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We talk to Dr Errin Johnson, the Electron Microscopy (EM) Facility Manager, about how the facility is being used by Oxford scientists to aid their research, and how you too can make use of it.

Extracted from Issue 12, August 2014 OxfordMedSci News.

Tell us a little about the Electron Microscopy Facility at the Dunn School

Dr Errin JohnsonBased in the Sir William Dunn School of Pathology, the Electron Microscopy (EM) Facility is part of the Dunn School Bioimaging Facility which, in association with the Micron Advanced Bioimaging Unit and the Oxford Particle Imaging Centre, provides Oxford University scientists with a broad range of light and electron microscopy options to facilitate their research. The EM Facility comprises a FEI Tecnai T12 Transmission Electron Microscope (TEM), a JEOL-6390 Scanning Electron Microscope (SEM), a Zeiss Merlin Compact Field Emission Gun (FEG)-SEM (with Oxford Brookes), an EM specimen preparation laboratory and dedicated computer stations for image analysis and 3D data reconstruction. People are often daunted by the specimen preparation techniques and complex instrumentation involved with EM, or may simply be unaware of the ways EM techniques can be applied to, and benefit, their research. The EM Facility has dedicated expert staff always available to advise and train its users, as well as to prepare and image specimens as a service, so that the wonderful world of EM is accessible to anyone!

How many people use the EM Facility in the Dunn School?

We currently have over 60 active users in the EM Facility, with projects covering an extremely diverse and exciting range of science, from fundamental cell biology and medicine, through to ecology and bio-manufacturing. Two thirds of our users are from external Departments, including Biochemistry, Earth Sciences, Zoology, STRUBI, DPAG, NDM, Physics and Engineering.

How is EM being applied to research in the Medical Sciences Division?

TEM imagesThe wavelength of accelerated electrons is up to 100,000x shorter than that of light and using an electron beam, which is manipulated by electro-magnetic lenses in the EM, allows objects smaller than 1 nm to be magnified and resolved. In TEM, the image is formed by electron interactions with the specimen as the beam transmits through it, enabling small particulate samples (eg: proteins, viruses and bacteria), cellular ultrastructure and tissue architecture to be visualised at ultra-high resolution. Examples of recent biomedical TEM projects include imaging negatively stained: synthetic virus-like particles, ribonucleoproteins, type IV pili in Neisseria gonorrhea and exosomes isolated from dendritic cells; as well as using resin embedded specimens to characterise the ultrastructure of: the flagellum during life cycle stage differentiation in Leishmania sp., the blood-brain barrier in mice models, centrosomes in Drosophila and chicken cells infected with coronavirus.

SEM imagesSEM works differently, with the image instead being formed by scattered electrons that result from interactions between the electron beam and the specimen as the beam is scanned across its surface. This permits specimen topography to be imaged in exquisite detail and is ideal for morphological characterisation of mutant or drug-treated cells, tissue or even whole organisms. Recent biomedical applications include assessing the rough-eye phenotype of Drosophila mutants, investigating the effect of growth media on the morphology of stem-cell derived blood cells, and characterising the morphology of: genetically modified mouse dendritic cells, E. coli, and malaria merozites after freezing.

What are some of the more advanced techniques you are using?

There are a number of advanced techniques available including 3D-SEM, correlative microscopy, cryo-ultramicrotomy, protein localisation using immunogold or new genetic tags for EM, and electron tomography. The latter is facilitated by our new user-friendly ISSF-funded dual-axis tomography holder, which allows organelles and structures to be reconstructed and modelled in 3D (eg: this is currently being used here to study the immunological synapse between melanoma cells and T cells).

An area of active technique development in the EM lab is correlative microscopy. Using this method, the same cell is imaged using two different techniques, commonly confocal and TEM. In this way, proteins and/or other sub-cellular components can be imaged on the confocal using fluorescent probes and then placed into ultrastructural context at the EM level. Though the specimen preparation involved is more demanding, this is an extremely powerful bridging technique and we look forward to assisting more correlative projects in future.

We also have a new BBSRC-funded Zeiss Merlin Compact Variable Pressure FEG-SEM, equipped with a Gatan 3view system (an in situ ultramicrotome). This instrument is capable of producing large, high resolution volumes of biological samples using a technique called serial block face sectioning, which enables the 3D architecture of whole cells and tissues to be reconstructed and analysed at the ultrastructural level. The microscope is based at Oxford Brookes University and is jointly run by the two departments under a shared access agreement. Please contact Dr Errin Johnson to learn more about this cutting-edge (excuse the pun) technique and to setup a collaboration.

 How can people access the EM Facility?

Joshua LongThe EM Facility is open to all members of Oxford University and multiple usage options are available: full/partial service (recommended for difficult, short term or one-off projects), training (free and comprehensive, a particularly good option for PhD students) and collaboration (for long-term technically challenging projects). Usage charges are available upon request and vary depending on the instrument and project. Service work involves an additional fee. I am also able to prepare quotes for grant applications.

Please contact Dr Errin Johnson to discuss how you can use the Dunn School EM Facility to further your research.

 Links:

Website: www.dunnschoolbioimaging.co.uk

Twitter: @DunnSchoolBIF

Images:

Top: Dr Errin Johnson, EM Facility Manager

Middle left: A variety of specimens imaged on the Tecnai 12 TEM: negatively stained protein aggregates (top left; image by E Johnson) and thin sections of a mouse embryonic fibroblast cell (image by E Johnson), the blood-brain barrier in mouse (bottom left; A Douglas, DPAG) and a very happy nucleus in a Leishmania sp. cell (J Valli, Dunn School).

Middle right: SEM micrographs of: Drosophila (top left), E. coli (top right), a monocyte and a macrophage (bottom left), and a T-cell killing a melanoma cell (bottom right). Imaged on the JEOL JSM-6390 by E Johnson.

Bottom: Joshua Long, a PhD student in the Fodor group in the Dunn School, using the TEM.