The goal of this thesis is to work out an introduction to the chip packaging and decapsulation. Further can be found a description of a method leading to dacapsulate concrete chips. Final part is devoted to getting chip pictures using microscope and analysis of the pictures afterwards.

This diploma thesis is focused on encapsulation probiotics and co-encapsulation with some types of prebiotics. In theoretical part is aimed to probiotics, their general characteristics and application of probiotics in food industry. There are described prebiotics and their classification, there is described principles of encapsulation and encapsulation techniques. Methods, which are used for analysis of particles and encapsulation components were introduced too. The experimental part describes methods of prebiotics characterization by high performance liquid chromatography, thin layer chromatography and spectrophotometric methods. Cultivation of probiotics with prebiotics - hydrolyzed and non-hydrolyzed wad tested. Using flow cytometry cell viability was measured too. Finally probiotics and prebiotics were encapsulated, mainly by encapsulator machine. Long-term stability of particles during 6 week storage was observed. The particles were exposed to effect of artificial intestinal, gastric and bile juices.

Cílem této diplomové práce je zlepšit chování nanomanipulátoru, používaného v elektronovém mikroskopu, při vykonávaní nejmenších kroků pohybu. První částí je analýza mechanismu za účelem nalezení možných řešení problému. Dále se práce zabývá testováním řešení jako je optimalizace tuhosti předepínacích pružin mechanismu anebo změna mazání šnekového převodu mechanismu. Výsledkem práce je výběr nejvhodnějšího řešení problému na základě výsledků testů a následná modifikace nanomanipulátoru.

Tato Diplomová práce je zaměřena na odstranění šumu ze snímků získaných pomocí Transmisního elektronového mikroskopu. V práci jsou popsány principy digitalizace výsledných snímků a popis jednotlivých šumových složek, které vznikají při digitalizaci snímků. Tyto nechtěné složky ovlivňují kvalitu výsledného snímku. Proto byly vybrány filtrační metody založené na minimalizaci totální variace, jejichž principy jsou v této práci popsány. Jako referenční filtrační metoda byla vybrána filtrace pomocí Non-local means filtru. Tento filtr byl vybrán, jelikož v dnešní dobře patří mezi nejvíce využívané metody, které mají vysokou účinnost. Pro objektivní hodnocení kvality filtrací byly použity tyto hodnotící kritéria – SNR, PSNR a SSIM. V závěru této práce, jsou všechny získané výsledky zobrazeny a jsou diskutovány účinnosti jednotlivých filtrační metod.

Present doctoral thesis is focused on electron microscopy and spectroscopy investigation of novel metal-oxide anode catalyst for fuel cell application. Catalyst based on Pt- doped cerium oxide in form of thin layers prepared by simultaneous magnetron sputtering deposition on intermediate carbonaceous films grown on silicon substrate has been studied. The influence of catalyst support composition (a-C and CNx films), deposition time of CeOx layer and other deposition parameters, as deposition rate, composition of working atmosphere and Pt concentration on the morphology of Pt-CeOx layers has been investigated mainly by Transmission Electron Microscopy (TEM). The obtained results have shown that by suitable preparation conditions combination we are able to tune final morphology and composition of catalyst. Composition of carbonaceous films and Pt-CeOx layers was examined by complementary spectroscopy techniques - Energy Dispersive X-ray Spectroscopy (EDX), Electron Energy Loss Spectroscopy (EELS) and X-ray Photoelectron Spectroscopy (XPS). Such prepared porous structures of Pt-CeOx are of promising as anode catalytic material for real fuel cell application. Keywords: cerium oxide, platinum, fuel cell, magnetron sputtering, Transmission Electron Microscopy

Die vorliegende Arbeit beschreibt die Implementierung ionenstrahlanalytischer Methoden zur Charakterisierung der Probenzusammensetzung in einem Helium-Ionen-Mikroskop mit einem auf unter einen Nanometer fokussierten Ionenstrahl. Zur Bildgebung wird dieser im Mikroskop über Probenoberflächen gerastert und die lokale Ausbeute an Sekundärelektronen gemessen. Obwohl sich damit ein hoher topografischer Kontrast erzeugen lässt, lassen sich weder aus der Ausbeute noch aus der Energieverteilung der Sekundärelektronen verlässliche Aussagen zur chemischen Zusammensetzung der Probe treffen. Daher wurden in dieser Arbeit verschiedene ionenstrahlinduzierte Sekundärteilchen hinsichtlich ihrer Eignung für die Elementanalytik im Helium-Ionen-Mikroskop verglichen. Zur Evaluation standen der Informationsgehalt der Teilchen, deren Analysierbarkeit sowie deren verwertbare Ausbeute. Die Spektrometrie rückgestreuter Teilchen sowie die Sekundärionen-Massenspektrometrie wurden dabei als die geeignetsten Methoden identifiziert und im Detail untersucht. Gegenstand der Untersuchung waren physikalische Limitierungen und Nachweisgrenzen der Methoden sowie deren Eignung zum Einbau in ein Helium-Ionen-Mikroskop. Dazu wurden verschiedene Konzepte von Spektrometern evaluiert, erprobt und hinsichtlich ihrer Effizienz, Energieauflösung und Umsetzbarkeit im Mikroskop bewertet. Die Flugzeitspektrometrie durch Pulsen des primären Ionenstrahls konnte als die geeignetste Technik identifiziert werden und wurde erfolgreich in einem Helium-Ionen-Mikroskop implementiert. Der Messaufbau, die Signal- und Datenverarbeitung sowie vergleichende Simulationen werden detailliert beschrieben. Das Spektrometer wurde weiterhin ausführlich hinsichtlich Zeit-, Energie- und Massenauflösung charakterisiert. Es werden ortsaufgelöste Rückstreuspektren vorgestellt und damit erstmalig die Möglichkeit zur Ionenstrahlanalytik im Helium-Ionen-Mikroskop auf einer Größenskala von ≤ 60 nm aufgezeigt. Das Pulsen des primären Ionenstrahls erlaubt es zudem, die Technik der Sekundärionen-Massenspektrometrie anzuwenden. Diese Methode bietet Informationen zur molekularen Probenzusammensetzung und erreicht für einige Elemente niedrigere Nachweisgrenzen als die Rückstreuspektrometrie. Damit konnten erstmalig im Helium-Ionen-Mikroskop gemessene Sekundärionen-Massenspektren sowie die ortsaufgelöste Elementanalyse durch spektrometrierte Sekundärionen demonstriert werden. Die Ergebnisse dieser Arbeit sind in der Fachzeitschrift Ultramicroscopy Band 162 (2016) S. 91–97 veröffentlicht. Ab Oktober 2016 werden diese auch in Form eines Buchkapitels in dem Buch „Helium Ion Microscopy“, Springer Verlag Heidelberg zur Verfügung stehen. / The present work describes the implementation of ion beam analysis methods in a helium-ion-microscope for the determination of sample compositions with a focused ion beam of < 1 nm size. Imaging in the microscope is realized by scanning the focused ion beam over the sample surface while measuring the local secondary electron yield. Although this procedure leads to a high topographical contrast, neither the yield nor the energy distribution of the secondary electrons deliver reliable information on the chemical composition of the sample. For this purpose, in this work different ion beam induced secondary particles were compared with respect to their suitability for the analysis of the chemical composition in the helium-ion-microscope. In particular the information content of the particles, their analysability and their yield were evaluated. As a result, the spectrometry of backscattered particles and the mass spectrometry of sputtered secondary ions were identified as the most promising methods and regarded in detail. The investigation focused on physical limitations and detection limits of the methods as well as their implementability into a helium-ion-microscope. Therefor various concepts of spectrometers were evaluated, tested and validated in terms of their efficiency, energy resolution and practicability in the microscope. Time-of-flight spectrometry by pulsing the primary ion beam could be identified as the most suitable technique and has been successfully implemented in a helium-ion-microscope. The measurement setup, signal processing and data handling as well as comparative simulations are described in detail. Further the spectrometer was characterized explicitly in terms of time, energy and mass resolution. Spatially resolved backscattering spectra will be shown demonstrating the feasibility of performing ion beam analysis in a helium-ion-microscope for the first time on a size scale of ≤ 60 nm. By pulsing the primary ion beam the technique of secondary ion mass spectrometry becomes automatically accessible. This method provides information on the molecular composition of samples and can reach higher detection limits than those from backscattering spectrometry. For the first time, in a helium-ion-microscope measured secondary ion mass spectra and spatially resolved elemental analysis by spectrometry of secondary ions, could be demonstrated. The results of this work are published 2016 in the scientific journal Ultramicroscopy, volume 162 on pages 91 to 971. In October 2016 there will be another publication as a book chapter in „Helium Ion Microscopy“ (publisher: Springer Verlag Heidelberg).

The objectives of the present project were to set up, optimise and characterise a digitalholographic microscopy (DHM) laboratory set-up designed for the study of eyetissue and to implement and optimise digital data processing and noise reductionroutines. This work is part of a collaborative project aiming to provide quantitativemethods for the in vitro and in vivo characterisation of human corneal transparency.The laboratory set-up is based on a commercial laboratory microscope with zoomfunction (a “macroscope”). In continuation of previous work, we completed and optimised,and extended a software for holographic signal processing and numericalpropagation of the wavefront.To characterise the set-up and quantify its performances for standard operationand in its DHM configuration, we compare the magnification and resolution to theoreticalvalues for a given set of parameters. We determined the magnification factorand the rotation angle between the object and camera planes. With a laser wavelengthof 532 nm, a x1 objective and a zoom setting of x2.9 (which corresponds to aplane sample wavefront), we measured a magnification of 1.68. With the same parameters,we measure a holographic resolution of about 11 m. The wavefront phasecould be determined with a precision of a fraction of the wavelength.We subsequently performed analysis of the relative contribution of coherent noiseand implemented and evaluated several noise reduction routines. While the impactof coherent noise remained visible in the amplitude image, interferometric precisionwas obtained for the phase of the wavefront and the set-up was considered qualifiedfor its intended use for corneal characterisation.A first test measurement was performed on primate cornea.Subsequent work will address the further quantitative characterisation of the setupfor the full set of parameters (objectives, zoom positions, wavelengths), test measurementson samples with known transmission and light scattering properties (e.g.solutions of PMMA beads) and the comparison of the results with the predictions ofa theoretical model, and measurements on animal and human tissue.

A key component of biological research is cell culture technology, which allows researchersto examine the behavior and functionality of cells in controlled environments. Conventionalcell culture monitoring frequently necessitates taking the cultures out of their incubators tomake observations under a microscope. This exposes them to pollutants and changes in thesurrounding environment and may jeopardize the integrity of the experiment.This thesis presents the development of a cost-effective, miniaturized microscope designedfor imaging of cell cultures directly within incubators. By integrating simple, inexpensiveglass lenses and 3D-printed components and focusing on the ESP32-CAM module for digitalimaging, the project explores various optical setups to optimize image quality while minimizingdisruption to cell environments.Central to the research was the identification and testing of diverse optical configurations todetermine the most effective arrangement for both brightfield and fluorescence microscopy.The design features a baseplate for stability, a filter plate for fluorescence imaging, and afocus adjustment mechanism using magnets. Iterative enhancements led to a side illuminationtechnique using an economical LED, removing the need for a beamsplitter and simplifying theoptical path.The final microscope demonstrated successful brightfield imaging and weak fluorescenceimaging of Madin-Darby Canine Kidney (MDCK) II cell cultures marked with Green FluorescentProtein (GFP), using a magnification ratio of 2.5:1 through an infinity-corrected optical system.The findings illustrate the potential of developing an economical, functional microscope thatcan be readily replicated and scaled for use in cell culture technology. / En central del av biologisk forsking är användningen av cellkulturer, vilket tillåter forskare attutforska beeendet och funktionerna av celler i kontrollerade miljöer. Konventionell bevakning avcellkulturer kräver ofta att de tas ut från deras inkubatorer för att observeras under ett mikroskop.Detta kan utsätta dem för föroreningar och förändringar i omgivningen vilket kan äventyra helaexperimentet.Det här examensarbetet beskriver utvecklingen av ett kostnadseffektivt, miniatyriserat mikroskopanpassat för att avbilda cellkulturer inuti inkubatorer. Genom att integrera enkla, billigaglaslinser och 3D-printade komponenter, samt ESP32-CAM modulen för bildtagning, utforskardetta arbete olika optiska system för att optimera bildkvalitet och minimera störningar i cellernasmiljö.En väsentlig del av forskningen involverade identifiering och testning av olika optiska konfigurationerför att bestämma det mest effektiva arrangemanget för både brightfield- och fluorescensmikroskopi.Designen inkluderar en basplatta för stabilitet, en filterplatta för fluorescensavbildning ochen fokusjustering som utnyttjar magneter. Iterativa förbättringar ledde till utvecklingen av enbelysningsteknik med en billig LED, vilket tog bort behovet av en stråldelare och förenkladeden optiska banan.Det slutgiltiga mikroskopet uppvisade framgångsrik avbildning med brightfield och begränsadavbildning med fluorescens av MDCK II cellkulturer märkta med GFP. En förstoringsfaktor2.5:1 användes genom ett oändlighetskorrigerat optiskt system. Resultaten demonstrerar potentialenav att utveckla ett ekonomiskt och funktionellt mikroskop som kan replikeras för användninginom cellkulturforskning.

The thesis deals with modifying and biulding of scintilation detector of secondary electrons for environmental scanning electron microscopy. It describes dilemma of environmental scanning electron microscopy, types of detectors and secondary electrons detection. The experimental part of this thesis focuses on the design and construction of new scintillation detector on the basis of simulations secondary electrons trajectories. Identifying the parameters, pressure dependencies and optimizations of electrode system of the detector realized.

This thesis describes the physical nature of corrections of an electron microscope and mathematical methods of image processing required for their complete automation. The corrections include different types of focusing, astigmatism correction, electron beam centring, and image stabilisation. The mathematical methods described in this thesis include various methods of measuring focus and astigmatism, with and without using the Fourier transform, edge detection, histogram operations, and image registration, i.e. detection of spatial transformations in images. This thesis includes detailed descriptions of the mathematical methods, their evaluation using an “offline” application, descriptions of the algorithms of their implementation into an actual electron microscope and results of their testing on the actual electron microscope, in the form of a video footage grabbed from its control computer’s screen.