The Electron Microscope Facility
The Electron Microscope Facility provides experienced staff members who work with the researchers in the Cell-Matrix Centre to obtain high-resolution 2D and 3D images of cells and the ECM. The facility houses three transmission electron microscopes and an environmental scanning electron microscope. We also have equipment for preparing samples for cryo-electron microscopy, which can be visualised on our 300kV G2 Polara. A recent acquisition is the Gatan 3view machine, which allows us to easily generate serial section reconstructions through significant volumes of tissues and constructs. The new addition of the Gatan 3view (serial section generation within an electron microscope) to the electron microscopy (EM) facility has increased the scale and scope of techniques available to users from within the Centre. Users are able to examine macromolecules at the sub-nanometre resolution using single particle techniques on our 300kV G2 Polara. Moving up to nanometre resolution; interactions between matrix molecules and membrane proteins can be examined using standard transmission electron microscopy (TEM) on our two 120kV T12s.
Standard TEM can be complimented by electron tomography to generate three-dimensional reconstructions of sections of tissue. The 3view machine offers the ability to examine longer distance interactions at near TEM resolutions. The addition of the 3view machine to the facility has allowed us to bridge the gap in resolution and scale between the images generated within the Bioimaging core facility and the high-resolution imaging generated on our transmission electron microscopes. The facility also has expertise in single particle reconstruction, offering insight into protein folding without the need for crystallisation; electron tomography provides high-resolution reconstruction of a tissue samples.
Supporting the Cell-Matrix Centre
Kadler: The Kadler laboratory makes use of most of the EM facility instruments. The G2 Polara has been used to generate tomographic reconstructions of dispersed collagen fibrils in order to examine the microfibrillar structure. The T12-Twin is used to generate scanning transmission electron microscopy (STEM) mass maps of dispersed fibrils in order to examine the number of molecules per unit length in a fibril. The T12-Biotwin has been used by the Kadler group to examine sections of developing tissues and artificial constructs in order to look at the cell matrix interface and to examine the distribution, organisation and overall morphology of the collagen fibrils. Examination of collagen fibril deposition has been the driving force for the development of improved sample preparation and staining for the 3view machine. Embryonic collagen fibrils range from a few μm to mm in length, but are only ~30nm in diameter. Techniques have been developed by a direct collaboration between EM facility staff and the Kadler group in order to generate imaging protocols that allow tracking of entire embryonic fibrils in developing tissues and artificial constructs. The ease with which we can now examine tissue architecture at resolutions sufficient to identify and track individual collagen fibrils has, for the first time, allowed 3-dimensional electron microscopy to be used as a readout for biochemical experiments.
Kielty: Transmission electron microscopy has been used to examine the deposition and organisation of elastic fibres and collagen VI at high resolution. Working closely with the EM facility staff the Kielty laboratory has developed stains to reveal the organisation and patterning 54 that occurs during elastic fibre deposition. TEM has recently been complimented by 3view studies that have used the increased resolution of the 3view machine to expand on observations that were first seen using light microscopes from the bioimaging facility.