The aim of this thesis is to investigate the methodology of three dimensional chemical imaging of nanoparticles through the use of scanning transmission electron microscope (STEM) – energy dispersive X-ray (EDX) spectroscopy. In this thesis, an absorption correction factor is derived for spherical nanoparticles that can correct X-ray absorption effects. Quantification of EDX spectra of nanoparticles usually neglects X-ray absorption within the nanoparticle but may lead to erroneous results, thus an absorption correction is important for accurate compositional quantification. The absorption correction presented is verified through comparison with experimental data of Au X-ray peaks in spherical Au nanoparticles and is found to agree excellently. This absorption correction allows accurate compositional quantification of large ( > 100 nm) particles with STEM-EDX.Three dimensional chemical mapping is achievable through the use of EDX spectroscopy with electron tomography. Here, the methodology of STEM-EDX tomography is fully explored, with a focus on how to avoid artefacts introduced through detector shadowing and low counts per pixel. A varied-time acquisition scheme is proposed to correct for detector shadowing that is shown to provide a more constant intensity over a series of projections, allowing a higher fidelity reconstruction. The STEM-EDX tomography methodology presented is applied to the study of AgAu nanoparticles synthesized by the galvanic replacement reaction. The elemental distribution as a function of the composition of the as-synthesized nanoparticles is characterised and a reversal in the element segregated to the surface of the nanoparticles is found. The composition at which the reversal takes place is shown to correlate with a peak in the catalytic yield of a three component coupling reaction. It is hypothesized that a continuous Au surface results in the optimum catalytic conditions for the reaction studied, which guides the use of galvanically prepared AgAu nanoparticles as catalysts.

Ce manuscrit décrit les travaux menés pour le développement et l'utilisation du microscope électrochimique (SECM) pour l'étude des surfaces fonctionnelles. Cette microscopie à sonde locale, utilisant les propriétés des ultramicroélectrodes (UMEs), permet l'analyse de la réactivité chimique à la surface d'un échantillon. Le but de ces travaux est alors l'analyse de surfaces fonctionnalisées par une couche organique. Dans un premier temps, nous avons développé la méthodologie du SECM par l'utilisation de sonde redox spécifique. D'abord, cela nous a permis de déterminer la distance entre l'électrode-sonde et la surface grâce à un médiateur redox irréversible comme les polyaromatiques. Ensuite, nous avons pu observer des propriétés physico-chimiques d'une surface, notamment afin de différencier les différents modes de transport électronique au travers une couche organique, grâce à l'utilisation des catéchols ou des dendrimères redox. Nous avons également commencé le développement d'un nouveau mode d'utilisation du SECM : le SECM transitoire. Dans un deuxième temps, nous avons utilisé le SECM afin d'analyser trois surfaces fonctionnalisées. La première surface est un substrat d'or fonctionnalisé par des calixarènes. La deuxième est une surface de verre sur laquelle il a été déposée des nanoparticules photosensibles, appelées « quantum rods ». Et la dernière est un substrat de silicium modifié par un récepteur anthracène. Finalement, la dernière étude qui a été menée concerne l'écriture et la lecture d'une monocouche organique grâce à la sonde du SECM. Ces modifications localisées de la couche organique sont alors de la taille de l'UME, soit de l'ordre du micromètre. Dans notre cas nous avons choisi la modification de monocouche organique auto-assemblée (SAM) sur un substrat d'or grâce à la réaction de couplage de Huisgen. En plus, de l'amélioration de nos conditions de fonctionnalisation, nous avons pu mettre en évidence un phénomène de propagation catalytique en surface grâce à un complexe spécial de cuivre (II) contenant une fonction éthynyle. / This manuscript describes the work done for the development and use of electrochemical microscope (SECM) for the investigations of functional surfaces. This local probe microscopy, using the properties of ultramicroelectrodes (UMEs), permits the analysis of chemical reactivity on the sample’s surface. The aim of this work is then analyzing surfaces functionalized with an organic layer. Initially, we have developed the SECM methodology by using specific redox probes. First, it allowed us to determine the absolute distance between the electrode and the surface, thanks to an irreversible redox mediator such as polyaromatic. Then, we could observe the physicochemical surface properties, in particular to differentiate the electronic transfer modes through an organic layer, by using catechols or redox dendrimers. We have also started the development of a new mode of SECM: transient SECM. Secondly, we used the SECM to analyze three functionalized surfaces. The first surface is a gold substrate functionalized by calixarenes. The second is a glass side on which it was deposed photosensitive nanoparticles, called "quantum rods" and the last sample is a substrate of silicon modified by anthracene receptors. Finally, the last study concerns the writing and reading on an organic monolayer with the SECM probe. These localized changes of the organic layer are about the size of the UME, of the order of a micrometer. In our case, we chose the modification of self-assembled organic monolayer (SAM) on a gold substrate by the Huisgen coupling reaction. In addition to the improvement of our conditions of functionalization, we were able to demonstrate a phenomenon of surface catalytic propagation due to a special complex of copper (II) containing ethynyl functions.

Magister Chirurgiae Dentium (MChD)

abstract: The cell is a dense environment composes of proteins, nucleic acids, as well as other small molecules, which are constantly bombarding each other and interacting. These interactions and the diffusive motions are driven by internal thermal fluctuations. Upon collision, molecules can interact and form complexes. It is of interest to learn kinetic parameters such as reaction rates of one molecule converting to different species or two molecules colliding and form a new species as well as to learn diffusion coefficients. Several experimental measurements can probe diffusion coefficients at the single-molecule and bulk level. The target of this thesis is on single-molecule methods, which can assess diffusion coefficients at the individual molecular level. For instance, super resolution methods like stochastic optical reconstruction microscopy (STORM) and photo activated localization microscopy (PALM), have a high spatial resolution with the cost of lower temporal resolution. Also, there is a different group of methods, such as MINFLUX, multi-detector tracking, which can track a single molecule with high spatio-temporal resolution. The problem with these methods is that they are only applicable to very diluted samples since they need to ensure existence of a single molecule in the region of interest (ROI). In this thesis, the goal is to have the best of both worlds by achieving high spatio-temporal resolutions without being limited to a few molecules. To do so, one needs to refocus on fluorescence correlation spectroscopy (FCS) as a method that applies to both in vivo and in vitro systems with a high temporal resolution and relies on multiple molecules traversing a confocal volume for an extended period of time. The difficulty here is that the interpretation of the signal leads to different estimates for the kinetic parameters such as diffusion coefficients based on a different number of molecules we consider in the model. It is for this reason that the focus of this thesis is now on using Bayesian nonparametrics (BNPs) as a way to solve this model selection problem and extract kinetic parameters such as diffusion coefficients at the single-molecule level from a few photons, and thus with the highest temporal resolution as possible. / Dissertation/Thesis / Source code related to chapter 3 / Source code related to chapter 4 / Doctoral Dissertation Physics 2020

The Diploma thesis deals with object surface reconstruction by means of confocal microscope. It includes part of mathematical theory which is associated with this problem. The mathematical algorithm of the object surface reconstruction is illustrated. This Diploma thesis result is the application development for this reconstruction. The thesis also include the results of specific object.

This diploma thesis deals with the possibilities of stress resistance analysis of the Cupriavidus necator H16 and PHB-4 bacterial cells by spectroscopic methods and by testing the suitability of acridine orange as a viable dye. Based on research in literature, suitable analytical methods have been proposed, namely flow cytometer and fluorescence microscope. The first part of the experimental work was focused on the fluorescence microscope, which confirmed the basic character of acridine orange. Three stress factors, 50% and 70% ethanol, and acidic pH (pH = 1) were selected for viability monitoring. The bacteria fluoresced with green color after exposure to ethanol and red spots were found next to the cells, indicating their loss of integrity. In an acidic environment, the bacteria fluoresced red because of a partial DNA breakdown. The results were verified by the combination of propidium iodide with SYTO9 and the acridine orange suitability proved to be useful in this method. Image records were processed using image analysis. In the second part, acridine orange was used to monitor fluorescence using a flow cytometer. The result of the measurement was fluorescence expressed as histograms for individual channels, where fluorescence was characterized by median and mean intensity. By comparing the methods used, the acridine orange appears to be a more suitable fluorescent dye for the microscope than for a flow cytometer in which it was more difficult to obtain cell viability information. In the last part of the experimental work interesting photophysical properties of acridine orange were investigated.

The aim of the diploma thesis is to clarify the topic of calibration samples used in eddy current testing on equipment in nuclear energy. The theoretical part of the diploma thesis is focused on the eddy current testing method, which belongs to the technology of non-destructive defectoscopy. This part of the work describes the principle of the method, its scope, limitations and dividing of sensors used in this test method. The work briefly describes the physical principle of the laser, its types and operating modes. The practical part of the work is focused on the production of a calibration samples by laser. Another point of the work is the comparison of data obtained from eddy current measurements on samples made by laser and on calibration samples used in practice.

In the Laboratory of the Experimental Biophotonics at Brno University of Technology, a new generation of the Coherence-Controlled Holographic Microscope (CCHM) is being developed. Because of the automation of certain microscope procedures, a fully motorized three axis computer controlled manipulator of the sample is to be employed. A fast and accurate lateral positioning in a range of several centimeters is required. For the focusing capability, very precise positioning in the optical axes is required as well. Initially, the basics of microscope manipulator design are presented. Then, commercially available solutions are introduced and compared with special focus on its possible implementation in the new generation of CCHM. However, due to the specific design of the new CCHM, available manipulators are not suitable. Therefore, a new optimized manipulator design is introduced in the second part of the thesis. A selection of its appropriate elements, especially actuators, is based on a review. For the new manipulator design, a technical documentation was made and a testing software was developed. A crucial part of the manipulator was assembled and tested.

The main aim of this thesis is to design a communication and control card for an electron microscope, eventually for other equipments of the company Delong Instruments a.s., which is dealing with its development and production. This card should replace existing communication card because of its low computational performance. Also, it should replace control and measure card manned with A/D and D/A converters. Thus, the new card will be providing not only the communication with a superior PC and other electronic systems, but also the control of other subsystems of the electron microscope, the determining of processional states and the measuring of internal physical quantities. At the beginning the requirements were determined and the main concept was made with the inclusion of the simple block diagram. It was very important to choose a suitable microcontroller, which will be an intelligence of the whole card and so it will be the most important component. The accent was set mainly on the sufficient computational performance, high modularity of its periphery, good vision of the future innovation and support and, of course, the price. The next requirement was the implementation of the Ethernet, which will be used for the communication between the card and the superior PC. The microcontroller which was chosen have the core architecture ARM Cortex-M3 and it is described in separate chapter. The next part of this thesis is dealing with the main communication standard of the card, which is the Ethernet. After the general description, the Ethernet was mainly discussed in context of the chosen microcontroller. The possibilities of the application of higher layer TCP/IP protocols were also discussed. Implied part of the thesis was the selection of other important components, especially A/D and D/A converters. Its characteristics will have a big effect on the characteristics of the card. In the last and the most important phase the electrical scheme was designed and it was described in detail. This scheme is the main result of this thesis and it is the main document for the future realization. Designed card will be able to process and control 16 differential analogue input signals, 16 differential analogue output signals, 8 digital inputs, 8 simple digital outputs and 4 digital outputs triggered by optocouplers. It will be able to communicate with the superior PC over the Ethernet with the maximum bit rate of 100 Mbit/s, with other electronic cards over the serial line UART through the optical fibers and with other internal and external equipments over the RS-485 and RS-232.

The aim of the paper is to introduce an optic microscope as well as microscopic photography producing. It further engages in sharpening issues and attaining a sharp image with extremely robust depth acuteness assuming using a series of images with different acuteness levels. The goal is to create a suitable algorithm to increase depth acuteness, to perform a visual processing of the series of images and to get one final image with high depth acuteness.