X-rays Reveal the Internal Structure of Keratin Bundles in Whole Cells

Abstract

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.

Investigating Cellular Nanoscale with X-rays

“…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).…”

“…The impact of salt ions on the formation of keratin bundles is described in chapter 4. It is followed by results from synchrotron experiments on the keratin networks and DNA/chromatin condensation over the cell cycle, which are presented in chapter 5 [78] and chapter 6, respectively. In chapter 5 the keratin network in whole cells is investigated by two combined X-ray techniques.…”

“…The sample preparation and different procedures are detailed in the appendix B, while a study on the structural changes induced by chemical fixatives to cells is presented in appendix C. The appendix D is composed of results from collaborations and from bachelor students I have supervised. Parts of this thesis have been published in Biomacromolecules [105] and ACS Nano [78] or have been prepared for publication. …”

“…Hémonnot et al ACS Nano 10, 2016 [78], and reproduced in this dissertation with permission and under copyright (2016) of the American Chemical Society. The table 5.1 summarises the acquisition parameters of each samples that were scanned during the beamtime.…”

“…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].…”