X-rays enable a direct view inside biological cells

14-04-2016

Structures within intact biological cells have been resolved quantitatively at the nanoscale using a combination of ptychography and scanning X-ray nanodiffraction. Employed sequentially, these techniques provided high resolution real space and reciprocal space images while exposing the cells to a comparatively small radiation dose. The structural arrangement and diameter of keratin protein bundles inside epithelial cells was revealed and filament diameters and distances could be derived.

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Biological cells contain a plethora of nanometre-sized objects and structures. Our knowledge about cellular function has been gained from direct imaging. Traditionally, fluorescence-based techniques and electron microscopy have been used to achieve micrometre to nanometre resolution in cellular imaging. More recently, X-ray imaging methods, which take advantage of the short wavelength and high penetration power, were developed and applied to the imaging of biological matter as well [1-3]. The small beams available at specialised synchrotron beamlines such as ID13 (Figure 1) enable high-resolution applications. One of the greatest challenges, however, remains radiation damage by the X-rays, which becomes more pronounced as the resolution reaches smaller values.

Experimental setup at ID13

Figure 1. Experimental setup at ID13.

By first recording ptychography overview images of whole cells at 65 nm resolution (Figure 2a and b), cellular structures were precisely located at a comparatively low dose of 103 Gy. Ptychography is a coherent diffractive imaging technique, in which the electron density of the scattering material is determined from a series of two-dimensional scattering patterns via iterative reconstruction algorithms and redundant information from overlapping scanning positions. Subsequently, scanning X-ray nanodiffraction (approximate dose 108 Gy) was employed to record reciprocal space scattering patterns from regions of interest (ROIs). For this technique, the sample is scanned through the beam at a step size comparable to the beam size. The intensity values in the individual scattering patterns can be integrated in order to derive dark-field-images (Figure 2c) at an intermediate resolution, which corresponds to the beam size on the order of 100 nm. Further analysis of each individual pattern reveals a local scattering signal (Figure 2d) which can be assigned to the specific position in the cell.

Ptychography and dark-field images

Figure 2. a) Ptychography image, scale bar 5 μm and b) detail from the ROI shown by the black box in a). c) Dark-field image computed from scanning X-ray nanodiffraction of the same ROI, and d) example of one pixel from (c) showing an anisotropic signal.

The mechanical properties of biological cells are to a great part governed by the so-called cytoskeleton, a composite network of fibrous proteins [4]. This present study focuses on keratin bundles, which in epithelial cells constitute one of the filament types. By fitting a hexagonally packed bundle model [5] to the data, the radius of individual filaments (r = 5.5 ± 0.7 nm), the inter-filament distance (a = 15.2 ± 1.4 nm) and the bundle diameter (D = 72.0 ± 5.5 nm) can be derived (Figure 3). These values are in agreement with results from electron microscopy [6], but were obtained without slicing or staining the cell and are thus at reduced risk for artifacts.

Cross section of a model of the keratin bundles

Figure 3. a) Cross section of a model of the keratin bundles [5] with 19 filaments per bundle. b) TEM image of the cross section of a keratin bundle in a hexagonal lattice, scale bar 50 nm. c) Radial intensity of the pattern shown in Figure 2d and results from the fit. d) 2D simulated pattern corresponding to the fit with 19 filaments in panel (c).

This study demonstrates the advantage of combining ptychography and scanning X-ray nanodiffraction in biological imaging since it provides nanometre resolution imaging of specific cellular structures while avoiding pronounced radiation damage of the samples.

 

Principal publication and authors
X-rays reveal the internal structure of keratin bundles in whole cells, C.Y.J. Hémonnot (a), J. Reinhardt (b), O. Saldanha (a), J. Patommel (c), R. Graceffa (a), B. Weinhausen (d), M. Burghammer (d,e), C.G. Schroer (b,f) and S. Köster (a), ACS Nano 10, 3553-3561 (2016); doi: 10.1021/acsnano.5b07871.
(a) Institute for X-ray Physics, University of Göttingen (Germany)
(b) DESY, Hamburg (Germany)
(c) Institute of Structural Physics, TU Dresden (Germany)
(d) ESRF
(e) Department of Analytical Chemistry, Ghent University (Belgium)
(f) Institute for Nanostructure and Solid State Physics, University of Hamburg (Germany)

 

References
[1] B. Weinhausen et. al., New J. Phys. 14, 085013 (2012).
[2] B. Weinhausen et. al., Phys. Rev. Lett. 112, 088102 (2014).
[3] V. Piazza et al., ACS Nano 8, 12228-12237 (2014).
[4] F. Huber et al., Curr. Opin. Cell Biol. 32, 39-47 (2015).
[5] M.P. Priebe et. al., Biophys. J. 107, 2662-2673 (2014).
[6] J.-F. Nolting et. al., Biophys. J. 107, 2693-2699 (2014).

 

Top image: Combination of ptychography and scanning X-ray nanodiffraction applied to whole cells. At positions, where bundles of filamentous proteins are found, the signal is highly anisotropic and exhibits modulation that is specific to the probed structure.