Electron Microscopy : Technologies

The Phenom ProX SEM is a powerful but compact SEM that is similar in size to a PC tower. It is user friendly, and capable of imaging specimens from single cells to Drosophila eyes to spheroids.

The Zeiss Sigma Variable Pressure SEM with Gatan 3View is an automated 3D EM. The system contains a minituarised microtome within the chamber that removes slices of the resin block using a diamond knife, prior to imaging the blockface with the electron beam. The SBF SEM provides large volume 3D imaging at high resolution: blockfaces up to 1 mm x 1 mm can be cut and imaged with a pixel resolution of ~6 nm and a slice thickness of 15-200 nm over thousands of serial images.

The Zeiss Crossbeam 540 FIB SEM is an automated 3D EM that uses a beam of gallium ions to cut slices from the sample, prior to imaging the blockface with the electron beam. The FIB SEM provides targeted 3D imaging at extremely high resolution: blockfaces up to 100 μm by 100 μm can be cut and imaged with a pixel resolution of ~5 nm and and a slice thickness of 15-200 nm over thousands of serial images.

The FEI Quanta SEM with DELMIC SECOM light microscope is a novel system that incorporates both light and electron imaging in the same microscope. The system was purchased via a Next Generation Optical Microscopy Award from the MRC/BBSRC/EPSRC in 2013. We have developed and implemented super resolution light microscopy in the system, which enables us to localise fluorescent protein constructs to cellular structures with an accuracy of approximately 100 nm.

The FEI G2 Spirit Twin TEM is mainly used for imaging proteins and DNA prepared by negative staining or low angle rotary shadowing. The microscope also has cryo-capability and has been used to screen plunge-frozen microtubules and proteins in preparation for imaging experiments on more powerful EMs.

The FEI G2 Spirit BioTwin TEM with iCorr light microscope is mainly used for imaging resin-embedded cells and tissues. The microscope can run electron tomography with single- and dual-tilt holders, and has a new integrated light microscope allowing localisation of fluorophores immediately prior to electron imaging.

The Zeiss Axioscope widefield light microscope with Linkam cryo-correlative stage allows us to screen vitrified (frozen) cells prior to further processing.

Cells are either grown on carbon-coated EM grids and plunge-frozen in liquid ethane, or pipetted into 1.5 mm diameter planchettes and high pressure frozen at liquid nitrogen temperature.

Vitrified cells on grids can be imaged in a cryo EM or in synchrotron-hosted cryo soft X-ray microscopes; cells in planchettes are processed into resin for immunolabelling or imaging of preserved fluorescent proteins in integrated light and electron microscopes.

The Zeiss Xradia 510 Versa X-ray microscope is used for 3D imaging of biological samples from soft tissues to bone, with sub-micron resolution.

Used for neurobiology, developmental biology, tumour biology and micro-anatomy, the microscope is linked to the focused ion beam SEM through novel correlative software that allow us to identify regions of interest inside the sample for targeted analysis by 3D electron microscopy at nanometer resolution.

Cryo-SXT is used to image the subcellular structure of whole vitrified cells, as close to their native state as possible. Resolution can reach ~25 nm, and the properties of the soft X-rays mean that no chemical fixation or heavy metal staining is required.

Images of cells resemble those acquired from resin-embedded cells in TEM due to the absorption of the X-rays by cell membranes, but in a fully hydrated state.

Correlative cryo-fluorescence and cryo-SXT workflows have been developed with our collaborator Dr Liz Duke at the Diamond Light Source (Oxfordshire) to enable fluorescent protein localisation in near native-state cells.

Imaging is performed at beamline U41-TXM at BESSY II in Berlin, beamline BL09-Mistral at ALBA in Barcelona, and at beamline B24 at the Diamond Light Source.