This diploma thesis describes the design of the illumination system for a Coherence--Controlled Holographic Microscope (CCHM). The theoretical part mentions the history of microscopy, the principle of holography and individual types of interference microscopy. To get closer to the topic, individual light sources and an overview of current illumination systems are mentioned. The diploma thesis also describes the procedure of designing a fiber-optic illumination module for microscopy from optical design through design of construction to the last step, which is assembly and testing of the module.

Magnetické materiály a z nich vyrobené nanostruktury jsou v průběhu posledních let studovány pro jejich aplikace v např. záznamových médiích a logických obvodech. Tato práce navazuje na náš předchozí výzkum tohoto oboru s hlavním zaměřením na statické a dynamické vlastnosti nanostrukturovaných magnetických materiálů, jako například NiFe, CoFeB a YIG. Práce začíná teoretickým úvodem s popisem mikromagnetických systémů, dynamiky magnetických vortexů, feromagnetické rezonance (FMR) a spinových vln včetně jejich disperzních vlastností. Následuje popis použitých experimentálních metod a první experimentální část zabývající se nukleačním procesem magnetického vortexu, jinými slovy procesem transformace ze saturovaného stavu do spinové konfigurace magnetického vortexu v průběhu snižování magnetického pole. Jsou použity mikroskopické metody zobrazující magnetickou strukturu materiálu, jmenovitě Lorentzova transmisní elektronová mikroskopie a rentgenová transmisní mikroskopie. Výsledky jsou poté korelovány s měřením elektrické odezvy pomocí jevu anizotropní magnetorezistence. Výhodou elektrických měření je, že plně elektrická detekce dovoluje použití tohoto systému v uzavřených systémech integrovaných obvodů. Výsledky oblasti nukleací magnetických vortexů ukazují, že při tomto procesu prochází magnetizace v nano- a mikrometrových magnetických discích několika fázemi s růsnými typy spinových konfigurací nazvaných nukleační stavy. Dále je představeno měření magnetických materiálů pomocí vektorového síťového analyzátoru (VNA), což je aplikováno na měření resonance magnetických vortexů (určení gyrotropické frekvence a měření vysokofrekvenčních módů), feromagnetické rezonance tenkých vrstev (získání základních magnetických materiálových parametrů) a spektroskopii spinových vln. Právě spektroskopie spinových vlna je rozvinuta za účelem měření disperzních relací tenkých magnetických vrstev, což je základní charakteristika, jejíž znalost je důležitá v návrhu aplikací. Nakonec je představeno anténní zařízení, díky kterému lze oddělit magnetické buzení od vzorku samotného bez nutnosti absolvovat proces elektronové litorafie, což je zapotřebí v klasickém přístupu antény na vzorku a kontaktování vysokofrekvenční sondou. Toto zařízení se skládá ze skleněného kantilívru, na kterém je vyrobena budící anténa, konektoru a spojovacího prvku v podobě plošného spoje. Celé zařízení je díky umístění na x-y-z stolek s náklonem pozicovatelné a lze tedy měřit v jakémkoliv místě vzorku. Umístění antény na sklo umožňuje navigaci pomocí mikroskopu a optické měření, např. metodou Brillouinova světelného rozptylu (BLS) nebo Kerrova jevu.

Tato Diplomová práce se zabývá povrchovou analýzou nanokompozitní folie polyetherimidu (PEI) vyztuženého exfoliovanými grafitickými nanodestičkami (xGnP). Analyzovány byly take vzorky nevyztužené PEI folie a samostatné nanodestičky. Vzorky nanokompozitu a PEI folie byly plazmaticky leptány s využitím argonového plazmatu po dobu 1, 3 a 10 hod. Skenovací elektronová mikroskopie (SEM) byla použita pro charakterizaci samostatných nanodestiček rozptýlených na křemíkovém substrátu, původních či leptaných vzorků PEI folie a nanokompozitu. Nanodestičky byly identifikovány při povrchu leptané nanokompozitní folie. Mikroskopie atomárních sil (AFM) byla použita pro zobrazení povrchové topografie separovaných nanodestiček a odkrytých destiček při povrchu leptaného kompozitu. Povrchová drsnost (střední kvadratická hodnota, vzdálenost nejnižšího a nejvyššího bodu) leptaného nanokompozitu narůstala s prodlužující se dobou leptání. Akustická mikroskopie atomárních sil (AFAM) byla použita pro charakterizaci elastické anizotropie leptaných kompozitních vzorků. Nanoindentační měření umožnila charakterizaci lokálních mechanických vlastností PEI a nanokompozitních folií.

The theoretical part of the diploma thesis deals with the basic principles of the scanning electron microscope and the environmental scanning electron microscope. A description of the signals generated by an electrons incidence onto a specimen and detection options of these signals in the mentioned microscopes is included in the work. The experimental part of the work concerns an examination of a quality of several joints soldered by different methods on the printed circuit boards after different stresses by use of a visual check and the instruments of the light microscope and the environmental scanning electron microscope. The acquired data are evaluated and summarized.

This graduation theses in introduction deals with principle and problems of electron signal detection in scanning electron microscopy and charging of insulating specimens in SEM. The experimental part of the thesis describe the methods of qualification and quantification of insulating specimen charging effect observed in environmental scanning electron microscope through the use of ionisation and scintillation detector in dependence on water vapour pressure in specimen chamber. The goal of the thesis is formation of the methodics useful to evaluate charging effect at insulating specimens observing and on the basis of measuring, determine optimal conditions for insulating specimen observation with ionisation and scintillation detector.

This diploma thesis deals with the utilization of physico-chemical analysis in study on microbial stress–response. The main content of this work was to propose and optimize techniques and methods which are generally used in different industries. Two bacterial strains, Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4 producing polyhydroxyalkanoates (PHA) under certain conditions, were used as model microorganisms. Initially, microscopic teques such as transmission electron microscopy (TEM), cryo scanning electron microscopy (cryo SEM) and atomic force microscopy (AFM) were proposed and tested for the characteriozation of morphological differences of bacteria. Furthermore, viscoelastic properties of bacteria were determined and compared by the oscillatory tests of the rheology method considering another type of samples. Thermal analysis methods, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) in particular, focused on water transport and behavior of intracellular water influenced by presence of the granules of PHB in bacteria. The last technique, utilized in the study, was dynamic and electrophoretic light dispersion during pH changes for characterization of the surface properties of bacteria such as size, zeta potential and the isoelectric point.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Karolína Růžičková Supervisor: PharmDr. Barbora Švecová, Ph.D. Consultant: Mgr. Anna Nováčková Title of thesis: Evaluation of preparation of monolayer lipid skin barrier models Skin, the protective barrier of human body, consists of several layers. The uppermost one is the stratum corneum, part of epidermis, whose extracellular matrix is composed mainly of ceramides, cholesterol and free fatty acids. The composition and arrangement of skin lipids are essential for the proper skin barrier function. Various multilayer and monolayer models are used to study skin lipids at the molecular level. Some of the evaluation methods are Langmuir monolayers at the air interface. In this work I dealt with the behavior of monolayer lipid models at four different pH values of the liquid subphase. Lipids isolated from human skin, lipid mixture prepared from the individual components, and a mixture of fatty acids were compared as well. Langmuir isotherms and the Brewster angle microscopy at different compression rates were used for this purpose. The results showed that pH of the subphase has no major effect on lipids arrangement. Lipids were most likely to form a tight monolayer at neutral pH 7,0, at a...

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.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127 / 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.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127

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...

13551398399831-171f83a418df200fec6f622f22b6fb3d.txt The Thesis describes new methods for automatic processing of image data in biology, physics and medicine. The developed methods reconstruct light microscopy images of growing microorganisms in intervals between observations, measure particles in atomic force microscopy images, and evaluate parameters of vocal fold vibrations in videokymographic images. All three problems have been hitherto solved primarily visually. The proposed methods allow automatic or computer-aided processing of the image data, and thus facilitate the evaluation process. Performance of the developed methods was tested on real images; the results were comparable with ground truth or results of visual evaluation. Application of the developed methods is not limited to the specific type of image data; the methods can be used in general for processing of images with similar characteristics. Page 1