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Class II cytokine receptors are important pleiotropic regulators of the immune system that play a central role in pathogen defense, tumor surveillance and immune system homeostasis. Most of these activities are very promising for biomedical applications, which, however, have so far failed to succeed due to severe undesired side effects resulting from the pleiotropic nature of these cytokine receptors. Controlling the functional plasticity of class I/II cytokine receptor signaling by engineered cytokines has recently emerged as a promising approach to selectively reduce such side effects. In this context, systematic studies on the IFNalpha/beta receptor and other systems have identified that the binding kinetics of the ligand-receptor interaction play an important role in defining signaling specificity. This has been explained by altered equilibrium and dynamics of the signaling complex in the plasma membrane. In this work, I have investigated how the spatiotemporal organization and dynamics of signaling complexes regulate activation and signaling specificity of other members of the class II cytokine receptors. I focused on the type II IFN and IL-10 systems that supposedly form hexameric ligand-receptor signaling complexes in the plasma membrane. To this end, we developed an orthogonal multicolor anti-GFP nanobody-based labeling strategy, that allowed imaging of up to four different class II cytokine receptor subunits simultaneously. Using this labeling strategy, I investigated the spatiotemporal dynamics of IFNGR and IL-10R complex assembly by co-localization and co-tracking of single receptor subunits. Thereby, I did show that unliganded receptor subunits of IFNGR and IL-10R remain monomeric at the cell surface, whereas binding of the ligand led to fast and efficient receptor homo- and hetero-dimerization, verifying a ligand-induced receptor complex assembly model for both cytokine receptors. Moreover, I verified the hexameric ligand-receptor complex structure in cellulo. Analysis of single molecule trajectories and co-trajectories revealed a decrease in mobility and diffusion of IFNGR and IL-10R subunits upon ligand stimulation indicating receptor confinement and endocytosis. In this context, I identified an abnormal diffusion behavior of IL-10R2 that was dependent on the length of its transmembrane helix. We used partial agonists for both receptor complexes to systematically alter receptor binding stoichiometry and complex stability in the plasma membrane and correlated these with downstream signaling responses. Our analysis revealed a minor contribution of the second low affinity receptor subunit and its associated kinase to the overall signaling activity. However, the second high affinity binding subunit was indispensable to acquire full signaling potential. We managed to obtained decoupling of gene expression for both hexameric class II cytokine receptors by utilizing engineered ligands with altered receptor binding affinities. Our findings could pave the way for new biomedical approaches with engineered IFNgamma and IL-10 in the future. Furthermore, we uncovered pathogenic mechanisms behind the IFNGR2-T168N mutant and auto-IFNgamma antibodies, both of which prominently cause the Mendelian Susceptibility to Mycobacteria Disease (MSMD) syndrome, showing that both interfere with IFNGR activation by preventing recruitment of IFNGR2 into receptor complexes.

Optical microscopy is the oldest form of microscopy that has been visually aiding scientific research. In our research, I have reported here two such optical microscopy techniques for two different projects. In the first project, we re-developed an instrumentation of a cost-effective, high-performing, single-molecular localization super-resolution microscopy setup that breaks the diffraction limitation barrier. Then we use a stochastic image capturing technique to capture the best precision image positions of gold nanoparticles. In our second project, we apply confocal microscopy technique to image DNA molecular nanoscale structural alterations of chromatin in cell nuclei of gut tissues caused by total body irradiation (TBI). We then quantify these alterations using a light localization technique called inverse participation ratio (IPR) using the confocal micrographs of the sample. Our results show radiation causes reduction and saturation of DNA spatial mass density fluctuations that were observed for different durations of post-irradiation.

Mitochondria are the power plant of most non-green eukaryotic cells. In order to understand mitochondrial functions and their regulation, knowledge of the spatiotemporal organization of their proteins is important. Mitochondrial membrane proteins can diffuse within membranes. They are involved in diverse functions e.g. protein import, cell respiration, metabolism, metabolite transport, fusion, fission or formation of the mitochondrial architecture. Furthermore, mitochondria compose of different subcompartments with different tasks. Especially, the inner mitochondrial membrane (IM), where the oxidative phosphorylation (OXPHOS) takes places, has a complex architecture with cristae extending into the matrix. The present work revealed the restricted localization of some mitochondrial proteins to specific membrane sections and linked it to their function or bioenergetic circumstances in the living cell. 
Single particle tracking (SPT) techniques like tracking and localization microscopy (TALM) allow to localize proteins with a precision below 20 nm. Additionally, tracking single proteins provides information about their mobility, dynamic and their spatiotemporal organization. TALM uses proteins, which were genetically tagged either with the HaloTag® (HaloTag) or the fSnapTag® (fSnapTag). These tags can be orthogonally and posttranslationally stained with specific and self-marking dyes. If the dyes are conjugated to the respective substrate of the tag. Single molecule labeling of mitochondrial proteins was performed substoichiometrically using membrane permeable rhodamine dyes, either tetramethylrhodamine (TMR) or silicon rhodamine (SiR). TALM allowed to localize proteins in different mitochondrial subcompartments. The gained trajectories and trajectory maps of mitochondrial proteins revealed their spatiotemporal organization. In the case of IM proteins like F1FO ATP synthase (Complex V - CV) a restricted diffusion in the CM, which is part of the continuous IM, was determined. The unimpeded diffusion of mitochondrial proteins in the outer mitochondrial membrane (OM) was compared with the mobility of IM proteins. The diffusion of mitochondrial IM proteins was restricted by the IM architecture and their diffusion coefficients were lower. Furthermore, significant differences of different mitochondrial IM proteins were compared, showing different localizations in the IM often coupled to their function, accompanied by different spatiotemporal organization and diffusion coefficients. Furthermore, a distinction was made between diffusion of proteins in the inner boundary membrane (IBM) and proteins that preferentially diffuse in the cristae membrane (CM). Evaluating trajectory maps, the different subcompartments in the IM were revealed by trajectories and the trajectory directionality, allowing the identification of mitochondrial proteins, which mark these subcompartments.
The morphology of mitochondria / mitochondrial networks and their bioenergetic parameters are linked to the metabolic states of the cell. In this work, the connection of the spatiotemporal protein organization of CV and the IM architecture was uncovered on the micro- and nanoscopic level and linked to the metabolic state of the cell. It was determined that the spatiotemporal organization of the CV was altered, when CV was inhibited. In addition, the bioenergetic influence of cells on the spatiotemporal behavior of CV and the reorganization of the IM architecture was investigated by TALM and compared with results of electron microscopy images. It was shown that starvation of cells led to a loss of cristae and thus to an increased mobility and spatiotemporal reorganization of CV. Taken together, the results presented in this work showed that a correctly functioning and active CV helps to maintain the IM architecture and both, the spatiotemporal organization of CV and the IM architecture were coupled to the metabolic state.. 
In order to investigate putative protein-protein interactions by colocalization and co-locomotion studies on single molecule level, dual color SPT is needed. Therefore, posttranslational and substoichimetric labeling as performed in TALM was tested for its potential of protein-protein interaction studies of mitochondrial membrane proteins. Here, a genetically double tagged translocase of the outer membrane subunit-20 (Tom20) (Tom20:HaloTag:fSnapTag) acted as a positive control. It turned out that substoichimetric, posttranslational labeling of mitochondrial proteins was not suitable for protein-protein interaction studies on mitochondrial proteins, because it was restricted by the low labeling degrees needed for TALM. However, dual-color TALM still allowed to study effects of proteins influencing the IM architecture and to study their influence on the spatiotemporal organization of CV. The co-transfection of Mic10, as the central protein of the mitochondrial inner membrane organizing system / mitochondrial contact site complex / mitochondrial organizing structure (MINOS / MICOS / MitOS (MINOS/MICOS)), altered the regular and aligned organization of the cristae. This was measured by a changed spatiotemporal organization of the CV, such as the loss of the perpendicular oriented of CV subunit-γ (CV-SUγ) cristae trajectories. In contrast to this, co-transfection of CV subunit-e (CV-SUe), important for dimerization of CV, increased the number of cristae trajectories. 
Mitochondria are three-dimensional (3D) cell organelles. Consequently, subcompartments like the IBM and CM are a 3D space in which CV is localized and diffuses. Thus, the diffusion of mitochondrial proteins is underestimated by two-dimensional SPT e.g. lateral confined diffusion can result from mitochondrial proteins diffusing along the z-axis of the microscope. In order to reveal the 3D spatiotemporal organization of CV, the potential of TALM to be extended to a 3D-SPT technique was investigated. Therto a cylindrical lens was installed in the emission path of a total internal reflection fluorescence (TIRF) microscope. This leads to an astigmatically distorted point spread function (PSF) of the fluorescent single molecule signals. This distortion allowed the reconstruction of single molecule localizations of CV to a superresolved image of the IM, in living cells. In addition, 3D-TALM enabled to display the 3D architecture of the IM by 3D trajectories of CV. 3D-TALM was able to detect whether CV diffuses in the IBM or in the CM, and extended the information about its mobility in the CM that it takes place in a disc-like manner. In this way it could be shown that CV is mobile within the cristae in all directions. Finally, 3D-TALM revealed an altered IM architecture caused by the metabolic state of the cell. As performed in two-dimensional TALM, the cells were kept under starving conditions. Here the now tubular IM architecture was revealed by 3D-TALM. The reversed metabolic state under improved respiratory conditions unexpectedly led to a more diverse IM architecture. These ultrastructural changes were also revealed by electron microscopy. Consequently, 3D-TALM enabled the study of IM architecture by tracking CV under different metabolic conditions, allowing an ultrastructural analysis of mitochondria in living cells. In addition, 3D TALM provided the spatiotemporal organization of CV under different metabolic conditions, so that the diffusion coefficients of CV could be related to changes in IM architecture caused by the metabolic condition.

La microscopie optique est un outil indispensable pour la recherche de la neurobiologie et médecine qui permet l’étude des cellules dans leur environnement natif. Les processus sous-cellulaires restent néanmoins cachés derrière les limites de la résolution optique, ce qui rend la résolution des structures plus petites que ~300nm impossible. Récemment, les techniques de la localisation des molécules individuelles (SML) ont permis le suivi des protéines de l’échelle nanométrique grâce à l’ajustement des molécules uniques à la réponse impulsionnelle du système optique. Ce processus dépend de la quantité de lumière recueilli et rend ces techniques très sensibles aux imperfections de la voie d’imagerie, nommé des aberrations, qui limitent l’application de SML aux cultures cellulaires sur les lamelles de verre. Un système commercial d’optiques adaptatives est implémenté pour compenser les aberrations du microscope, et un flux de travail est défini pour corriger les aberrations dépendant de la profondeur qui rend la 3D SML possible dans les milieux biologiques complexes. Une nouvelle méthode de SML est présentée qui utilise deux objectifs pour détecter le spectre d’émission des molécules individuelles pour des applications du suivi des particules uniques dans 5 dimensions (x,y,z,t,λ) sans compromis ni de la résolution spatiotemporelle ni du champ de vue. Pour faciliter les analyses de manière quantitative des Go de données générés, le développement des outils biochimiques, numériques et optiques est présenté. Ensemble, ces approches ont le but d’amener l’imagerie quantitative des molécules uniques dans les échantillons biologiques complexes / Optical microscopy is an indispensable tool for research in neurobiology and medicine, enabling studies of cells in their native environment. However, subcellular processes remain hidden behind the resolution limits of diffraction-limited optics which makes structures smaller than ~300nm impossible to resolve. Recently, single molecule localization (SML) and tracking has revolutionized the field, giving nanometer-scale insight into protein organization and dynamics by fitting individual fluorescent molecules to the known point spread function of the optical imaging system. This fitting process depends critically on the amount of collected light and renders SML techniques extremely sensitive to imperfections in the imaging path, called aberrations, that have limited SML to cell cultures on glass coverslips. A commercially available adaptive optics system is implemented to compensate for aberrations inherent to the microscope, and a workflow is defined for depth-dependent aberration correction that enables 3D SML in complex biological environments. A new SML technique is presented that employs a dual-objective approach to detect the emission spectrum of single molecules, enabling 5-dimensional single particle imaging and tracking (x,y,z,t,λ) without compromising spatiotemporal resolution or field of view. These acquisitions generate ~GBs of data, containing a wealth of information about the localization and environment of individual proteins. To facilitate quantitative acquisition and data analysis, the development of biochemical, software and hardware tools are presented. Together, these approaches aim to enable quantitative SML in complex biological samples.

Computational methods in single molecule localization microscopy Abstract Fluorescence microscopy is one of the chief tools used in biomedical research as it is a non invasive, non destructive, and highly specific imaging method. Unfortunately, an optical microscope is a diffraction limited system. Maximum achievable spatial resolution is approximately 250 nm laterally and 500 nm axially. Since most of the structures in cells researchers are interested in are smaller than that, increasing resolution is of prime importance. In recent years, several methods for imaging beyond the diffraction barrier have been developed. One of them is single molecule localization microscopy, a powerful method reported to resolve details as small as 5 nm. This approach to fluorescence microscopy is very computationally intensive. Developing methods to analyze single molecule data and to obtain super-resolution images are the topics of this thesis. In localization microscopy, a super-resolution image is reconstructed from a long sequence of conventional images of sparsely distributed single photoswitchable molecules that need to be sys- tematically localized with sub-diffraction precision. We designed, implemented, and experimentally verified a set of methods for automated processing, analysis and visualization of data acquired...

Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects. This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.

Computational methods in single molecule localization microscopy Abstract Fluorescence microscopy is one of the chief tools used in biomedical research as it is a non invasive, non destructive, and highly specific imaging method. Unfortunately, an optical microscope is a diffraction limited system. Maximum achievable spatial resolution is approximately 250 nm laterally and 500 nm axially. Since most of the structures in cells researchers are interested in are smaller than that, increasing resolution is of prime importance. In recent years, several methods for imaging beyond the diffraction barrier have been developed. One of them is single molecule localization microscopy, a powerful method reported to resolve details as small as 5 nm. This approach to fluorescence microscopy is very computationally intensive. Developing methods to analyze single molecule data and to obtain super-resolution images are the topics of this thesis. In localization microscopy, a super-resolution image is reconstructed from a long sequence of conventional images of sparsely distributed single photoswitchable molecules that need to be sys- tematically localized with sub-diffraction precision. We designed, implemented, and experimentally verified a set of methods for automated processing, analysis and visualization of data acquired...

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Le domaine de la microscopie de fluorescence a connu une réelle révolution ces dernières années, permettant d'atteindre des résolutions nanométriques, bien en dessous de la limite de diffraction prédite par Abbe il y a plus d’un siècle. Les techniques basées sur la localisation de molécules individuelles telles que le PALM (Photo-Activation Light Microscopy) ou le (d)STORM (direct Stochastic Optical Reconstruction Microscopy) permettent la reconstruction d’images d’échantillons biologiques en 2 et 3 dimensions, avec des résolutions quasi-moléculaires. Néanmoins, même si ces techniques nécessitent une instrumentation relativement simple, elles requièrent des traitements informatiques conséquents, limitant leur utilisation en routine. En effet, plusieurs dizaines de milliers d’images brutes contenant plus d’un million de molécules doivent être acquises et analysées pour reconstruire une seule image. La plupart des outils disponibles nécessitent une analyse post-acquisition, alourdissant considérablement le processus d’acquisition. Par ailleurs la quantification de l’organisation, de la dynamique mais aussi de la stœchiométrie des complexes moléculaires à des échelles nanométriques peut constituer une clé déterminante pour élucider l’origine de certaines maladies. Ces nouvelles techniques offrent de telles capacités, mais les méthodes d’analyse pour y parvenir restent à développer. Afin d’accompagner cette nouvelle vague de microscopie de localisation et de la rendre utilisable en routine par des expérimentateurs non experts, il est primordial de développer des méthodes de localisation et d’analyse efficaces, simples d’emploi et quantitatives. Dans le cadre de ce travail de thèse, nous avons développé dans un premier temps une nouvelle technique de localisation et reconstruction en temps réel basée sur la décomposition en ondelettes et l‘utilisation des cartes GPU pour la microscopie de super-résolution en 2 et 3 dimensions. Dans un second temps, nous avons mis au point une méthode quantitative basée sur la visualisation et la photophysique des fluorophores organiques pour la mesure de la stœchiométrie des récepteurs AMPA dans les synapses à l’échelle nanométrique. / The field of fluorescence microscopy has witnessed a real revolution these last few years, allowing nanometric spatial resolutions, well below the diffraction limit predicted by Abe more than a century ago. Single molecule-based super-resolution techniques such as PALM (Photo-Activation Light Microscopy) or (d)STORM (direct Stochastic Optical Reconstruction Microscopy) allow the image reconstruction of biological samples in 2 and 3 dimensions, with close to molecular resolution. However, while they require a quite straightforward instrumentation, they need heavy computation, limiting their use in routine. In practice, few tens of thousands of raw images with more than one million molecules must be acquired and analyzed to reconstruct a single super-resolution image. Most of the available tools require post-acquisition processing, making the acquisition protocol much heavier. In addition, the quantification of the organization, dynamics but also the stoichiometry of biomolecular complexes at nanometer scales can be a key determinant to elucidate the origin of certain diseases. Novel localization microscopy techniques offer such capabilities, but dedicated analysis methods still have to be developed. In order to democratize this new generation of localization microscopy techniques and make them usable in routine by non-experts, it is essential to develop simple and easy to use localization and quantitative analysis methods. During this PhD thesis, we first developed a new technique for real-time localization and reconstruction based on wavelet decomposition and the use of GPU cards for super-resolution microscopy in 2 and 3 dimensions. Second, we have proposed a quantitative method based on the visualization and the photophysics of organic fluorophores for measuring the stoichiometry of AMPA receptors in synapses at the molecular scale.