This thesis concentrates on the methodology of semiconductor samples preparation for low voltage scanning electron microscopy. In the first part a detailed theory of sample imaging using electron beam and difference between classical scanning electron microscopy (SEM) and low voltage scanning electron microscopy (LVSEM) is described. It is given a description of a contrast formation in SEM and LVSEM and theories describing a contrast formation of differently doped semiconductors. The second part contains experimental data. The advantages and disadvantages of cleavage and focused ion beam (FIB) milling as sample preparation techniques are discussed. FIB was found as the best method for sample preparation for the analysis of precisely defined location on the sample. It is necessary to use the lowest possible FIB accelerating voltage for final polishing, ideally 1 kV.

Dizertační práce je obecně zaměřena na problematiku mikroskopie atomárních sil (AFM), a to jak vývoje částí těchto mikroskopů, tak i jejich obecnému využití v oblasti výzkumu povrchů, ultratenkých vrstev a nanostruktur. Na Ústavu fyzikálního inženýrství jsou vyvíjena zařízení umožňující aplikovat uvedenou mikroskopickou metodu. V těchto mikroskopech jsou využívány piezoelektrické motory pro zajištění pohybu vzorku a ladicích zrcátek v optickém detekčním systému. Práce se v části věnované vývoji AFM zabývá studiem parametrů řídicích pulzů za účelem optimalizace funkce těchto komponent. Měřením vlivu tvaru pulzů a opakovací frekvence byl jejich pohyb optimalizován z hlediska stability a rychlosti posuvu. V části věnované výzkumu povrchů byly experimentálně zkoumány morfologické změny ultratenkých vrstev zlata na povrchu oxidu křemičitého za zvýšených teplot. Bylo zjištěno, že vhodná povrchová modifikace způsobuje vznik preferenčních trhlin ve vrstvě zlata. Řízeným rozdělením polykrystalické vrstvy na oddělené oblasti je možné významně ovlivnit proces tvorby ostrůvků zlata vznikajících při žíhání. S využitím metod elektronové litografie je možná příprava uspořádaných polí zlatých ostrůvků o velikostech 50 – 400 nm. Dále bylo ukázáno, že zvýšením teploty žíhání na 1000 °C dochází k postupnému zanořování ostrůvků zlata do povrchu. Tento jev je pravděpodobně způsoben přesunem oxidu křemičitého z oblasti pod zlatým ostrůvkem do těsného okolí vzniklého kráteru, kde tvoří tzv. límec. V těchto studiích vedle metody AFM byla s výhodou používána rovněž elektronová mikroskopie (SEM).

Fluorescence microscopy is an indispensable tool for biology to study the spatio-temporal dynamics of cells, tissues, and developing organisms. Modern imaging modalities, such as light-sheet microscopy, are able to acquire large three- dimensional volumes with high spatio-temporal resolution for many hours or days, thereby routinely generating Terabytes of image data in a single experiment. The quality of these images, however, is limited by the optics of the microscope, the signal-to-noise ratio of acquisitions, the photo-toxic effects of illumination, and the distortion of light by the sample. Additionally, the serial operation mode of most microscopy experiments, where large data sets are first acquired and only afterwards inspected and analyzed, excludes the possibility to optimize image quality during acquisition by automatically adapting the microscope parameters. These limits make certain observations difficult or impossible, forcing trade-offs between imaging speed, spatial resolution, light exposure, and imaging depth. This thesis is concerned with addressing several of these challenges with computational methods. First, I present methods for visualizing and processing the volumetric data from a microscope in real-time, i.e. at the acquisition rate of typical experiments, which is a prerequisite for the development of adaptive microscopes. I propose a low-discrepancy sampling strategy that enables the seamless display of large data sets during acquisition, investigate real-time compatible denoising, convolution, and deconvolution methods, and introduce a low-rank decomposition strategy for common deblurring tasks. Secondly, I propose a computational tractable method to simulate the interaction of light with realistically large biological tissues by combining a GPU-accelerated beam propagation method with a novel multiplexing scheme. I demonstrate that this approach enables to rigorously simulate the wave-optical image formation in light-sheet microscopes, to numerically investigate correlative effects in scattering tissues, and to elucidate the optical properties of the inverted mouse retina. Finally, I propose a data-driven restoration approach for fluorescence microscopy images based on convolutional neural networks (Care) that leverages sample and imaging specific prior knowledge. By demonstrating the superiority of this approach when compared to classical methods on a variety of problems, ranging from restoration of high quality images from low signal-to-noise-ratio acquisitions, to projection of noisy developing surface, isotropic recovery from anisotropic volumes, and to the recovery of diffraction-limited structures from widefield images alone, I show that Care is a flexible and general method to solve fundamental restoration problems in fluorescence microscopy.

This dissertation, entitled »Control of the morphology of biodegradable polymer blends«, has been running parallel with the grant project »Multiphase biodegradable polymer systems« and it represents a new research direction in the Department of morphology and rheology of polymer materials at the Institute of Macromolecular Chemistry. The main idea was to employ our long-lasting work and experience in the field of morphology control of synthetic polymer blends in the very analogous field of the biodegradable polymer blends. We have chosen three most common, widely used and relatively cheap bio-based polymers - starch, poly(lactic acid) and poly(ε-caprolactone) - in order to investigate how the properties of their blends might be improved if we control the blend morphology in targeted, reproducible and well-defined way from the very beginning. It has been well established that morphology (phase structure, supramolecular structure) is one of the key factors influencing final properties of polymer blends, including mechanical performance, rate of (bio)degradation, gas permeability etc. In this work, numerous preliminary experiments showed that there are two systems in which the morphology control could significantly help in the improving of their end-use properties: PLA/PCL/TiX (where PLA = poly(lactic acid),...

Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...

Many freshwater, terrestrial or marine microalgae contain various microcrystalline inclusions that they use in their life cycle. However, the identification of the molecular composition of these inclusions via many physical or chemical methods is often very difficult and susceptible to many measurement errors. Therefore, the chemical composition of these microcrystals in many microalgae has not been determined at all or may be incorrect. One of the high precision methods capable of determining the composition of microcrystalline bodies within microalgae is Raman confocal microscopy. This very promising method of optical vibrational spectroscopy allows rapid and non-destructive molecular analysis of objects. Raman microscopy does not require chemical extraction, modification or other color marking or staining of the sample. Therefore, it can directly measure living cells at various stages of their natural development. The chemical composition of the sample is then characterized by its corresponding Raman vibrational spectrum. The aim of this diploma thesis is to determine the presence of microcrystals in different species of microalgae, study the conditions of their occurrence and identify their chemical composition via Raman microscopy.

The aim of this thesis is to investigate applicability of regulariza- tion by Krylov subspace methods to discrete inverse problems arising in single particle analysis (SPA). We start with a smooth model formulation and describe its discretization, yielding an ill-posed inverse problem Ax ≈ b, where A is a lin- ear operator and b represents the measured noisy data. We provide theoretical background and overview of selected methods for the solution of general linear inverse problems. Then we focus on specific properties of inverse problems from SPA, and provide experimental analysis based on synthetically generated SPA datasets (experiments are performed in the Matlab enviroment). Turning to the solution of our inverse problem, we investigate in particular an approach based on iterative Hybrid LSQR with inner Tikhonov regularization. A reliable stopping criterion for the iterative part as well as parameter-choice method for the inner regularization are discussed. Providing a complete implementation of the proposed solver (in Matlab and in C++), its performance is evaluated on various SPA model datasets, considering high levels of noise and realistic distri- bution of orientations of scanning angles. Comparison to other regularization methods, including the ART method traditionally used in SPA,...

Title: Investigation of spin structure and dynamics in magnetically ordered thin films Author: Tomáš Janda Abstract: This thesis is devoted to the development of methods for imaging and ultrafast manipulation of magnetic textures, such as magnetic domains and domain walls (DWs), and to the investigation of the corresponding magnetiza- tion dynamics. We focused on techniques that are, besides ferromagnets (FMs), applicable also to antiferromagnets (AFs), in particular, AF thin films. The employed excitation techniques were mostly based on direct or indirect effects of ultrashort laser pulses. We studied the DW motion induced by a transfer of angular momentum from circularly polarized femtosecond laser pulses in a FM GaMnAsP and we found that the observed macroscopic DW displacement is only possible due to its inertia. In a distinct experiment, picosecond current pulses were generated by an absorption of the ultrashort laser pulses in a verti- cal PIN diode-based photoconductive switch and used to excite a magnetic film deposited directly on top of the diode. For imaging of AF domain patterns, we developed a simple table-top laser-scanning technique, which is based on a magneto-thermoelectric response of the AF textures in the presence of a local laser-induced heating. We first used the method to image the...

Material und Methoden: Insgesamt 40 Proben glatter und rauer Oberflächen von Titanimplantaten wurden mit Parodontalsonden aus Metall oder Kunststoff in einem Winkel von 20° oder 60° bearbeitet. Zur Bestimmung verschiedener standardisierter 2D- und 3D-Rauheitsparameter wurden die Titanoberflächen mit einem konfokalen Laser-Scanning-Mikroskop (CLSM) vor und nach der Durchführung evaluiert. Ergebnisse: Die durchschnittliche Profil- und Oberflächenrauheit (Ra und Sa) zeigten unabhängig vom Sondenmaterial keinen signifikanten Unterschied zwischen behandelten und unbehandelten Proben auf glatten und rauen Oberflächen. Auf glatten Oberflächen führten Metallsonden zu erhöhten Amplitudenrauheiten wobei nur für Rp eine Signifikanz (p = 0,007) erreicht wurde. Raue Oberflächenbereiche wurden nach dem Kontakt mit Metallsonden leicht aber nicht signifikant geglättet. Nach Verwendung von Kunststoffsonden blieb die Oberflächenrauhigkeit von glatten und rauen Bereichen nahezu unverändert. Unabhängig des Sondenmaterials zeigte sich kein Zusammenhang zum Applikationswinkel. Fazit: Das Sondieren von Titanimplantaten mit Kunststoffsonden aber auch mit Metallsonden verursacht nur geringe Veränderungen der Oberflächenrauhigkeit. Die klinische Bedeutung dieser Veränderung bleibt ungeklärt. Klinische Relevanz: Ultrastrukturelle Veränderungen an Titan-Oberflächen durch periimplantäre Untersuchungen könnten durch die Verwendung von Kunststoffsonden vermieden werden.

Background: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. Methods: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. Results: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. Conclusion: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders. / Hintergrund: Die Vitalitätsbestimmung von Blutplättchen ist sowohl für die Analyse angeborener Plättchendefekte als auch für die Qualitätsbestimmung von Plättchenkonzentraten von zentraler Bedeutung. Methoden: In der vorliegenden Arbeit stellen wir eine Methode vor, die mittels einer Kombination von Vitalfarbstoffen und konfokaler «Real time»-Mikroskopie neue Einblicke in die Vitalitätsbestimmung lebender Plättchen ermöglicht. Mittels der Zugabe von FITC-gekoppeltem Weizenkeimlektin (WGA), Tetramethylrhodamin-Methylesterperchlorat (TMRM) und Acetoxymethylester (Rhod-2) wurde bei lebenden Blutplättchen deren Morphologie, mitochondriale Aktivität und Veränderungen im Calcium-Haushalt im Rahmen der Lagerung analysiert. Für die Mikroskopie wurde ein Nipkow-System gewählt, das eine konfokale Mikroskopie lebender Zellen ermöglicht. Ergebnisse: Der Vergleich von 10 humanen Blutplättchenproben zu Beginn bzw. nach 5 und 7 Tagen Lagerung zeigte einen Anstieg der Rhod-2-positiven Plättchen von 3,6 über 47 auf 71%. Die Anzahl der Blutplättchen mit TMRM-positiven Mitochondrien hingegen lag vor der Lagerung bei 95,4% und nach den 7 Tagen Lagerung bei 92,5%. Schlussfolgerung: Die hier vorgestellte Methodik der Bildgebung zur Bestimmung vitaler Parameter von Blutplättchen eignet sich als ergänzende Analysemodalität für eine bessere Bestimmung der Blutplättchenqualität. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.