The Oxford Consortium for Three Dimensional Electron Microscopy

Lead Research Organisation: Oxford Brookes University
Department Name: Faculty of Health and Life Sciences

Abstract

High resolution cell ultrastructure has been studied by transmission electron microscopy for more than 40 years and this method has revolutionised our understanding of the shape and form of organelles, the number in a cell, how they divide, how individual cells divide and the organisation of tissue. One of the biggest problems with these methods are that only very thin slices of cells can be imaged so a three dimensional view was very difficult to produce without complex serial sectioning and reconstruction techniques and it is also impossible to produce quantitative data. More recently transmission electron tomography has become the technique of choice for extracting high resolution 3-dimensional information from EM sections and is a very powerful technique for limited areas of a cell or tissue. The technique is still complex, and although serial tomograms can be made to observe the 3-D structure of whole cells, there are severe technical and time limitations if large areas of specimen in considerable depth need to be studied.

In the last decade two new techniques have emerged that we believe to be the next revolutionary change in how we image cell and tissue ultrastructure. These are focussed ion beam milling (FIBSEM), physically etching away the surface of a sample and serial block face microscopy (SBFM), cutting into the sample by removing thin sections from its face. Both techniques use a scanning electron microscope to image the surface of a resin block in which the sample is embedded. By continued removal of slices of the resin follwed by imaging the resin surface a series of images are collected that can be reconstructed into a three dimensional volume.

In this proposal we will establish a three dimensional imaging consortium in the Thames Valley area. Oxford Brookes University and Sir William Dunn School of Pathology, University of Oxford both have expertise in three dimensional cellular electron tomography with three members of staff trained at the internationally renowned centre for three dimensional imaging at University of Colorado, Boulder, USA and each PI on the grant have publications on three dimensional imaging. To date there are only 6 instruments in the UK, Manchester (x2), Leicester, UCL, CR-UK London Res. Inst. One is dedicated to materials science and 5 to biomedical research. Increasing the capacity and training of staff in these techniques and increasing the output of excellent research in these techniques should be considered essential to UK bioscience research. Most of our applications data has been carried out in Manchester where demand on the instrument is extremely high. There is no such facility in the Oxford region, nor a facility with expertise to handle plant material and advise plant scientists on specimen preparation.

There are 4 key projects from the PI's Departments on this grant application that we will undertake over the next three years and the underlying research for several of these is currently supported by BBSRC grants. Immediately a number of specific projects will be carried out on the structure of membrane systems within plant cells, the structure of the swimming apparatus of trypanosome (sleeping sickness) parasites, development of nuclear division mechanisms and membrane systems within the nucleus.

Technical Summary

Recent developments in SEM technology have permitted the development of high resolution instruments capable of imaging smooth block faces of resin embedded material at a resolution comparable with that of a conventional thin section biological electron microscope. The further development of the incorporation of an ultramicrotome inside the SEM chamber has revolutionised the collection of three-dimensional data for biological material through a technique known as serial block face microscopy SEM (SBF-SEM). Thus, it is now possible quantitatively extract 3-D data from large volumes or numbers of cells at high resolution.
This application is for the establishment of a regional three dimensional SEM facility in the Oxford area to serve the Oxford cell biology community, the UK trypanosome/cilia community and the UK plant cell community. Alongside this are 4 key projects from the PI and COI's Departments that will make extensive use of the system to establish a centre of excellence in the technique. SBFM-SEM will be used within existing grant funded work as follows. A: Selective membrane staining techniques will be used to investigate the 3-D organisation of endoplasmic reticulum (ER) at the developing cell plate in dividing plant cells, the organisation of the ER-plasma membrane-cell wall interface and the relationships between the ER and Golgi throughout the cytoplasm. B: The three dimensional organisation of the Trypanosome cytoskeleton will be elucidated as will the flagellar basal body in a range of mutants. C: SBFM-SEM will be used in a study of centriole/centrosome dynamics and organisation in Drosophila spermatocytes and embryos. D: A study of the function and three dimensional organisation of the nucleoplasmic reticulum.

Planned Impact

Potential impact from this programme will vary with each different project that the microscope is used in. In the longer term we also expect industrial users to have access to the facility which may result in direct commercial impact.
However, there are a number of beneficiaries of this research. The UK bioscience community will benefit as this is an emerging 3D imaging technology that could be used in most biological research communities. We believe that this an important technology for UK scientists to be trained in for future applications and the UK research community needs to build a good set of people that are able to train future scientists and disseminate knowledge of these techniques. There are potential beneficiaries within the commercial private sector as use of this technique could lead to new or modified cell preparation, imaging or reconstruction methods and these technical advances in microscopy could be transferred into commercial usage.

Three of main research proposals have impact in terms of wider Human and animal health. Abnormalities in flagella and cilia are now implicated in a number of Human diseases called the ciliopathies, which includes retinal degeneration, polydactyly, cystic kidneys, Bardet-Biedl syndrome, Kartagener's syndrome. Knowledge of basal body biogenesis and assembly of a eukaryotic cilium or flagellum is critical to understanding the basis of these diseases. The protozoan parasite Trypanosoma brucei, which causes African sleeping sickness in Humans and Nagana in cattle. The disease affects domestic animals, particularly cattle and is recognised as a major obstacle to the economic development of the rural areas affected in Africa. The flagellum of this parasite is now recognised as a major virulence factor in the maintenance and spread of the parasite. Knowledge of how the flagellum is assembled, maintained and regulated as cells divide and differentiate through the life cycle is important to fully understanding how this parasite spreads. Abnormalities of the nuclear envelope and lamina are also linked to a range of human and animal pathologies and as such a full undertanding of thier structure function relationships is required.
Finally the plant secretory pathway is vital to the biosynthesis of many food proteins, carbohydrates and lipids. As such an in depth knowledge of it structure-function relationships is necessary for full agricultural and biotechnological exploitation in terms of both increase capacity and for the production of novel products.

Publications


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Scheuring D (2016) Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression. in Proceedings of the National Academy of Sciences of the United States of America
 
Description This grant is to develop a facility for three dimensional electron microscopy using high resolution scanning elelctron microscopy. We have been using a specific osmium-based staining technique in plant material that highlights one set of cellular structures, the organelles of the secretory pathway. We are now able to reconstruct one organelle, the endoplasmic reticulum in three dimensions throughout a whole cell and for the first time in leaf epidermal cells. Work is now being undertaken to study the changes in the 3-D organisation of the endoplasmic reticulum when key proteins in controlling its organisation are disrupted or mutated.
Exploitation Route Collaborators in other plant cell biology labs. are starting to use this technique on material they will be imaging at the Brookes' facility.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology