High-resolution x-ray imaging techniques offer a variety of possibilities for studying the nanoscale structure of biological cells. A challenging task remains the study of cells by x rays in their natural, aqueous environment. Here, we overcome this limitation by presenting scanning x-ray diffraction measurements with beam sizes in the range of a few hundred nm on living and fixed-hydrated eukaryotic cells in microfluidic devices which mimic a native environment. The direct comparison between fixed-hydrated and living cells shows distinct differences in the scattering signal, pointing to structural changes on the order of 30 to 50 nm.

In recent years, X-ray imaging of biological cells has emerged as a complementary alternative to fluorescence and electron microscopy. Different techniques were established and successfully applied to macromolecular assemblies and structures in cells. However, while the resolution is reaching the nanometer scale, the dose is increasing. It is essential to develop strategies to overcome or reduce radiation damage. Here we approach this intrinsic problem by combing two different X-ray techniques, namely ptychography and nanodiffraction, in one experiment and on the same sample. We acquire low dose ptychography overview images of whole cells at a resolution of 65 nm. We subsequently record high-resolution nanodiffraction data from regions of interest. By comparing images from the two modalities, we can exclude strong effects of radiation damage on the specimen. From the diffraction data we retrieve quantitative structural information from intracellular bundles of keratin intermediate filaments such as a filament radius of 5 nm, hexagonal geometric arrangement with an interfilament distance of 14 nm and bundle diameters on the order of 70 nm. Thus, we present an appealing combined approach to answer a broad range of questions in soft-matter physics, biophysics and biology.

Vimentin intermediate filaments (IFs) are part of a family of proteins that constitute one of the three filament systems in the cytoskeleton, a major contributor to cell mechanics. One property that distinguishes IFs from the other cytoskeletal filament types, actin filaments and microtubules, is their highly hierarchical assembly pathway, where a lateral association step is followed by elongation. Here we present an innovative technique to follow the elongation reaction in solution and in situ by time-resolved static and dynamic light scattering, thereby precisely capturing the relevant time and length scales of seconds to minutes and 60-600 nm, respectively. We apply a quantitative model to our data and succeed in consistently describing the entire set of data, including particle mass, radius of gyration, and hydrodynamic radius during longitudinal association.I ntermediate filaments (IFs) constitute one of the three protein filament systems in the cytoskeleton of metazoa. Together with actin filaments and microtubules they form a sophisticated composite network, which has been identified as a main player in cell mechanics (1). By contrast to actin filaments and microtubules, which are conserved across cell types and organisms, IFs comprise a large family of proteins, encoded by 70 genes in humans (2), and they are expressed in a cell-type-specific manner. Vimentin is an IF protein expressed in fibroblasts, the eye lens, and cells of mesenchymal origin. The monomers with a molecular weight M w of 53.5 × 10 3 g/mol share their tripartite structure consisting of an α-helical rod flanked by intrinsically disordered "head" and "tail" domains, as shown in Fig. 1A, with all other IFs. These monomers are stable in denaturing conditions, such as 8 M urea, and assemble into coiled-coil dimers and subsequently into antiparallel tetramers with M w = 214 × 10 3 g/mol, a length of 60 nm, and a diameter of 5 nm upon stepwise dialysis into low-salt buffers, such as 2 mM sodium phosphate (3). Thus, in buffer conditions without urea, these tetramers with a mass per unit length M tet L of 3,570 g/(mol·nm) are the smallest subunits and starting precursors for vimentin IF assembly. In vitro the assembly of tetramers into full-length filaments can be initiated by the addition of, e.g., monovalent salts such as potassium chloride (KCl) at concentrations of a few tens of millimolars. It has been shown by time-lapse electron microscopy (4) and more recently by real-time small-angle X-ray scattering (SAXS) in combination with microfluidic techniques (5-7) that a lateral assembly step into unit-length filaments (ULFs) consisting of typically eight tetramers (Fig. 1B) is followed by an elongation reaction where ULFs and short filaments join to form micrometer-long filaments (Fig. 1C). However, the exact molecular mechanism of the elongation reaction remains elusive. It is clear that the tail domains are not needed (4). The way that the IF consensus domains at either end of the rod interact, overlapping (8) vs. interdigitating (9), is n...

The combination of microfluidics and X-ray methods attracts a lot of attention from researchers as it brings together the high controllability of microfluidic sample environments and the small length scales probed by X-rays. In particular, the fields of biophysics and biology have benefited enormously from such approaches. We introduce a straightforward fabrication method for X-ray compatible microfluidic devices made solely from cyclic olefin copolymers. We benchmark the performance of the devices against other devices including more commonly used Kapton windows and obtain data of equal quality using small angle X-ray scattering. An advantage of the devices presented here is that no gluing between interfaces is necessary, rendering the production very reliable. As a biophysical application, we investigate the early time points of the assembly of vimentin intermediate filament proteins into higher-order structures. This weakly scattering protein system leads to high quality data in the new devices, thus opening up the way for numerous future applications.

The structural organization of metazoan cells and their shape are established through the coordinated interaction of a composite network consisting of three individual filament systems, collectively termed the cytoskeleton. Specifically, microtubules and actin filaments, which assemble from monomeric globular proteins, provide polar structures that serve motor proteins as tracks. In contrast, intermediate filaments (IFs) assemble from highly charged, extended coiled coils in a hierarchical assembly mechanism of lateral and longitudinal interaction steps into non-polar structures. IF proteins are expressed in a distinctly tissue-specific way and thereby serve to generate the precise plasticity of the respective cells and tissues. Accordingly, in the cell, numerous parameters such as pH and salt concentration are adjusted such that the generation of functional networks is ensured. Here, we transfer the problem for the mesenchymal IF protein vimentin to an in vitro setting and combine small angle x-ray scattering with microfluidics and finite element method simulations. Our approach is adapted to resolve the early assembly steps, which take place in the sub-second to second range. In particular, we reveal the influence of ion species and concentrations on the assembly. By tuning the flow rates and thus concentration profiles, we find a minimal critical salt concentration for the initiation of the assembly. Furthermore, our analysis of the surface sensitive Porod regime in the x-ray data reveals that the formation of first assembly intermediates, so-called unit length filaments, is not a one-step reaction but consists of distinct consecutive lateral association steps followed by radial compaction as well as smoothening of the surface of the full-width filament.