Methods to expose subsurface objects of interest identified from 3D imaging: The intermediate sample preparation stage in the correlative microscopy workflow.

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X-ray CT, FIB-SEM), scales (cm to nm) and dimensions (2D-3D-4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X-ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad-beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF-SEM), and ion milling and laser approaches such as FIB-SEM, Xe plasma FIB-SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non-correlative workflows.

Correlative Microscopy: a tool for understanding soil weathering in modern analogues of early terrestrial biospheres.

Correlative imaging provides a method of investigating complex systems by combining analytical (chemistry) and imaging (tomography) information across dimensions (2D-3D) and scales (centimetres-nanometres). We studied weathering processes in a modern cryptogamic ground cover from Iceland, containing early colonizing, and evolutionary ancient, communities of mosses, lichens, fungi, and bacteria. Targeted multi-scale X-ray Microscopy of a grain in-situ within a soil core revealed networks of surficial and internal features (tunnels) originating from organic-rich surface holes. Further targeted 2D grain characterisation by optical microscopy (OM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (SEM-EDS), following an intermediate manual correlative preparation step, revealed Fe-rich nodules within the tunnels. Finally, nanotomographic imaging by focussed ion beam microscopy (FIB-SEM) revealed coccoid and filamentous-like structures within subsurface tunnels, as well as accumulations of Fe and S in grain surface crusts, which may represent a biological rock varnish/glaze. We attribute these features to biological processes. This work highlights the advantages and novelty of the correlative imaging approach, across scales, dimensions, and modes, to investigate biological weathering processes. Further, we demonstrate correlative microscopy as a means of identifying fingerprints of biological communities, which could be used in the geologic rock record and on extra-terrestrial bodies.

Quantification of sample thickness and in-concentration of InGaAs quantum wells by transmission measurements in a scanning electron microscope.

High-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) images of electron-transparent samples show dominant atomic number (Z-) contrast with a high lateral resolution. HAADF STEM at low electron energies <30 keV is applied in this work for quantitative composition analyses of InGaAs quantum wells. To determine the local composition, normalized experimental image intensities are compared with results of Monte Carlo simulations. For verification of the technique, InGaAs/GaAs quantum-well structures with known In concentration are used. Transmission electron microscopy samples with known thickness are prepared by the focused-ion-beam technique. The method can be extended to other material systems and is particularly promising for the analysis of materials that are sensitive toward knock-on damage.