Nowadays, electron microscopy is a widespread method used in many biological branches like medicine, physical science, mikrobiology or material technology. Scanning electron microscope is a type of an electron microscope that shows the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The signal of electrons reveals information about the sample such as morphology, chemical composition and structure and materials. Gold nanoparticles are widely used as a marker for immunolabeling in these days. The main point of this research was to find other nanoparticles, which can be used for multiple immunolabeling. The main task of this study is to conjugate these particles with atibodies, to use them for immunolocalisation on the convenient biological sample and to observe it in FESEM JEOL JSM-7401F.

This work deal with problems of investigation materials electron beam. This project is focuses on investigation materials by the help of method environmental scan electron microscopy (ESEM) and describes her patterns. The perfection of ESEM excel above all at studies non - conducting or water containing, often biological samples. These samples not to be in no way prepared or cut – and – dries, in contradistinction to classical scanning electron microscope and thanks is under investigation their native surface structure without desiccation breaking. The general aim is determination of contrast in image acquired by ionization detector.

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).

Among students, interest in Science is decreasing, despite the fact that knowledge of Science can be useful in many real life situations, such as medicine, conservation etc. Science education can also simply help to form a positive attitude towards nature. There are many possibilities of increasing students' interest in Science that could increase their motivation to study. Apart from Inquiry Based Education or Prescribed Practical Activities, working with devices used in research practice is one of the possibilities. If a school doesn't own such devices, there is a possibility to borrow them, or to work with them via the Internet. Possibilities of remote access to these devices are subject to research abroad, and this trend is also starting to appear in the Czech Republic. One example of a device that can be borrowed by schools is a fluorescence microscope at the Department of Didactics and Education of Biology, Faculty of Science, Charles University in Prague. In my thesis, I was investigating the influence of the presence or absence of this fluorescence microscope on the knowledge of students. Research was carried out in four different classes at two secondary schools in Prague. Students who took part in this research were from the first year of a four year secondary school or from the third...

This thesis deals with the topic of 3D image processing for digital holographic microscopy - numerical refocusing. This method allows to perform mathematically accurate defocus correction on image of a sample captured away from the sample plane and it was applicable only for images that were made made using coherent illumination source. It has been generalized to a form in which it is also applicable to devices that use incoherent (non-monochromatic or extended) illumination sources. Another presented achievement concerns hologram processing. The advanced hologram processing method enables obtaining more data mainly concerning precision of quantities from one hologram — normally, one would have to capture multiple holograms to get those. Both methods have been verified experimentally.

This diploma thesis deals with common parasites of game animals, animals and human occurring in the territory of the Czech Republic. It focuses on their fixation and general preparation of such specimens for usage in teaching of Science. The first part is devoted to the phenomenon of parasitism, and the history and definition of this concept. Systematic hierarchy of various unrelated parasitic groups is clarified. The second part provides the list of parasites, from the evolutionary original to the highly derived groups. We give an overview of their morphology and anatomy with the emphasis on their life cycle. The detailed knowledge of life cycle is essential for obtaining specimens. In each group, we indicate methods of collection and fixation in detail. The co called smartmicroscope represent the main point of the third part of proposed diploma thesis. Text of the thesis is supplemented with pictures taken by smart microscope, as well as by common microscope. KEY WORDS parasite, parasitism, mikropredatore, vector, fixation, preparation, smart mikroscope

Mobility of pigment-protein complexes (phycobilizomes and photosystem II playing a key role in photosynthesis) was studied by FRAP method (Fluorescence Recovery After Photobleaching). FRAP represents a fluorescence based microscopy method enabling measurement of protein mobility in living systems. The protein complexes are bleached by a laser pulse. And mobility of unbleached proteins is measured as a fluorescence recovery in the bleached area. Currently we have only limited knowledge about the mobility of photosynthetic proteins. This work was aimed at optimization of the photosynthetic protein mobility measurement by FRAP. I have performed several methodological experiments which led to the successful assessment of phycobilisome and chlorophyll-containing proteins diffusion coefficients in selected red algae (Porfyridium cruentum, Cyanidium caldarium) and cyanobacteria (Synechocystis PCC6803, Acaryochloris marina). The methodology developed and validated in my thesis was then applied in further research projects.

The main aim of my thesis was to come up with a manual for microscopic practices for elementary school's biology classes that are useful and easy to understand. The practices were drawn from ordinary textbooks (such as Fortuna, Fraus, Jinan, Natura, Nová Škola, Prodos, Scientia, SPN) that are often used for teaching pupils from 6th to 9th grade of elementary school. I also added interesting facts and practices which were drawn from the internet. I also focused on the pupil's attitude to microscoping using special questionnaires. Pupils wrote that their relationship with microscoping is positive and it helps them to understand the biology curriculum more. Furthermore, I gained information about the frequency that they practice microscoping at school. Although there are not enough biology lessons overall, I find out that most teachers usually manage to practice microscoping in class once every two weeks. In the theoretical part of the thesis, I focused on the structure of the optical microscope, which is used in classes and on the making of a temporary and permanent preparation. I also clarified the importance of microscoping practices and the pupil's opinions about it. Lastly, I added an overview of biology textbooks used in primary schools and grammar schools. Keywords: microscope, laboratory...

This paper investigates noise characteristics of detector preamplifier in electron scanning microscopes. Various preamplifiers are measured and the lowest noise level amplifier with good level of speed is used. Further measurement of individual levels of amplifier is done and proposed the most suitable way how to achieve the total amplification. By detailed measuring of amplifier structure are found all unpleasant factors that affect the image quality. On the basis of these dates amplifier hardware is adjusted, software solutions to improve image quality designed and model control implemented, which directly controls active elements of preamplifier so that noise for given amplification was the lowest and reached required amplification.

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.