1. Introduction
Transmission (TEM) and scanning (SEM) electron microscopy techniques provide high-resolution images across a range of natural sciences. Through destructive serial sectioning, stacks of 2D images can be collected and aligned in sequence to build up three dimensional (3D) volume images [1], [2]. Typically, 3D serial block-face scanning electron microscopic (SBF-SEM) methods involving focused ion beam (FIB) milling [3] or mechanical sectioning [4] are used to provide high resolution information on very small regions of material, and the volumes interrogated by ultramicrotomy and FIB serial sectioning are typically around (1 mm)3 and (0.03 mm)3, respectively [5]. Therefore, there is a requirement to be able to select the region of interest (ROI) for study with a high degree of site specificity.
It is possible to use information gleaned from inspecting the surface of the sample prior to serial block-face imaging to provide contextual information about the region selected for detailed investigation, thus increasing the chances that the region selected will contain the features of interest prior to embarking on time consuming slicing and imaging workflows. Despite the fact that the volumes interrogated by ion beam methods have been increased somewhat by the advent of plasma FIB microscopes [5] to around (0.3 mm)3, capturing the feature of interest is still challenging when the internal morphology of a sample is not known.
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In this paper we examine how non-destructive X-ray micro computed tomography (microCT) can be incorporated into multiscale correlative microscopy workflows to ensure high efficiency and quality of electron microscopy. MicroCT is well suited to the imaging of samples of many millimetre dimensions at resolutions approaching a micron and some nanoCT instruments can image sub-millimeter samples at 50 nm resolution [6]. As a non-destructive method, X-ray CT thus provides an opportunity to establish a map of the sample prior to more detailed, guided investigation by electron microscopy methods [7], [8].
In the current work we examine the experimental issues associated with developing a simple correlative workflow. Our approach utilises the freely available and open-source software package IMOD [9] although other software, such as ImageJ/Fiji [10] could be used following the same principles. This workflow is then applied to a number of demonstrator case studies where prior microCT could be a useful preliminary step, namely:
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Other potential advantages include using microCT to first generate a coarser scale overview of the sample in order to put into context the regions of interest (ROIs) from which higher resolution electron microscopy images are acquired (multiscale correlative imaging), to orient the sample to optimise subsequent sectioning/excision, or to quantify levels of shrinkage/damage to the sample during sample preparation (fixing, staining or slicing) [11].
Finally we consider future refinements, such as automated sample registration procedures, that would enable correlative imaging or experimental steering by prior CT to be routinely incorporated into experimental workflows.
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