Fibromyalgia syndrome (FMS) is a largely heterogeneous chronic pain syndrome of unclear pathophysiology, which lacks objective diagnostics and specific treatment. An immune-related shift towards a pro-inflammatory profile is discussed at a systemic level. Small fiber pathology (SFP) and local participation of non-neuronal skin cells like keratinocytes in cutaneous nociception are potential peripheral contributors. Small RNAs, particularly microRNAs (miRs) and newly described tRNA fragments (tRFs) act as posttranscriptional key regulators of gene expression and may modulate systemic and peripheral cell pathways. On cellular level, the exact mechanisms of keratinocyte-intraepidermal nerve fiber (IENF) interaction in the skin are insufficiently understood. Via small RNA sequencing and quantitative real-time PCR, we investigated miR and tRF signatures in whole blood cells and skin biopsy-derived keratinocytes of female FMS patients versus healthy controls. We applied gene target prediction analysis to uncover underlying cellular pathways affected by dysregulated small RNAs. Altered FMS small RNAs from blood were compared with their expression in disease controls, i.e. Parkinson`s patients and patients with major depression and chronic pain. Association of SFP with small RNAs was investigated via correlation with clinical parameter. To explore keratinocyte-nerve fiber interactions with high relevance for SFP and cutaneous nociception, we adapted a super-resolution array tomography (srAT) approach and expansion microscopy (ExM) for human skin samples. Further, we created a fully human 2D co-culture model of primary keratinocytes and induced pluripotent stem cell derived sensory neurons. Blood miR deregulation indicated systemic modulation of immune processes exerted by CholinomiRs and by miRs targeting the FoxO signaling pathway. Short sized tRFs were associated with mRNA metabolism and splicing. This supports the hypothesis of an inflammatory/autoimmunity component in FMS. Expression of blood small RNAs in FMS were discriminative against disease controls, highlighting their potential as objective biomarker. Blood small RNAs were predominantly upregulated and correlations between miR and clinical parameter reflected rather pain in general than SFP. In FMS keratinocytes, a downregulation of miRs and tRFs was evident. Pathways for adenosine monophosphate-activated protein kinase (AMPK), adherens junction, and focal adhesion were predicted to be affected by miRs, while tRFs may influence proliferation, migration, and cell growth. Similar to blood miRs, altered miRs in keratinocytes correlated mostly with widespread pain and pain severity parameter. TRFs were partially associated with more severe IENF loss. Small RNAs in FMS keratinocytes may modulate pathways that define how keratinocytes interact with each other and with IENF. These interactions include nerve fiber ensheathment, a conserved epithelial mechanism, which we visualize in human epidermis and a fully human co-culture model. Additionally, we revealed plaques of connexin 43, a pore forming protein involved in intercellular communication, at keratinocyte- nerve fiber contact sites. Objective quantification of these morphological findings in FMS and other diseases with SFP may inherit diagnostic value similar to IENF density. We provide evidence for distinct miR and tRF signatures in FMS with implications for systemic immune regulation and local cell-cell interaction pathways. In the periphery we explored novel keratinocyte-nerve fiber interactions relevant for SFP and cutaneous nociception. / Das Fibromyalgie Syndrom (FMS) umfasst ein sehr heterogenes chronisches Schmerzsyndrom mit ungeklärter Pathophysiologie, ohne objektive Diagnostik und gezielt wirkende Behandlungsmöglichkeiten. Auf systemischer Ebene wird eine entzündungsfördernde Verschiebung von Immunprozessen diskutiert. In der Peripherie stellen die Kleinfaserpathologie (SFP) und Beteiligungen nicht-neuronaler Hautzellen, beispielsweise Keratinozyten, an kutaner Nozizeption potenziell beitragende Faktoren dar. Kleine RNAs, vor allem microRNAs (miRs) und die kürzlich beschriebenen tRNA Fragmente (tRFs) agieren als posttranskriptionelle Schlüsselregulatoren der Genexpression und könnten daher systemische und periphere Zellprozesse modulieren. Die genauen zellulären Mechanismen bei der Interaktion von Keratinozyten mit intraepidermalen Nervenfasern (IENF) in der Haut sind nur unzureichend verstanden. Mittels Sequenzierung von kleinen RNAs und quantitativer Real-Time PCR untersuchten wir miR und tRF Signaturen in Vollblutzellen und in durch Hautbiopsie gewonnene Keratinozyten von FMS Patientinnen im Vergleich zu gesunden weiblichen Kontrollen. Um zugrundeliegende Zellprozesswege aufzudecken, die von der Deregulierung kleiner RNAs betroffen sind, verwendeten wir Vorhersageprogramme für regulierte Gene. In FMS verändert vorliegende kleine RNAs im Blut verglichen wir mit ihrer Expression in Krankheitskontrollen, d.h. Parkinson Patientinnen und Patientinnen mit schwerer Depression und chronischem Schmerz. Die Beziehung zwischen SFP und kleinen RNAs wurde mittels der Korrelation mit klinischen Parametern untersucht. Zur Erforschung von Keratinozyten-Nervenfaser Interaktionen, mit großer Relevanz für SFP und kutane Nozizeption, adaptierten wir eine superauflösende Array-Tomographie (srAT) Methodik und Expansionsmikroskopie (ExM) für humane Hautproben. Außerdem entwickelten wir ein rein humanes 2D Ko-Kultur Zellmodell, bestehend aus primären Keratinozyten und sensiblen Neuronen, die aus induzierten pluripotenten Stammzellen generiert wurden. MiR Deregulierungen in Blut wiesen auf systemische Modulierung von Immunprozessen hin, ausgeübt durch CholinomiRs und miRs, die auf den FoxO Signalweg einwirken. Die tRFs mit kurzer Fragmentlänge waren mit mRNA Metabolismus und Splicing verknüpft. Diese Ergebnisse unterbauen die Hypothese einer entzündungsfördernden/autoimmunen Komponente in FMS. Die Expression kleiner RNAs aus FMS Blut war unterschiedlich zu Krankheitskontrollen, was ihr Potenzial als objektive Biomarker hervorhebt. Kleine RNAs im Blut waren überwiegend erhöht exprimiert und Korrelation zwischen miRs und klinischen Parametern spiegelten eher Schmerzen im Allgemeinen wider als SFP. In Keratinozyten von FMS Patientinnen war eine Herunterregulierung von miRs und tRFs ersichtlich. Der Signalweg der Adenosinmonophosphat aktivierten Proteinkinase (AMPK), sowie Adherens Junction und Fokale Adhäsion waren prognostiziere Prozesse unter Einfluss von miRs. Ähnlich wie bei den Blut miRs, korrelierten veränderte miRs in Keratinozyten vor allem mit der Verbreitung des Schmerzes über den Körper und der Schmerzintensität. TRFs waren teilweise mit einem höheren Verlust an IENF verknüpft. Kleine RNAs in Keratinozyten von FMS Patientinnen könnten jene Prozesse modulieren, die festlegen, wie Keratinozyten miteinander und mit IENF interagieren. Diese Interaktionen beinhalten den konservierten Mechanismus der Nervenfaserumhüllung, den wir in humaner Epidermis und einem komplett humanen Ko-Kultur Modell auflösen konnten. Zusätzlich zeigten wir Anhäufungen von Connexin 43, einem an interzellulärer Kommunikation beteiligten porenformenden Protein, an Keratinozyten-Nervenfaser Kontaktstellen. Eine objektive Quantifizierung dieser morphologischen Befunde in FMS und weiteren Erkrankungen mit SFP könnte einen diagnostischen Wert vergleichbar mit dem der IENF Dichte innehaben. Wir liefern Belege für klare miR und tRF Signaturen in FMS mit Bedeutung für systemische Immunregulation und lokale Zell-Zell Interaktionsprozesse. In der Peripherie erkundeten wir neueartige Keratinozyten-Nervenfaser Interaktionen relevant für SFP und kutane Nozizeption.

Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis. / Diffusion erleichtert zahlreiche Reaktionen im biologischen Kontext einer Zelle. Es ist bemerkenswert, wie der kosteneffiziente Zufallsprozess der Brownschen Bewegung schnelle Reaktionen fördert. Mit Hilfe der Narrow Escape Theorie kann die mittlere erste Durchgangszeit (mean first passage time) solcher Prozesse auf der Grundlage ihres Reaktionsraums und des Diffusionskoeffizienten bestimmt werden. Die Narrow Escape Theorie von Brownschen Teilchen wird durch einen begrenzten Bereich mit reflektierenden Grenzen und einen kleinen Reaktionsraum gekennzeichnet. In dieser Arbeit wurde die mittlere erste Durchgangszeit in einer Scheibe systematisch als Funktion der Größe der Fluchtöffnung in vitro und in silico untersucht. Fur die in vitro-Experimente wurde ein Modellsystem aus strukturierten, Glas gestützten Lipiddoppelschichten (SLB) erstellt. Dieses Modell wurde durch einen kombinierten Kolloid-Metallisierungsansatz hergestellt, bei dem eine strukturierte Goldschicht auf Glas die Bildung von SLB-Scheiben unterschiedlicher Größe durch die Vesikelfusion ermöglicht. Es wurde festgestellt, dass das Modell alle Anforderungen erfüllt, um das Narrow escape problem in vitro zu testen. Insbesondere wurde das Reflexionsvermögen der Grenzen, die ungehinderte, freie Diffusion der Lipide und die Präzision der Fläche bewertet. Die beobachteten Ergebnisse der mittleren ersten Durchgangszeit stimmen mit der NEP-Theorie ̈uberein. Es besteht eine hervorragende ̈Ubereinstimmung sowohl bei den absoluten Werten als auch systematisch ̈uber einen Bereich von kleinen Fluchtöffnungsgrößen. Außerdem zeige ich eine einfache Methode, einen Korrekturfaktor, zur Berechnung der mittleren ersten Durchgangszeit aus unvollstandigen Lipid Trajektorien des Experimentes. Indem ich die mittlere erste Durchgangszeit auf den Anteil der entkommenen Par- tikel skalierte, konnte ich die Einschränkungen der Lebensdauer von fluoreszenten Farbstoffen ausgleichen. Mit dieser Technik werden bisher unzugängliche Messungen der mittleren ersten Durchgangszeit auf der Grundlage von fluoreszenten Farbstoffen möglich. In einem umfassenden Ansatz wurden die in vitro-Experimente durch verschiedene in silico-Experimente ergänzt. Letztere basierten auf Random- Walk-Simulationen in Scheiben, die die in vitro-Situation mit ihren Unsicherheiten nachahmten. Die Lebensdauer einzelner Partikel wurde entweder so lang angesetzt, dass alle Partikel entkommen konnten, oder sie wurde so angepasst, dass sie den in den in vitro-Experimenten beobachteten Lebensdauerbeschränkungen entsprach. Ein Vergleich der mittleren ersten Durchgangszeit von unsterblichen Partikeln mit den korrigierten, lebensdauerbegrenzten Partikeln hat die Verwendung des Korrekturfaktors bestätigt. In ̈Ubereinstimmung mit der Narrow Escape Theorie wurde experimentell festgestellt, dass die mittlere erste Durchgangszeit unabhängig vom Startpunkt des Teilchens innerhalb der Domäne ist. Dies ist dann der Fall, wenn das Teilchen einen Mindestabstand zur Austrittsstelle einhält. Im Allgemeinen bilden die vorgestellten Random-Walk-Simulationen die in dieser Studie durchgeführten Experimente genau ab. Die erforderliche Hardware für die Einrichtung eines auf Astigmatismus basierenden 3D-Systems wurde in ein vorhandenes Mikroskop eingebaut. Erste Versuche, die gewonnenen 3D-Bilddaten zu analysieren, gaben einen Einblick in das Potenzial der Methode zur Untersuchung der Moleküldynamik im lebenden Trypanosom. Die volle Funktionalität wird mit der laufenden Verbesserung der Bildanalyse außerhalb dieser Arbeit realisiert werden. Ein Teil der Ergebnisse und Methoden dieser Dissertation wurde zur Veröffentlichung unter dem Titel ’Experiments in micro-patterned model membranes support the narrow escape theory’ eingereicht.

Aluminium Al-Mg-Si alloys are the most commonly used in automotive and construction industry. Hexagonal ’-phase is one of the metastable phases occured in this type of alloys. Unlike classic square -phase, this ’-phase is characterized by different crystalographic orientation to the matrix and shape. Standard method used for identification of aluminium alloys is scanning electron microscopy (SEM), because of its quickness and efficiency, but in case of very thin or damaged structures (as a result of metallographic process) it’s insufficient. Scanning low energy electron microscopy (SLEEM) can be appropriate for identification of mentioned precipitates due to its physical principles resulting in many advantages compared to SEM. So the most important benefits are interaction volume reduction (which leads to improvement of surface sensitivity), increase of material contrast (ability to change matrix / precipitates contrast) as well as crystalographic contrast.

The dissertation presents theoretical and experimental studies on the physical origin of the signal in photothermal microscopy of single particles. This noninvasive optical far field microscopy scheme allows the imaging and detection of single absorbing nanoparticles. Based on a heat-induced pertur- bation in the refractive index in the embedding medium of the nanoscopic absorber, a corresponding probe beam modification is measured and quantified. The method is well established and has been applied since its first demonstration in 2002 to the imaging and characterization of various absorbing particle species, such as quantum dots, single molecules and nanoparticles of different shapes. The extensive theoretical developments presented in this thesis provide the first quantitative assess- ment of the signal and at the same time enlarge its phenomenology and thereby its potential. On the basis of several approximation schemes to the Maxwell equations, which fundamentally gov- ern the interaction of light with inhomogeneities, several complementing models are devised which describe the photothermal signal both qualitatively and quantitatively. In succession an interdepen- dent and self-consistent set of theoretical descriptions is given and allows important experimental consequences to be drawn. In consequence, the photothermal signal is shown to correspond to the action of a nanoscopic (thermal) lens, represented by the spherically symmetric refractive index pro- file n(r) which accompanies the thermal expansion of the absorber’s environment. The achieved quantification allows the direct measurement of absorption cross-sections of nanoparticles. Further, a qualitatively new phenomenology of the signal is unraveled and experimentally demonstrated. The separate roles of the probing and the heating beams in photothermal microscopy is dismantled and the influence of their relative alignment shown to allow for a controlled adjustment of the effective detection volume. For the first time, both positive and negative signals are demonstrated to occur and to be the characteristic signature of the lens-like action on the probe beam. The detection of the probe beam’s modification is also shown to sensitively depend on the aperture used in the detection chan- nel, and a signal optimization is shown to be feasible. Also, a generalization of the detectable signal via the use of a quadrant photodiode is achieved. Specifically, measuring the far field beam deflec- tion the result of the beam passing the lens off-center manifests in a laterally split detection volume. Hereby, finally each classical photothermal spectroscopic techniques has been shown to possess its microscopic counterpart. Central to the understanding of this generalized and new phenomenology is a scalar wave-optical model which draws an analogy between the scattering of a massive particle wave-packet by a Coulomb potential and the deflection of a focused beam by a photonic potential connected with the thermal lens. The significance of the findings is demonstrated by its methodological implications on photother- mal correlation spectroscopy in which the diffusion dynamics of absorbing colloidal particles can be studied. The unique split focal detection volumes are shown to allow the sensitive measurement of a deterministic velocity field. Finally, the method is supplemented by a newly introduced sta- tistical analysis method which is capable of characterizing samples containing a heterogeneous size distribution.:Contents Bibliographic description Abbreviations 1 Introduction 2 Theoretical Background 2.1 The current literature on the subject of the photothermal signal 2.2 Thermal conduction, and the temperature field around heated nanoparticles 2.3 The linear thermo-refractive response and the thermal lens 2.4 MAXWELL equations and approximation schemes 2.4.1 The MAXWELL equations 2.4.2 HELMHOLTZ equations 2.4.3 Paraxial HELMHOLTZ equation for the field components 2.4.4 Geometrical optics and the eikonal ansatz 2.5 Diffraction and the optical resolution limit in far field microscopy 2.5.1 Transmission scanning microscopy 2.5.2 Point spread functions and aberrations 2.5.3 Scalar diffraction approximation for weakly focused beams 2.5.4 Vectorial diffraction for highly focused electromagnetic fields 2.5.5 Theoretical description of transmission signals 2.6 Elastic scattering of light 2.6.1 Overview of optical elastic scattering theory 2.6.2 The integral equation of potential scattering and the BORN approximation 2.6.3 The generalized LORENZ-MIE theory 2.6.4 The electromagnetic fields 2.6.5 Description of the incident field: beam shape coefficients 2.6.6 Multilayered scatterers 2.6.7 POYNTING vector and field decomposition 2.6.8 Energy balance & total cross-sections 2.6.9 Optical theorem & the extinction paradox 2.6.10 Small particle scattering: the RAYLEIGH-limit 2.7 Optical properties of gold nanoparticles & Surface plasmon resonances 2.7.1 Dielectric function of gold 2.7.2 Total cross-sections of plasmonic nanoparticles properties of gold nanoparticles & Surface plasmon resonances 2.8 (Hot) BROWNian motion, diffusion and their statistical analysis 2.8.1 (Hot) BROWNian motion 2.8.2 Diffusion and correlation analysis 2.8.3 Methods regarding the signal statistics of diffusing tracer particles 2.9 RUTHERFORD scattering of charged particles 2.9.1 Classical RUTHERFORD scattering 2.9.2 Quantum mechanical COULOMB scattering 3 Experimental Setup 3.1 Sample preparation 3.2 Photothermal microscopy setup 4 Photothermal Imaging: Results and Discussion 4.1 MAXWELL equations: Exact treatment of the PT signal 4.1.1 Angularly resolved powers: Fractional cross-sections 4.1.2 Incident power and background normalization 4.1.3 Fractional scattering and extinction cross-sections (off-axis) 4.1.4 Fractional scattering and extinction cross-sections (on-axis) 4.1.5 Small particle approximation(on-axis) 4.1.6 General properties of transmission scans 4.1.7 The thermal lens n(r) in the MIE-scattering framework 4.1.8 The photothermal signal F in the MIE scattering framework 4.2 Geometrical optics: Photonic RUTHERFORD scattering (ray optics) 4.2.1 FERMAT’s principle for a thermal lens medium 4.2.2 Gaussian beam transformation by a thermal lens 4.2.3 Experiments using weakly focused, i.e. nearly Gaussian beams 4.3 HELMHOLTZ equation: Photonic RUTHERFORD scattering (wave optics) 4.3.1 Plane-wave scattering 4.3.2 Focused beam scattering 4.3.3 Connection to the far field 4.3.4 Photothermal Rutherford scattering microscopy 4.3.5 Photothermal half-aperture measurements 4.4 Paraxial HELMHOLTZ equation: FRESNEL diffraction by a thermal lens 4.4.1 The diffraction integral and the phase mask for a thermal lens 4.4.2 The photothermal signal expressed via the image plane field 4.4.3 Experimental demonstration of the signal inversion 4.4.4 Connection to photothermal RUTHERFORD scattering 4.5 Plane-wave extinction & scattering by a thermal lens 4.5.1 The BORN approximation for the ideal and time-dependent thermal lens 4.5.2 The eikonal approximation for the ideal thermal lens and x>>1 4.5.3 Lessons to be learned from plane-wave scattering by thermal lenses 4.6 What is a lens? And is n(r) a lens? 5 Methodological Applications of the Results 5.1 Generalized photothermal correlation spectroscopy (incl. twin-PhoCS) 5.2 Photothermal signal distribution analysis (PhoSDA) 6 Summary and Outlook 6.1 Summary of the results 6.2 Outlook 7 Appendix 7.1 Material parameters 7.2 Calculation parameters 7.3 Interactive simulation scripts (Processing) 7.4 Vectorial scattering in the BORN-approximation 7.5 Details regarding the scattering framework 7.5.1 Connection between Gmn,TE,TM of Ref.1 and gmn,TE,TM in the GLMT 7.5.2 Off-axis BSCs including aberration (single interface) 7.5.3 Details on the incidence power Pinc 7.5.4 Details on the incidence power Pinc for arbitrary beams 7.5.5 Explicit expressions for the spherical field components of Es,i and Hs,i 7.5.6 Note on the time-dependence and the corresponding sign-conventions in M 7.5.7 Recurrence relation for Pn and tn 7.5.8 Gaussian beam shape coefficients: Off-axis 7.5.9 Multilayered Scatterer 7.5.10 POYNTING-vector and energy flow fields 7.5.11 Convergence 7.5.12 Further evaluations in the GLMT framework 7.5.13 Diffraction model: Comparison of angular PT signal pattern to the GLMT 7.6 Details on geometrical optics models 7.6.1 Geometrical optics: Exact solution r(f) for |bx|<1 7.6.2 Correspondences in photonic and partile RUTHERFORD scattering 7.6.3 On the difference in the definition of optical energy 7.6.4 Ray-opticsphotothermalsignal 7.6.5 Thick lens raytracing and the equivalent lens shape for a given aberration 7.7 Thermal lens around a wire of radius R 7.8 Twin-PhoCS: Graphic illustration of the CCF integrand Curriculum Vitae Publications Declaration Acknowledgements List of Tables List of Figures Bibliography

The thesis focuses on the development of instruments used for surfaces and nanostructures characterization. Individual techniques of scanning probe microscopy provide different information of the sample surface. The resolution of scanning probe microscopy, providing 3D topography information, reaches subnanometer values or even an atomic level. Therefore, the scanning probe microscopy is one of the most employed method in the field of nanotechnology. The thesis describes the details of development of two scanning probe microscopes intended for measurement under ultra high vacuum conditions. As for the first one, many changes were proposed leading to its better variability, extended functionality and increased user comfort. The second microscope is being design with the aim of its combination with other analytic techniques, especially with scanning electron microscopy. An integral part of scanning probe microscopes is a precise positioning system for navigation of the probe to the selected site. Therefore, the thesis also deals with the development of linear piezoceramic actuators used not only in the ultra high vacuum compatible microscopes but also as a general purpose nanomanipulators.

4 Abstract The present thesis focuses on testate amoebae (TA) and their relationship to their natural environment, as well as on relevant microscopic imaging methods. The bulk of the data has been published in original scientific papers and is compiled into three separate chapters (Pt I, Pt II and Pt III), each annotated by a brief introduction. (Pt I) The methods section is devoted to specialized microscopic techniques employed to broaden the scope of the ecological analyses. In particular, precise discrimination between live and dead individuals, biomass determination inside individual tests and a multi-modal visualization of the cytoplasm and organelles enhance the data. Laser scanning confocal microscopy and two-photon microscopy are the main imaging modalities employed to study TA morphology in detail. The data have implications for taxonomy and ecophysiology, including the use of TA as bioindicators of pollution. (Pt II) An actuoecological analysis focuses on the seasonal variability of TA species composition in a freshwater ecosystem, namely the Komo any ponds in Prague, during the course of the year. The species composition variation is correlated to simultaneously recorded limnological parameters such as temperature, pH, contamination by (heavy) metals (As, Cd, Mn, Ni, Fe, Pb), polycyclic aromatic...

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...

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren. / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.

This master’s thesis deals with the design of methodology for measurement of refractive index and thickness of living cells by coherence-controlled holographic microscope. The theoretical part summarises the holographic microscopy and its development at IPE FME BUT in Brno. The thesis focuses on the multimodal holographic microscope, its description, the principle, the procedure of work and data processing. Confocal microscopy is also described, which serves to compare the acquired values with the proposed methodology. The last part of the theoretical part deals with the testing of statistical hypotheses, which is needed for the processing of measured data. Experiments were designed for the verification of methodology for determination of the refractive index and cell thickness. The experimental part of the thesis deals with the sample preparation and measurement. The procedure and results of the proposed experiments and their evaluation follows.

The Coherence Controlled Holographic Microscope (CCHM) was developed at BUT Brno for a quantitative phase imaging of living cells. Nowadays it ocurres that its imaging properties are enhanced by the use of additional modules. In the present the microscope is equipped with the epifluorescence module, which allows observation of fluorescently marked living cells. This thesis is going to follow up on the development of this module and is going to extend its options by confocal imaging. The disadvantage of current multi-channel confocal microscopes is a mechanical rotation of the Nipkow discs, which causes undesired mechanical vibrations. That is why in this thesis it is replaced by Digital Micromirror Device. With its use was developed optical system of the whole confocal model, whose correct funcion was simulated in optical CAD. The experimentally verified prototype serves to test the imaging properties. On this basis is designed an application idea of the fluorescence confocal module, which will be possible to connect to the CCHM microscope.