The Advanced Microscopy team specialise in imaging microbes, animal and human tissue including live bacterial microscopy and super-resolution microscopy.

Light Microscopy

We have many different light microscopes to fulfil a variety of experimental needs.

• Olympus BX53: Upright brightfield and widefield fluorescent microscope with colour camera and a stage with -195 °C to 420 °C temperature controls.
• Zeiss AxioImager M2: Upright brightfield, phase contrast, DIC and widefield fluorescent microscope with monochrome and colour cameras. Stage controls for sample tiling.
• Zeiss LSM880 with AiryScan: Upright pinhole confocal microscope with monochrome camera. Stage and environmental controls for live cell imaging.
• Leica M165c Stereoscope: Dissecting stereoscope with colour camera.
• DeltaVision Elite: Inverted brightfield and fluorescence microscope with a monochrome camera. Equipped with quantitative deconvolution and full environmental controls.
• DeltaVision OMX FLEX: Inverted brightfield, DIC and fluorescent microscope with four monochrome cameras dedicated to individual light sources. Equipped with deconvolution, IRIS line-scanning confocal, EDGE confocal, super-resolution (SIM, TIRF and Localization Microscopy). Stage and environmental controls for live cell imaging.
• INCell 6500HS HCA Analyzer: High content analyser for imaging microplates and slides. Imaging modes include Brightfield, DIC, Phase Contrast, Fluorescent wide-field, IRIS line-scanning confocal, and EDGE confocal.

Electron Microscopy

Unlike optical microscopy, which uses photons to illuminate samples, electron microscopes (EMs) use a focussed beam of electrons to image the sample. Due to the shorter wavelength of electrons than photons, some EMs can resolve objects of about <1nm, compared with 200 nm for optical microscopes. This enables detailed visualisation of structures such as viruses, proteins and extracellular vesicles (EVs) as well as bacterial cell wall surface structures, channels through cell membranes etc.

Electron microscopy is typically divided into transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The main differences being that TEM can be used to image inside a specimen (after sectioning and staining) and SEM creates an image of the exposed surface of a specimen. Although there is some overlap between the two techniques. TEM, with negative staining preparation, creates images of whole structures, such as EVs, viruses and flagellated bacteria. Immunolabelling can be carried out of TEM specimens (sections or whole cells) using a secondary antibody labelled with a gold ball (~10nm) to identify the location of specific proteins. SEM micrographs give the appearance of a 3D image with a much greater depth of field than an optical microscope.

We make use of the equipment at the John Innes Centre BioImaging Facility. The EMs available are:

• FEI Talos F200C TEM: a 200 KV, FEG (field emission gun) TEM, fitted with 3 cameras. It can be used for cryo-EM, micro-ED, tomography, viewing resin sections and negative stained samples.
• Zeiss Gemini 300 SEM: a FEG SEM, with Quorum cryo system (cryo-fixed samples/freeze-fracture work) and Gatan 3View system (serial block face SEM) to allow collection of 3D volume data. High-vacuum only, 4 detectors (secondary electron (SE) detector, in-lens detector, standard back-scatter detector and Gatan OnPoint back-scatter detector). Beam deceleration stage. Automated image acquisition, image tiling, array tomography applications.
• FEI Nova NanoSEM: a FEG SEM, with Gatan cryo-system. Advanced optics (including two-mode final lens), therefore capable of sub-nanometre resolution. Optimised for low kV work, beam deceleration, high and low vacuum modes, CBS (backscatter detector), EDS and STEM detectors.

Sample preparation

To assist in the preparation of your samples, we have a fully equipped preparation laboratory located close to the imaging facility. We can offer dyes, fixatives and some consumables to help, particularly if you want to test a technique, get some preliminary data or try a new microscope.

Training and useful information

We regularly offer training days and workshops on various techniques in the facility.

We also recommend the two online resources which are free to undertake:

• MyScope by Microscopy Australia is an interactive tutorial site that gives information on both light and electron microscopy.
• Image Processing and Analysis for Life Scientists is an interactive seminar course run by EdX. You will need to register but the course is free if you complete it within 7 weeks. It details how to analyse microscopy images.

Banner image credit: Glowing guts by Dr Aimee Parker and the Advanced Microscopy Facility