This work deals with tool steel Vanadis 6. It describes the preparation of samples for the three-point bending test, with which it examines the influence of surface roughness, the effect of nitriding, coating and duplex coatings on fracture behaviour of the steel. First, it examines bending strength and the total energy required to work of fracture. The work also includes mapping and measurement of surface roughness on different layers. This measurement is performed on a confocal microscope.

Master‘s thesis deals with the methods and system for measurement of concentration of intracellular calcium in LabVIEW. The thesis covers the theory of fluorescence principles, fluorescence methods and indicators for measurement of concentration of intracellular calcium. The procedure of measurement by means of fluorescence microscope, camera and indicators is also mentioned. The theory dealing with confocal microscopy is presented and two techniques are compared. Literary solution is presented. The practical part describes the program with a graphical user interface used for acquisition and analysis of data measuring dynamics of intracellular calcium concentration. The experimental part describes the preparation and measurement of cells and evaluation of experiments.

No description available.

<p dir="ltr">Cancer biological mechanisms are a vastly researched area in the field, yet they are not well understood in the various contexts in which cancer is found. Cancerous tumors often exist in harsh, stressful environments for normal cells, but cancer cells can thrive in these conditions. The tumor microenvironment (TME) typically has low oxygen levels (hypoxia), high acidity, and low nutrition. Exposure to the TME leads to many metabolic changes in the cells, enabling cancer to continue proliferating and migrating. However, these metabolic changes are not well understood, especially at the single-cell level. The ability to monitor cells in real time to determine the physical characteristics they undergo is critical to understanding the impact of these metabolic changes. Conventional methods focus on determining the genomic and proteomic changes in large numbers of cells, which may be overlooked if the changes are homogeneous across samples. In this work, we demonstrate the power of using multiple imaging techniques in combination with biochemical methods to visualize metabolic changes and determine the causes in various cancer cells under extreme hypoxia conditions.</p><p dir="ltr">The changes in the microtubule network that occur under hypoxia at the single-cell level are not widely researched. The use of confocal fluorescence microscopy can determine microtubule polymerization in conjunction with eGFP-transfected EB3, a protein that assists in microtubule polymerization. We have determined that hypoxic HeLa cells produce finger-like protrusions when exposed to hypoxia that help with cell migration and, ultimately, cancer cell metastasis. The formation of these protrusions is facilitated by localized mitochondria activities in the protrusions.</p><p dir="ltr">The metabolic changes in lipid droplets (LDs) under hypoxia at the single-cell level remain an elusive topic. The use of stimulated Raman spectroscopy (SRS) and coherent anti-Stokes Raman scattering (CARS) can determine the quantity and spatial-temporal distribution of LDs in cancer cells. We have found that LDs redistribute to the endoplasmic reticulum (ER) and increase in intensity in hypoxic MIA PaCa-2 and A549 cells. Time-lapse CARS microscopy revealed a release-accumulate process of these LDs on ER in hypoxia. We also studied the impact of carbon sources on LD formation and found that MIA PaCa2 cells prefer direct lipid uptake while glucose is also essential to reduce lipotoxicity. The use of hyperspectral stimulated Raman scattering (hSRS) also reveals that the content of the LDs changes to include less cholesteryl ester and a decrease in lipid saturation level.</p><p dir="ltr">Collectively, these findings shed new light on the understanding of cytoskeleton dynamics and lipid metabolism in hypoxic conditions. The discoveries made within this research would lead to better treatment strategies for effective treatment of hypoxia-resistant cancer cells.</p>

In this thesis, we investigate biological molecules on a micron scale in the ultraviolet spectral region through the non-destructive confocal absorption microscopy. The setup involves a combination of confocal microscope with a UV light excitation beam to measure the optical absorption spectra with spatial resolution of 1.4 μm in the lateral and 3.6 μm in the axial direction. Confocal absorption microscopy has the benefits of requiring no labels and only low light intensity for excitation while providing a strong signal from the contrast generated by the attenuation of propagating light due to absorption. This enables spatially resolved measurements of single live cells and bio-molecules with less than 10^9 molecules in the probe volume. Employing a multichannel detection system, the absorption spectrum of hemoglobin in a single red blood cell is measured on the timescale of seconds. We also extend the spectral range from the visible range to the experimentally more challenging ultra-violet region where characteristic absorption bands of bio-molecules are observed. Exploiting the ultra-violet range, amino acids, nucleic acids solutions, and plant cells are investigated. We measure the spatially resolved absorption spectra at the nucleus of an onion cell and cytoplasm to probe DNA base-pair absorption. Small variations in our micro-absorption data are seen around 260 nm, possibly due to the abundance of DNA in the nucleus. This thesis contributes to the goal of spectroscopic identification of spatial heterogeneities at the single cell level and the label-free detection of proteins and nucleic acids.