This work deals with the study of fluorescence lifetime and spectral changes of nanoparticles in cell biology. It describes the principle of fluorescence, fluorescence microscopy and laser confocal microscope Leica TCS SP8. The classic FLIM method, the Lambda Square mapping and the division of nanoparticles are introduced there. In the practical part, the created program for the evaluation of fluorescence lifetime and spectral changes is described. The program can show two-dimensional lambda maps, the fluorescence lifetime and spectral shift in the space area. In the final part of the thesis, an experiment with rhodamine nanoparticles is carried out and it is evaluated using the created program and then discussed.

This thesis deals with the performance of the measurement accuracy in the optical measuring systems. The first part describes basic principles of metrology and the guidelines of the expression of uncertainty and the factors which influence measurement result. The second part classifies optical measuring systems and describes some measuring principles of them. The final part compares two optical gauges and the evaluation of some of their characteristics through the use of very simple experiments.

Digital holographic microscopy is an interferometric imaging technique, the principle of which is the off-axis image plane holography. The principle of this technique enables to reconstruct both the image intensity and the image phase from the output interferencesignal. The reconstruction can be carried out on the basis of a single image plane hologram. This leads to the possibility of a realtime image reconstruction. The speed of the reconstruction depends on the detection and the computing process. The aim of this diploma thesis is to develop user software for the control of the detection camera and for the image plane hologram reconstruction. The effort was to achieve the highest number of image reconstructions per time unit, with the maximum utilization of the data transfer between the camera and the computer.The next aim of this thesis is the stabilization of the optical table position. The method of stabilization is based on the image phase information, which is used for the control loop feedback between reconstructed image phase and the piezoelectric actuator placed inside of the optical table. Experimental results, which prove the functionality of the stabilization, are presented.

The diploma thesis deals with the switching of spin vortices in magnetic nanodisks. First, the basic concepts of (micro)magnetism are defined and existing theoretical and experimental achievements in the field of switching of the two basic characteristics (chirality and polarity) of magnetic vortex are summarized. Then the principle of dynamic switching of magnetic vortex chirality using in-plane magnetic field pulse with a well defined amplitude and duration is presented. There is no need to use a certain shape of nanodisks or asymmetry in magnetic field distribution. Nanostructures were prepared by the multi-step electron beam lithography and ion beam sputtering. Individual steps of sample preparation and optimization for the magnetization dynamics measurements are described. Finally, the experimental measurements of the dynamic switching of chirality on prepared samples obtained by transmission x-ray microscopy at the synchrotron Advanced Light Source at Berkeley, USA are presented and discussed.

The project, developed under the terms of engineering studies of the M-STG Manufacturing Technology branch, presents the analytical assessment of the cutting surface of the metal sheet by two most commonly used modern technologies of cutting, which is the laser beam division and the exact cutting. The evaluation of this area was mainly focused on the level of the penetration of the zinc surface layer in the area of the given cutting. With the help of modern methods of electron microscopy, these values of penetration were then estabilished for each technology. The analysis itself was preceded by literary studies of all individual parts participating in this experiment.

Magnetic vortices in ferromagnetic disks are curling magnetization structures characterized by the sense of the spin circulation in the plane of the disk and by the direction of the magnetization in the vortex core. Concepts of memory devices using the magnetic vortices as multibit memory cells have been presented, which brought the high demand for their research in many physical aspects. This work investigates the magnetostatic coupling in pairs of ferromagnetic disks to clarify the influence of nearby disks or other magnetic structures to the vortex nucleation mechanism. To ensure that the vortex nucleation is influenced only by the neighbouring magnetic structures, the randomness of the nucleation process was studied in single disks prior to the work on pairs of disks. We had to ensure that the vortex nucleation is influenced only by the neighbouring magnetic structures and not by an unwanted geometrical asymmetry in the studied disk. Lithographic capabilities were inspected in order to achieve the best possible geometry. Further we present a concept of electrical readout of the spin circulation using the anisotropic magnetoresistance, which allows automated measurements to provide sufficient statistics. To explain the magnetoresistance behaviour, numerical calculations together with magnetic force microscopy measurements are presented.

This diploma thesis deals with phase and amplitude objects observation through scattering media by means of a coherence-controlled holographic microscope (CCHM). A brief history of development and construction of the microscope, its advantages compared to the classical light microscopy and hologram processing are described. Quantitative phase imaging through scattering media by means of ballistic as well as diffuse light is verificated in the experimental part. A comparison of an image obtained through a scattering layer by means of CCHM and a classical microscopy in the light field is demonstrated.

This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.

Diese Arbeit beschreibt die Untersuchung der Dynamik in eingeschränkten und gescherten Flüssigkeitsfilmen mit Hilfe der optischen Detektion einzelner Farbstoffmoleküle. Speziell dafür wurde ein modifizierter Surface-Forces- Apparatus konstruiert und aufgebaut. Dieser erlaubt es, im Flüssigkeitsfilm eine Scherströmung zu erzeugen. Mit der Analyse der Schrittweitenverteilung wird ein Werkzeug vorgestellt, welches räumlich aufgelöst und richtungssensitiv eine Analyse für Daten des Einzelmolekültrackings erlaubt. Angewandt auf Experimente in gescherten Flüssigkeitsfilmen ergibt sich die Möglichkeit, die strömungsinduzierte Bewegung einzelner Moleküle von deren diffusiver Bewegung zu unterscheiden. Für Experimente mit Tetrakis-(2-ethylhexoxy)-silane zeigt sich in der Nähe fester Substrate (Mica, Quarz) eine deutlich verlangsamte Diffusion einzelner Farbstoffmoleküle. Außerdem wird direkt an der Oberfäche ein Haftpotential (Tiefe etwa 600 meV ) gefunden, welches einzelne Farbstoffmoleküle in einer dünnen Grenzschicht (Dicke etwa 5 nm) an der Oberfläche hält, ohne deren laterale Beweglichkeit deutlich zu verringern. Dieses Verhalten lässt sich durch die Ausbildung von Flüssigkeitsschichten entlang der festen Oberfläche erklären. Für alle untersuchten Filmdicken und Scherraten wurde die selbe Diffusionskonstante gefunden, welche um etwa eine Größenordnung kleiner als die Diffusionkonstante für Farbstoffmoleküle in der Volumenflüssigkeit ist. Diese Beobachtung kann durch die in der Nähe fester Substrate vergrößerte effektive Viskosität der Flüssigkeit erklärt werden.

The mechanisms of invazivity and regulation of transcription of cancer cells Cancer originates in cells that overcome the control mechanisms of the organism. Cancer cells can be eventually released from the site of origin and spread through tissues. Cancer cells can acquire certain mechanisms that enable them to more effectively invade surrounding tissue or layers of other cells. The research on the migration of cancer cells is important for the understanding of the origin and spreading of metastases and consequently for anticancer therapy. In my Ph.D. work, I participated in the research of the properties of invasive metastatic cells. We compared non-invasive rat sarcoma cell line with a higly metastatic cell line derived from it. We showed that cells of the invasive cell line use amoeboid mode of migration, have upregulated Rho/ROCK signaling, and have accumulated actin and myosin at the leading edge. It is at the leading edge where the cells generate their traction forces. Cells of non-invasive cell line use mesenchymal mode of migration and generate forces mainly at their retracting end. We also compared two breast cancer cell lines derived from a single carcinoma. We showed that the more invasive cell line, derived from its parental line by neoplastic transformation, displayed elevated cytoskeletal...