The Electron Microscope Facility

FEI G2 Polara, 300kV transmission electron microscope. The Polara has a dedicated cryo-stage and a coherent, electron beam source, which gives the ability to generate high resolution, high quality, tomographic and single particle data of extracellular matrix assembliesThe 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

The electron microscope facility is a core facility open for use by any user who wishes to visualize their samples in the nanometer to micrometer scale. The facility supports the research of several research groups within the Cell-Matrix Centre, for example:

ImmunoEM: Immuno-electron microscopy. Immuno localization of type I collagen in a section through embryonic tendon. The immuno-gold has labeled the collagen fibrils in the extra cellular matrix (black dots on top of striated collagen fibres), but it also labels some of the intracellular compartments (black dots within vesicles in the central cell).

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.

An example data set generated by the Gatan 3view. The volume is made from one thousand 100nm slices each imaging an area 40µm x 40µm. This gives a total volume of 40x40x100µm. A cube of data has been removed to demonstrate the perfect alignment between the sections.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.

Baldock: Electron microscopy with image analysis is used as part of a multi-technique approach by the Baldock lab to study the nanoscale structure of ECM molecules. A range of electron microscopic techniques is being used. Single particle TEM is being used to study the structure of regulators of TGFbeta and BMP signalling combined with solution biophysical approaches such as X-ray scattering. Large microfibrillar assemblies formed by collagen VI and fibrillin have previously been studied using negative staining TEM. The Baldock group is now furthering these studies with cryo-electron microscopy with help from experienced EM staff. Cryo-electron microscopy allows images to be collected at liquid nitrogen temperatures in order to examine the proteins in a near-native state. In addition a range of techniques are being used to examine collagen VI in the pericellular matrix surrounding chondrocytes. The 3view machine has been used in order to map overall cell matrix architecture then combined with electron tomography to examine individual collagen VI microfibrils surrounding the cartilage cells.

Tomographic reconstruction of the base of a fibripositor made from three 300nm thick sections. The colours have been chosen to highlight the collagen-containing compartment (cyan) and the cytoskeletal elements (red). The insert shows a virtual slice from the reconstruction.

Streuli: Electron microscopic studies of mammary glands at the early stages of lactation, in both wild type mice and those with deleted integrin signalling genes. The distribution and organisation of cells in lactating alveoli and collecting ducts are being examined by a combination of 3view serial section reconstruction and standard TEM. The combination of these techniques provides an overview of the 3D structure of individual alveoli at nanometre resolution, and allows examination of cell-matrix and cell-cell junctions within the malformed alveoli that arise from altered integrin signalling.
Woolner: Studies of spindle pole bodies using three-dimensional electron microscopy. The structure and organisation of spindle pole bodies are being examined using a combination of TEM and 3view. The increased resolution of electron microscopy allows detailed examination of the position of the centrioles, the organisation of cell-cell and cell-matrix junctions and an appreciation of the overall morphology of the dividing cells. The work in the EM facility compliments work in the Bioimaging facility; examination of spindle poles by fluorescence microscopy is currently limited by the resolution of the light microscope and the need for specific fluorophores/antibodies, but the specific stains allow clear identification of the features being examined. In electron microscopy everything is visible so the skill of the EM facility staff is used to identify the organelles by their structure and morphology.