X-ray nano-diffraction on cytoskeletal networks

Abstract

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.

Investigating Cellular Nanoscale with X-rays

“…This is computed by looking at the orientation of the scattering patterns. The patterns are fitted by an ellipse, then the minor and major axis as well as the orientation were retrieved [76].…”

“…This algorithm has the advantage not to use a median filtering of the scattering patterns or a thresholding parameter to binarize the scattering patterns as used in ref. [76]. The results are shown in Fig.…”

“…This technique has been improved and benefits nowadays from nanometre size beams and high brilliance of synchrotron sources. It has shown great success on cytoskeletal proteins such as keratin intermediate filaments [76,78] (panel c of Fig. 1.2).…”

“…c) Scanning nano-diffraction of keratin rich cells SK8K18 (shown in the inset) from ref. [76]. d) Phase map of a ptychographical reconstruction of a SK8K18 cell from ref.…”

“…In the past, we and others have successfully employed scanning X-ray nano-diffraction on whole cells [40,47,59,61]. Taking advantage of the nano-focused X-ray beam, it was possible to resolve the local orientation and structure of the keratin network in full unstained cells [76,78].…”