TY - JOUR A1 - Trinks, Nora A1 - Reinhard, Sebastian A1 - Drobny, Matthias A1 - Heilig, Linda A1 - Löffler, Jürgen A1 - Sauer, Markus A1 - Terpitz, Ulrich T1 - Subdiffraction-resolution fluorescence imaging of immunological synapse formation between NK cells and A. fumigatus by expansion microscopy JF - Communications Biology N2 - Expansion microscopy (ExM) enables super-resolution fluorescence imaging on standard microscopes by physical expansion of the sample. However, the investigation of interactions between different organisms such as mammalian and fungal cells by ExM remains challenging because different cell types require different expansion protocols to ensure identical, ideally isotropic expansion of both partners. Here, we introduce an ExM method that enables super-resolved visualization of the interaction between NK cells and Aspergillus fumigatus hyphae. 4-fold expansion in combination with confocal fluorescence imaging allows us to resolve details of cytoskeleton rearrangement as well as NK cells' lytic granules triggered by contact with an RFP-expressing A. fumigatus strain. In particular, subdiffraction-resolution images show polarized degranulation upon contact formation and the presence of LAMP1 surrounding perforin at the NK cell-surface post degranulation. Our data demonstrate that optimized ExM protocols enable the investigation of immunological synapse formation between two different species with so far unmatched spatial resolution. KW - biological fluorescence KW - fluorescence imaging KW - imaging the immune system KW - infectious diseases KW - super-resolution microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-264996 VL - 4 IS - 1 ER - TY - JOUR A1 - Mrestani, Achmed A1 - Pauli, Martin A1 - Kollmannsberger, Philip A1 - Repp, Felix A1 - Kittel, Robert J. A1 - Eilers, Jens A1 - Doose, Sören A1 - Sauer, Markus A1 - Sirén, Anna-Leena A1 - Heckmann, Manfred A1 - Paul, Mila M. T1 - Active zone compaction correlates with presynaptic homeostatic potentiation JF - Cell Reports N2 - Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier. KW - active zone KW - Bruchpilot KW - RIM-binding protein KW - compaction KW - homeostasis KW - presynaptic plasticity KW - super-resolution microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265497 VL - 37 IS - 1 ER - TY - JOUR A1 - Eiring, Patrick A1 - McLaughlin, Ryan A1 - Matikonda, Siddharth S. A1 - Han, Zhongying A1 - Grabenhorst, Lennart A1 - Helmerich, Dominic A. A1 - Meub, Mara A1 - Beliu, Gerti A1 - Luciano, Michael A1 - Bandi, Venu A1 - Zijlstra, Niels A1 - Shi, Zhen-Dan A1 - Tarasov, Sergey G. A1 - Swenson, Rolf A1 - Tinnefeld, Philip A1 - Glembockyte, Viktorija A1 - Cordes, Thorben A1 - Sauer, Markus A1 - Schnermann, Martin J. T1 - Targetable conformationally restricted cyanines enable photon-count-limited applications JF - Angewandte Chemie Internationale Edition N2 - Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance. KW - biology KW - super-resolution microscopy KW - conformational restriction KW - cyanine dyes KW - DNA nanotechnology KW - fluorescent dyes KW - single-molecule fluorescence spectroscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256559 VL - 60 IS - 51 ER - TY - JOUR A1 - Lukeš, Tomáš A1 - Glatzová, Daniela A1 - Kvíčalová, Zuzana A1 - Levet, Florian A1 - Benda, Aleš A1 - Letschert, Sebastian A1 - Sauer, Markus A1 - Brdička, Tomáš A1 - Lasser, Theo A1 - Cebecauer, Marek T1 - Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging JF - Nature Communications N2 - Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells. KW - biology KW - fluorescence imaging KW - imaging the immune system KW - super-resolution microscopy Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172993 VL - 8 ER - TY - JOUR A1 - Markert, Sebastian Matthias A1 - Britz, Sebastian A1 - Proppert, Sven A1 - Lang, Marietta A1 - Witvliet, Daniel A1 - Mulcahy, Ben A1 - Sauer, Markus A1 - Zhen, Mei A1 - Bessereau, Jean-Louis A1 - Stigloher, Christian T1 - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome JF - Neurophotonics N2 - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses. KW - caenorhabditis elegans KW - localization micoscopy KW - fluorescent-probes KW - junction proteins KW - resolution limit KW - direct stochasticoptical reconstruction microscopy KW - structured illumination microscopy KW - correlative light and electron microscopy KW - gap junction KW - neural circuits KW - nervous-system KW - image data KW - reconstruction KW - innexins KW - super-resolution microscopy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292 VL - 3 IS - 4 ER - TY - JOUR A1 - Schlegel, Jan A1 - Peters, Simon A1 - Doose, Sören A1 - Schubert-Unkmeir, Alexandra A1 - Sauer, Markus T1 - Super-resolution microscopy reveals local accumulation of plasma membrane gangliosides at Neisseria meningitidis Invasion Sites JF - Frontiers in Cell and Developmental Biology N2 - Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection. KW - Neisseria meningitidis KW - sphingolipids KW - gangliosides and lipid rafts KW - super-resolution microscopy KW - single-molecule tracking Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201639 VL - 7 IS - 194 ER - TY - JOUR A1 - Ehmann, Nadine A1 - Sauer, Markus A1 - Kittel, Robert J. T1 - Super-resolution microscopy of the synaptic active zone JF - Frontiers in Cellular Neuroscience N2 - Brain function relies on accurate information transfer at chemical synapses. At the presynaptic active zone (AZ) a variety of specialized proteins are assembled to complex architectures, which set the basis for speed, precision and plasticity of synaptic transmission. Calcium channels are pivotal for the initiation of excitation-secretion coupling and, correspondingly, capture a central position at the AZ. Combining quantitative functional studies with modeling approaches has provided predictions of channel properties, numbers and even positions on the nanometer scale. However, elucidating the nanoscopic organization of the surrounding protein network requires direct ultrastructural access. Without this information, knowledge of molecular synaptic structure-function relationships remains incomplete. Recently, super-resolution microscopy (SRM) techniques have begun to enter the neurosciences. These approaches combine high spatial resolution with the molecular specificity of fluorescence microscopy. Here, we discuss how SRM can be used to obtain information on the organization of AZ proteins KW - excitation-secretion coupling KW - Ca\(^{2+}\) channels KW - structure-function relationships KW - super-resolution microscopy KW - active zone KW - presynaptic calcium KW - neurotransmitter release Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148997 VL - 9 IS - 7 ER - TY - THES A1 - Aufmkolk, Sarah T1 - Super-Resolution Microscopy of Synaptic Proteins T1 - Hochauflösende Mikroskopie von Synaptischen Proteinen N2 - The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit. The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples. In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity. The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm. These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data. Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM. In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes. N2 - Das Zusammenspiel von synaptischen Proteinen organisiert präzise die Funktion eines der komplexesten Organe, dem Gehirn. Die Vielfalt der molekularen Bestandteile, die diese neurologischen Beziehungen beeinflussen, verkomplizieren den Bildgebungsprozess, da die konventionellen Fluoreszenzmikroskopiemethoden Strukturen, die kleiner sind als das Beugungslimit, nicht auflösen können. Die Implementierung der hochauflösenden Fluoreszenzmikroskopie in das Gebiet der Neurowissenschaften ermöglicht die Visualisierung feiner Details neurologischer Verbindungen. Die hochauflösende Mikroskopietechnik dSTORM (direct Stochastic Optical Reconstruction Microscopy) und dessen Optimierung als Mehrfarbenanwendung sind Schlüsselelemente meiner Doktorarbeit. Mit der Möglichkeit mehr als ein Protein zu messen, ist es mein Ziel die Verteilung synaptischer Proteine mit nanometer Genauigkeit zu entschlüsseln und diese in ein strukturelles und quantitativ Verhältnis zueinander zu setzen. Aus diesem Grund wurden dSTORM spezifische Protokolle den Besonderheiten der jeweiligen neuronalen Proben angepasst und optimiert. In einem Projekt wird der neurotrophe Faktor BDNF (brain derived neurotrophic factor) in primären hippocampalen Neuronen untersucht. Mit einer Auflösungspräzision von unter 15 nm kann durch eine Färbung der Proteine Bassoon und Homer in der aktiven Zone die prä- und postsynaptische Seite identifiziert werden. Daraus resultierend können Kennzeichen für vollentwickelte Synapsen erfasst werden. Die Einzelmolekülsensitivität von dSTORM ermöglicht erstmalig die Messung von endogenem BDNF und zeigt, dass die BDNF Gruppierungen entlang von glutamatergen Präsynapsen verteilt sind. Diese Daten beweisen, dass hippocampale Neuronen die Möglichkeit haben, BDNF in der vollausgebildeten glutamatergen Präsynapse anzureichern und somit möglicherweise synaptische Plastizität beeinflussen. Die Verteilung des metabotropen Glutamatrezeptors mGlu4 wird in physiologischen Gehirnschnitten untersucht. Das ermöglicht den Rezeptor in seiner natürlichen Umgebung zu analysieren. Mit Zweifarben-dSTORM Messungen wird das räumliche Arrangement der mGlu4 Rezeptoren in der Präsynapse der parallelen Fasern der molekularen Schicht des Mauskleinhirns visualisiert und eine vier- bis sechsfache erhöhte Dichte des Rezeptors in der aktiven Zone, verglichen mit dem näheren Umfeld, aufgezeigt. Vorausgegangende funktionale Messungen zeigen, dass metabotrope Glutamatrezeptoren spannungsgesteuerte Calciumkanäle und Proteine, die in synaptische Vesikelgrundierung involviert sind, beeinflussen. Entsprechende dSTORM Daten deuten darauf hin, dass ein Teil der mGlu4 Rezeptoren mit dem spannungsgesteuerten Calciumkanal Cav2.1 auf einer Distanz von circa 60 nm korreliert ist. Diese Ergebnisse basieren auf der Verbesserung der direkten Analyse der Lokalisationsdatensätze. Werkzeuge, wie die Koordinaten basierte Korrelationsanalyse und die Nächste Nachbaranalyse von Clusterschwerpunkten werden sich ergänzend benutzt, um ein umfassendes Bild von Proteinverbindungen in der Synapse zu erzeugen. Die Grenzen und die Verbesserungsmöglichkeiten dieser Werkzeuge werden diskutiert, um die quantitative Analyse von Einzelmoleküldatensätzen voranzubringen. Die Durchführung von hochauflösender Mikroskopie an komplexen Proben, wie Gehirnschnitten, wird begünstigt durch die Maximierung der Aufnahmefläche in Kombination mit der Möglichkeit mehr als zwei Zielstrukturen zu visualisieren, um somit das Protein von primären Interesse in einen zellulären Zusammenhang zu setzen. Diese Herausforderung hat als Motivation gedient, ein Messprotokoll für korrelierte Strukturierte Beleuchtungsmikroskopie (SIM) und dSTORM zu etablieren. Die Entwicklung der Visualisierungssoftware coSIdSTORM erleichtert die Kombination dieser beiden leistungsstarken, hochauflösenden Techniken, sogar wenn diese auf getrennten Mikroskopieaufbauten umgesetzt werden. Als ein Beispiel werden Synapsen, die zwischen den parallelen Fasern in der molekularen Schicht des Cerebellums und den Purkinje-Zellen ausgebildet werden, mit SIM identifiziert und das Protein Bassoon in diesen Präsynapsen wird mit einer nanometergenauen Präzision drei-dimensional mit dSTORM Messungen visualisiert. In meiner Arbeit habe ich den Fokus auf die Weiterentwickelung von hochauflösender Mehrfarbenmikroskopie und die damit verbundenen analytischen Werkzeuge gelegt, sodass die Untersuchung von synaptischen Proteinen ermöglicht wird. Die Herausarbeitung des strukturellen Arrangements der untersuchten synaptischen Proteine unterstützt den Aufbau eines Models der Synapse und erweitert somit das Verständnis des Zusammenhangs von Struktur und Funktion in neuronalen Übertragungsvorgängen. KW - Hochauflösende Mikroskopie KW - correlative methods KW - Fluoreszenzmikroskopie KW - Synaptische Proteine KW - Korrelative Mikroskopie KW - dSTORM KW - SIM KW - fluorescence KW - super-resolution microscopy KW - localization microscopy KW - two-color microscopy KW - synapse KW - synaptic proteins Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151976 ER - TY - THES A1 - Wolter, Steve T1 - Single-molecule localization algorithms in super-resolution microscopy T1 - Einzelmoleküllokalisierungsalgorithmen in der superauflösenden Mikroskopie N2 - Lokalisationsmikroskopie ist eine Methodenklasse der superauflösenden Fluoreszenzmikroskopie, deren Methoden sich durch stochastische zeitliche Isolation der Fluoreszenzemission auszeichnen. Das Blinkverhalten von Fluorophoren wird so verändert, dass gleichzeitige Aktivierung von einander nahen Fluorophoren unwahrscheinlich ist. Bekannte okalisationsmikroskopische Methoden umfassen dSTORM, STORM, PALM, FPALM, oder GSDIM. Lokalisationsmikroskopie ist von hohem biologischem Interesse, weil sie die Auflösung des Fluoreszenzmikroskops bei minimalem technischem Aufwand um eine Größenordnung verbessert. Der verbundene Rechenaufwand ist allerdings erheblich, da Millionen von Fluoreszenzemissionen einzeln mit Nanometergenauigkeit lokalisiert werden müssen. Der Rechen- und Implementationsaufwand dieser Auswertung hat die Verbreitung der superauflösenden Mikroskopie lange verzögert. Diese Arbeit beschreibt meine algorithmische Grundstruktur für die Auswertung lokalisationsmikroskopischer Daten. Die Echtzeitfähigkeit, d.h. eine Auswertegeschwindigkeit oberhalb der Datenaufnahmegeschwindigkeit an normalen Messaufbauten, meines neuartigen und quelloffenen Programms wird demonstriert. Die Geschwindigkeit wird auf verbrauchermarktgängigen Prozessoren erreicht und dadurch spezialisierte Rechenzentren oder der Einsatz von Grafikkarten vermieden. Die Berechnung wird mit dem allgemein anerkannten Gaussschen Punktantwortmodell und einem Rauschmodell auf Basis der größten Poissonschen Wahrscheinlichkeit durchgeführt. Die algorithmische Grundstruktur wird erweitert, um robuste und optimale Zweifarbenauswertung zu realisieren und damit korrelative Mikroskopie zwischen verschiedenen Proteinen und Strukturen zu ermöglichen. Durch den Einsatz von kubischen Basissplines wird die Auswertung von dreidimensionalen Proben vereinfacht und stabilisiert, um präzisem Abbilden von mikrometerdicken Proben näher zu kommen. Das Grenzverhalten von Lokalisationsalgorithmen bei hohen Emissionsdichten wird untersucht. Abschließend werden Algorithmen für die Anwendung der Lokalisationsmikroskopie auf verbreitete Probleme der Biologie aufgezeigt. Zelluläre Bewegung und Motilität werden anhand der in vitro Bewegung von Myosin-Aktin-Filamenten studiert. Lebendzellbildgebung mit hellen und stabilen organischen Fluorophoren wird mittels SNAP-tag-Fusionsproteinen realisiert. Die Analyse des Aufbaus von Proteinklumpen zeigt, wie Lokalisationsmikroskopie neue quantitative Ansätze jenseits reiner Bildgebung bietet. N2 - Localization microscopy is a class of super-resolution fluorescence microscopy techniques. Localization microscopy methods are characterized by stochastic temporal isolation of fluorophore emission, i.e., making the fluorophores blink so rapidly that no two are likely to be photoactive at the same time close to each other. Well-known localization microscopy methods include dSTORM}, STORM, PALM, FPALM, or GSDIM. The biological community has taken great interest in localization microscopy, since it can enhance the resolution of common fluorescence microscopy by an order of magnitude at little experimental cost. However, localization microscopy has considerable computational cost since millions of individual stochastic emissions must be located with nanometer precision. The computational cost of this evaluation, and the organizational cost of implementing the complex algorithms, has impeded adoption of super-resolution microscopy for a long time. In this work, I describe my algorithmic framework for evaluating localization microscopy data. I demonstrate how my novel open-source software achieves real-time data evaluation, i.e., can evaluate data faster than the common experimental setups can capture them. I show how this speed is attained on standard consumer-grade CPUs, removing the need for computing on expensive clusters or deploying graphics processing units. The evaluation is performed with the widely accepted Gaussian PSF model and a Poissonian maximum-likelihood noise model. I extend the computational model to show how robust, optimal two-color evaluation is realized, allowing correlative microscopy between multiple proteins or structures. By employing cubic B-splines, I show how the evaluation of three-dimensional samples can be made simple and robust, taking an important step towards precise imaging of micrometer-thick samples. I uncover the behavior and limits of localization algorithms in the face of increasing emission densities. Finally, I show up algorithms to extend localization microscopy to common biological problems. I investigate cellular movement and motility by considering the in vitro movement of myosin-actin filaments. I show how SNAP-tag fusion proteins enable imaging with bright and stable organic fluorophores in live cells. By analyzing the internal structure of protein clusters, I show how localization microscopy can provide new quantitative approaches beyond pure imaging. KW - super-resolution microscopy KW - fluorescence KW - scientific computing KW - dSTORM KW - localization microscopy KW - PALM KW - 3D microscopy KW - two-color microscopy KW - Fluoreszenzmikroskopie KW - Bildauflösung KW - Bioinformatik Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-109370 ER -