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. 2018 Sep 24;24(5):526–544. doi: 10.1017/S1431927618015015

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© Microscopy Society of America 2018

This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Correlative light and electron microscopy of immobilized and pelleted prokaryotic cells. Light and scanning electron micrographs of Magnetobacterium bavaricum cells, within a mixed-culture prepared for correlative light microscopy and focused ion beam (FIB)/scanning electron microscopy (SEM). A slide with critical point dried bacteria was labeled with a water-resistant stamp for reference coordinates (a1). A target cell of was documented by phase contrast (a2; framed area) and re-localized in SEM with secondary electrons (SE) at low kV (a3) and with backscattered electrons (BSE) at higher kV, for detection of the magnetosomes by material contrast (a4). Energy dispersive X-ray analysis mapping of iron distribution (L-line of Fe at 3 kV) was superimposed to the SE image, confirming the characteristic Fe component of magnetosomes (a5; red). After coating the cells with platinum by ion beam deposition (b1) and FIB/SEM milling, in both SE (b2) and BSE images (b3) the magnetosome chains were clearly visible by material contrast, longitudinally (b2; rectangle) or cross-sectioned (b3; circle). For better milling properties, critical point dried cells were flat embedded in epoxy resin (c1) and milled again with significantly better image quality (c2). Besides the magnetosome chains, storage granules (poly-β-hydroxy butyrate (PHB) and sulfur) were slightly distinguishable (c2). Embedded cells are still beam sensitive: little holes formed during milling, even at low ion-currents (c2; circle). A rough 3D visualization was achieved in a short time using the threshold tool (c3). Best results were obtained if a pellet of high pressure frozen and freeze substituted magnetobateria is spread and embedded on slides in small droplets of resin (d1) for FIB/SEM-tomography (d2). Several structural details were reconstructed with high resolution in 3D (d3; d4) such as the number and arrangement of the magnetosomes and their chains, storage granules (sulfur=yellow; PHB=white), cellular envelope (blue) and flagella (brown). EsB, energy selective backscattered electron.