Facility for Electron Microscopy City Campus
The Electron Microscopy City Campus is performing ultrastructural research in collaboration with scientists within the Max-Planck-Institute for Multidisciplinary Sciences and the Göttingen research campus and from 2012 to 2018 also within the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain. Since 2004 the Unit is headed by Wiebke Möbius. Current members of the team are the two technicians Torben Ruhwedel and Boguslawa Sadowski and the Postdoc Anna Steyer.
Within the Electron Microscopy City Campus, we routinely apply transmission electron microscopy (TEM) to analyse ultrastructure at subcellular resolution. Depending on the question under investigation, we use methods for morphological analysis on plastic embedded samples, or we perform immunoelectron microscopy on ultrathin cryosections to localize molecules within the context of the cell or tissue (Möbius, 2016). For ultrastructural analysis, samples are either fixed chemically and processed by conventional embedding or prepared by high-pressure-freezing (HPF) and embedded in resin after freeze substitution (FS). While conventional preparations are more suited for quantitative comparison of phenotypes, HPF-frozen samples often reveal fine structural details that are often lost in conventional preparations due to chemical fixation and embedding. This is especially relevant in ultrastructural myelin research (exemplified in Möbius et al., 2016). We continuously develop and modify protocols of sample preparation such as microwave-assisted embedding for specialized applications like large area or volume imaging using focussed ion beam scanning electron microscopy (e.g. Steyer et al., 2019a and 2019b).
We are equipped with two transmission electron microscopes for routine applications: a Zeiss EM 900 and a LEO 912AB Omega. Since 2011 a Leica HPM100 high pressure freezing machine is available for fast cryo-immobilization of samples which are prone to fixation artefacts. For the possibility to visualize fast events such as synaptic vesicle fusion with a time resolution of milliseconds, the Department of Molecular Neurobiology (Nils Brose) acquired a Leica HPM100 with light stimulation and a Leica HPM ICE with the option of light- or electrical stimulation in the EM Core Unit. These instruments are operated by Ben Cooper and Cordelia Imig from the Department of Molecular Neurobiology.
In 2012 the Electron Microscopy City Campus joined the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) as EM Platform. With the support of the CNMPB in 2014 a Zeiss Crossbeam 540 was installed. This instrument is a focussed ion beam scanning electron microscope (FIB-SEM) in which the ion beam is used for in-situ removal of material by “milling”. With this approach a defined volume of the sample becomes accessible for imaging and 3D reconstruction. The surface area that can be milled is limited, but the milling can be targeted to multiple areas in the same sample. Removal of material as thin as 5 nm is possible, thereby increasing the resolution in Z and creating data stacks with almost isotropic voxel size.
Using FIB milling as a micromachining tool, TEM lamellae of almost any material can be manufactured. Applying this under cryo-conditions (-150°C), vitrified frozen TEM lamellae can be prepared for cryo-TEM tomography of biological samples.
Möbius W (2016) Immunoelectron Microscopy: High-Resolution Immunocytochemistry. In: Ralph A Bradshaw and Philip D Stahl (Editors-in-Chief), Encyclopedia of Cell Biology, Vol 2, Waltham, MA: Academic Press, 2016, pp. 32-43
Möbius W, Nave KA, Werner HB (2016) Electron microscopy of myelin: Structure
preservation by high-pressure freezing. Brain Res. 2016 Jun 15;1641(Pt A):92-100.
doi: 10.1016/j.brainres.2016.02.027. Review. PubMed PMID: 26920467.
Steyer AM, Ruhwedel T, Möbius W. Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging. (2019a) J Vis Exp. 2019 Mar 19;(145). doi: 10.3791/59156. PubMed PMID: 30958469
Steyer AM, Schertel SA, Nardis C, Möbius W: FIB-SEM of mouse nervous tissue: Fast and slow sample preparation (2019b). Methods in Cell Biology, Volume 152, pp.1-21, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.03.009