|dc.description.abstracteng||The cytoskeleton of eukaryotic cells is a complex and dynamic network of different biopolymers, which plays an important role in, e.g., the determination of cellular shape, mechanical properties, and consequently specific cellular functions. Cytoskeletal protein networks are mainly composed of three different classes of proteins: actin filaments, microtubules and intermediate filaments. These proteins can further form higher-order structures like bundles or paracrystalline arrays of filaments. One example for the higher-order organization of cytoskeletal proteins are bundles and networks of keratin intermediate filaments, which occur mainly in epithelial cells and provide these cells with the necessary mechanical properties to withstand external stress. Detailed knowledge about the filament arrangement inside keratin bundles in cells is still lacking. The small structure sizes in the order of tens of nanometers require high resolution imaging techniques, which are compatible with soft matter samples and non-invasive sample preparations.
X-rays provide an ideal probe for studying structures at the nano-scale and are routinely employed for investigating the structure and the composition of biological systems, making use of the variety of different techniques. The small wavelength in principle allows for structure determination with atomic precision and comparatively thick samples can be investigated non-invasively due to the high penetration depth of hard X-rays. By raster scanning the sample with a small beam, structural information obtained from individual scattering patterns in reciprocal space can be combined with positional information in real space.
In this work, scanning X-ray diffraction using a nano-focused beam was applied to samples of biological cells in order to probe the structure of cytoskeletal bundles and networks of keratin intermediate filaments. As a model system the cell line SK8/18-2 was employed, which expresses fluorescently labeled keratins that assemble in these cells into complex networks. Cellular samples were prepared using different methods, starting from well-established freeze-dried samples and going on to fixed-hydrated and finally living cells. Using this approach, potentially invasive and structure altering steps during the sample preparation could be reduced or completely avoided, which allowed for probing the native sample structure. However, the requirements of the different sample types on the sample environment and the sample handling during the experiments were more complex for the hydrated and particularly living cells as compared to the freeze-dried cells. In this context, the development of X-ray compatible microfluidic devices allowing for measurements on living cellular samples was an important aspect. Comparing the scattering signal from freeze-dried, fixed-hydrated and living cells, differences between the sample types at length scales of several tens of nanometers were determined. The successful application to hydrated and living cells further demonstrates the potential for structural analysis at hardly accessible length scales in native samples.||de