Three-Dimensional Imaging of Crystalline Inclusions Embedded In

OPEN Three-dimensional Imaging of Crystalline SUBJECT AREAS: Inclusions Embedded in Intact Maize IMAGING TECHNIQUES BIOFUELS Stalks BIOPHYSICS John Badger1, Jyotsana Lal2, Ross Harder2, Hideyo Inouye3, S. Charlotte Gleber2, Stefan Vogt2, TECHNIQUES AND Ian Robinson4,5 & Lee Makowski3 INSTRUMENTATION 1DeltaG Technologies, San Diego, California 92122, USA, 2X-ray Science Division, Advanced Photon Source, Argonne National Received Laboratory, Argonne, Illinois 60439, USA, 3Dept. of Electrical and Computer Engineering, Northeastern University, Boston, 4 June 2013 Massachusetts 02115, USA, 4London Center for Nanotechnology, University College London, London WC1E 6BT, UK, 5Research Complex at Harwell, Oxford, OX11 0FA, UK. Accepted 4 September 2013 Published Mineral inclusions in biomass are attracting increased scrutiny due to their potential impact on processing methods designed to provide renewable feedstocks for the production of chemicals and fuels. These 3 October 2013 inclusions are often sculpted by the plant into shapes required to support functional roles that include the storage of specific elements, strengthening of the plant structure, and providing a defense against pathogens and herbivores. In situ characterization of these inclusions faces substantial challenges since they are Correspondence and embedded in an opaque, complex polymeric matrix. Here we describe the use of Bragg coherent diffraction requests for materials imaging (BCDI) to study mineral inclusions within intact maize stalks. Three-dimensional BCDI data sets were collected and used to reconstruct images of mineral inclusions at 50–100 nm resolution. Asymmetries should be addressed to in the intensity distributions around the Bragg peaks provided detailed information about the deformation L.M. (makowski@ece. fields within these crystal particles revealing lattice defects that result in distinct internal crystal domains. neu.edu) haracterization of a heterogeneous sample such as corn stover involves dealing with material that is organized on multiple length scales and contains discrete domains with different compositions, orienta- C tions and order. Investigations into the structural components of these types of sample are of considerable interest since the presence of mineral inclusions in plant tissues is a factor in the development of industrial processes used to produce chemicals and fuel1. In addition, the inclusion of crystals in plant tissues has been identified with several distinct biological functions that include providing sinks for chemical accumulation, adding structural support to plant tissues and creating a defense against pathogens and herbivores2–4. A novel approach for non-destructive in situ imaging of the sizes and morphologies of micro crystals embedded in opaque media is provided by Bragg coherent diffraction imaging (BCDI). This approach has previously been used to characterize manufactured samples of simple crystals5–8. Here we demonstrate the applicability of BCDI to more complex crystals embedded in biological samples and, more generally, the use of the technique for analyzing sample types in which crystalline particles are concealed within an amorphous media. Results In order to survey the nature of x-ray scattering from the vascular bundles embedded in maize stalks, we collected wide angle x-ray scattering (WAXS) data using an 180 mm sample-to-detector distance at the BioCAT beam line at the Advanced Photon Source (APS). These patterns exhibited the characteristic intensity distribution expected for cellulose Ib9 as well as occasional, very sharp reflections of uncertain origin (Fig. 1). These sharp reflections exhibited no apparent preferred orientation relative to the fiber axis of the stover. Thirty WAXS patterns from maize stalks were examined and the scattering angles of nearly 100 of these Bragg peaks were measured from these data sets. A comparison of the measured scattering angles with the d-spacings of reflections predicted from the crystal structures of naturally occurring minerals commonly observed in plant tissues concluded that almost all of these reflections could be indexed using the crystal lattices of calcium oxalate monohydrate (whewellite) and dihydrate (weddelite). The strongest reflections expected10,11 from whewellite (5.93, 3.64 and 2.98 A˚ ) and weddelite (6.19, 4.42, 3.68 and 2.75 A˚ ) were all observed in this survey, indicating that both crystal forms exist in these samples. The observation of Bragg peaks on most of these images shows that these microcrystals occur at a density SCIENTIFIC REPORTS | 3 : 2843 | DOI: 10.1038/srep02843 1 www.nature.com/scientificreports the resolution of the XFM images is relatively low, the inclusions appear to have well-defined shapes and edges. Silicon-rich inclusions were also observed, particularly in the hollow centers of fiber cells. However, Bragg peaks observed by WAXS could not be indexed on the basis of any known naturally occurring silicon-rich mineral. We concluded that the silicon-rich inclusions were either amorphous or constituted crystals sufficiently small that the scattering peaks were not detectable in the WAXS patterns. BCDI diffraction data were collected on a CCD detector mounted on a 6-circle diffractometer at beam line 34-ID-C at the Advanced Photon Source at Argonne National Laboratory using a beam size of approximately 2 mm6,12. Early observations at room temperature indicated that radiation damage to the surrounding organic material was leading to movement of the crystals in the beam. Consequently, all data were collected with the samples held at liquid nitrogen temperature. Vascular bundles teased from dried maize stalks were Figure 1 | WAXS Pattern from vascular bundles of maize. This WAXS adhered to the cold finger of a cryostage with their fiber axes per- pattern shows diffraction characteristic of cellulose fibers and containing pendicular to the incident x-ray beam. The individual vascular bun- several Bragg reflections (magnified in the insets) that are due to dles, typically 1–3 mm in diameter, were broken from the bulk of the diffraction from crystallites embedded in the material. At the point-to- stalk and laid parallel to each other on the copper surface of the cold point resolution of this pattern, taken at a sample to detector distance of finger (see Supplementary Information, S1). The detector was placed 180 mm, little detail within the Bragg reflections can be discerned. at a scattering angle corresponding to a spacing of 3.65 A˚ where scattering from both forms of calcium oxalate can be observed. that is typically high enough to find crystals in orientations that place The samples were translated across the x-ray beam with the detector a few strong reflections on the detector surface. For the x-ray beam at 0.75 m from the sample in order to provide a relatively large field size of 140 3 40 mm and assuming a sample thickness of ,1 mm the of view for detection of Bragg reflections. Once a Bragg reflection was sample volume illuminated by x-rays in these experiments is ,6 3 observed, the detector was typically translated to either 1.0 m or 106 mm3. The calculations of three-dimensional diffraction patterns 1.8 m from the sample in order to collect the data on the finest from the crystal structures10 of whewellite and weddelite showed that possible grid. By rotating the sample over small increments a they produce very few strong reflections and, therefore, the probabil- ity of detecting diffraction from a specific randomly oriented crystal is small. If the mosaic spread of a crystal is assumed to be ,0.05u the crystal number density probably must exceed one crystal per 104 mm3 sample volume to produce detectable Bragg peaks on the majority of WAXS images. X-ray fluorescence microscopy (XFM) revealed occasional calcium-rich inclusions with linear dimensions of ,1 micron (Fig. 2). Since the samples used for the XFM analysis were from the same source as the stover used for the WAXS analysis these observations are consistent with the presence of calcium oxalate crystals. Although Figure 3 | BCDI data. (a) Sections through a three-dimensional BCDI data set collected as a series of diffraction patterns in which each successive pattern is separated from the previous by a small, constant angular rotation Figure 2 | XFM images of maize. XFM images of a fiber cell teased from a of the detector, dh. This illustration shows every fifth image taken as the sample of corn stover. Cell walls around the fiber cells are typically 10 detector rotated through the reflection. The stack of almost parallel images microns in diameter and hollow. The distribution of most elements is then assembled into a three-dimensional data set that maps the detailed corresponds to nearly uniform distribution within the cell walls. The diffracted intensity around the position of a single Bragg reflection. exceptions are calcium which exhibits several micron-sized calcium-rich (b) Central sections through five different Bragg peaks corresponding to inclusions, and silicon which appears to concentrate within the hollow the five reconstructions shown in Figure 4. The intensity measurement in interior of the fiber cells. all of the diffraction images are displayed on a logarithmic scale. SCIENTIFIC REPORTS | 3 : 2843 | DOI: 10.1038/srep02843 2 www.nature.com/scientificreports three-dimensional data set was assembled as a set of two-dimen- objects13–21. The implementation was checked by reconstructing sional slices that map the intensity through the reflection

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