3D Chemical Imaging with STXM Spectro-Tomography

Abstract

Three dimensional (3D) chemical imaging using angle scan spectro-tomography in a soft X-ray scanning transmission X-ray microscope (STXM) has been developed and applied to quantitative 3D chemical mapping of fully hydrated biological and polymer systems as well as non-hydrated environmental and biological samples. Soft X-ray tomography using full field X-ray transmission microscopy [1,2] or cryo-STXM [3] has been performed at only one energy which provides density/thickness contrast with limited and potentially ambiguous chemical information. In contrast, spectro-tomography [4-6] acquires tomograms at multiple photon energies to provide true, quantifiable 3D chemical mapping through element-and species-specific, near edge X-ray absorption (NEXAFS) contrast of the components. Fig. 1 shows the in-situ tomography rotator and the sample mounting used, which include pulled glass capillaries, carbon nanopipettes [7], grid strips, and needle frames with wet samples enclose in thin polyimide films. The latter two approaches enable spectro-tomography at energies as low as 150 eV, thus accessing the S 2p edge. Fig. 2d shows a spectro-tomography derived rendering of the distribution of low density (4% dry weight) acrylate polyelectrolyte ionomer inside 800 nm diameter hollow polystyrene micro-spheres in an aqueous medium in a ~3 micron diameter glass micro-capillary [4]. The uniformity and statistical distribution of filled versus unfilled microspheres was the goal of the study. Specificity to the acrylate filling was achieved from the difference of tomograms recorded at 532.2 eV (O 1s → π* C=O peak) and 530 eV (Fig. 2c). A 3D spatial resolution better than 50 nm was documented. The color coding ( fig. 2d) reflects the density of acrylate in each voxel which is quantifiable because the differential absorption signal is quantitatively related to the amount of acrylate. Fig. 3 presents renderings of the protein, all-calcium and intense-Ca signals from a spectrotomogram recorded at 6 energies at the C 1s and Ca 2p edges. The sample is a single Synechococcus leopoliensis PCC 7942 bacterium grown under controlled supersaturation of Ca ++ and CO 3 2-ions in order to study the initial stages of nucleation and the role of aragonite-like CaCO 3 which forms as an intermediate in biomineralization of calcite by this species. Detailed analysis of the 3D spatial and compositional correlations of the Ca ++ and organic carbon signal (Fig 3b,c) in the region of the cell envelope showed that the aragonite layer is rather uniformly distributed in the extra-cellular polymer matrix whereas calcite forms at specific Ca-rich hot spots [6].

Imaging and Analysis of Encapsulated Objects through Self‐Assembled Electron and Optically Transparent Graphene Oxide Membranes

“…Usually, to obtain a 3D reconstruction of an object, a set of 2D images of the sample pivoted within a wide range of angles ± 70° is recorded [42] . This technique requires mounting the sample on a pivoting axis with an open access for the X-rays in the aforementioned angle range.…”