The nano-scale structure of cytoskeletal biopolymers as well as sophisticated superstructures determine the versatile cellular shapes and specific mechanical properties. One example is keratin intermediate filaments in epithelial cells, which form thick bundles that can further organize in a crosslinked network. To study the native structure of keratin bundles in whole cells, high-resolution techniques are required, which do at the same time achieve high penetration depths. We employ scanning x-ray diffraction using a nano-focused x-ray beam to study the structure of keratin in freeze-dried eukaryotic cells. By scanning the sample through the beam we obtain x-ray dark-field images with a resolution of the order of the beam size, which clearly show the keratin network. Each individual diffraction pattern is further analyzed to yield insight into the local sample structure, which allows us to determine the local structure orientation. Due to the small beam size we access the structure in a small sample volume without performing the ensemble average over one complete cell.

The assembly of intermediate filaments (IFs) is a complex process that can be recapitulated through a series of distinct steps in vitro. The combination of microfluidics and small angle X-ray scattering (SAXS) provides a powerful tool to investigate the kinetics of this process on the relevant timescales. Microfluidic mixers based on the principle of hydrodynamic focusing allow for precise control of the mixing of proteins and smaller reagents like ions. Here, we present a multi-layer device that prevents proteins from adsorbing to the channel walls by engulfing the protein jet with a fluid layer of buffer. To ensure compatibility with SAXS, the device is fabricated from UV-curable adhesive (NOA 81). To demonstrate the successful prevention of contact between the protein jet and the channel walls we measure the distribution of a fluorescent dye in the device by confocal microscopy at various flow speeds and compare the results to finite element method (FEM) simulations. The prevention of contact enables the investigation of the assembly of IFs in flow by gradually increasing the salt concentration in the protein jet. The diffusion of salt into the jet can be determined by FEM simulations. SAXS data are collected at different positions in the jet, corresponding to different salt concentrations, and they reveal distinct differences between the earliest assembly states. We find that the mean square radius of gyration perpendicular to the filament axis increases from 13 nm(2) to 58 nm(2) upon assembly. Thereby we provide dynamic structural data of a complex assembly process that was amenable up to now only by microscopic techniques.

Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed.

A visible BaB2O4 optical parametric oscillator (OPO) pumped by a single-axial-mode 355-nm source has been demonstrated. An average output power of 140 mW with a signal wave conversion efficiency of 13% and an idler conversion efficiency of 11% for a total conversion efficiency of 24% has been achieved. The oscillator has continuously tuned from 412 nm to 2.55 μm limited by the infrared transmission range of the crystal. Through injection seeding we obtained single-axial-mode OPO operation with a corresponding OPO linewidth of less than 3 GHz.