion states of the acceptor. In proof-of-principle experiments, we demonstrate a significant resolution increase using probes with different numbers of acceptor fluorophores. Quantitative description by a theoretical model paves the way for the development of fluorescent probes that can more than double the resolution of essentially any confocal microscope in all three dimensions.

Research Article A Structurally Variable Hinged Tetrahedron Framework from

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nanometer-sized polyhedral wire-frame objects hold a wide range of potential applications both as structural scaffoldsaswellas a basis for synthetic nanocontainers. The utilization of DNA as basic building blocks for such structures allows the exploitation of bottom-up self-assembly in order to achieve molecular programmability through the pairing of complementary bases. In this work, we report on a hollow but rigid tetrahedron framework of 75 nm strut length constructed with the DNA origami method. Flexible hinges at each of their four joints provide a means for structural variability of the object. Through the opening of gaps along the struts, four variants can be created as confirmed by both gel electrophoresis and direct imaging techniques. The intrinsic site addressability provided by this technique allows the unique targeted attachment of dye and/or linker molecules at any point on the structure’s surface, which we prove through the superresolution fluorescence microscopy technique DNA PAINT. 1

Make them Blink: Probes for Super-Resolution Microscopy

In recent years, a number of approaches have emerged that enable far-field fluorescence imaging beyond the diffraction limit of light, namely super-resolution microscopy. These techniques are beginning to profoundly alter our abilities to look at biological structures and dynamics and are bound to spread into conventional biological laboratories. Nowadays these approaches can be divided into two categories, one based on targeted switching and readout, and the other based on stochastic switching and readout of the fluorescence information. The main prerequisite for a successful implementation of both categories is the ability to prepare the fluorescent emitters in two distinct states, a bright and a dark state. Herein, we provide an overview of recent developments in super-resolution microscopy techniques and outline the special requirements for the fluorescent probes used. In combination with the advances in understanding the photophysics and photochemistry of single fluorophores, we demonstrate how essentially any single-molecule compatible fluorophore can be used for super-resolution microscopy. We present examples for super-resolution microscopy with standard organic fluorophores, discuss factors that influence resolution and present approaches for calibration samples for super-resolution microscopes including AFM-based single-molecule assembly and DNA origami.