High resolution 3-D reconstructions from brain tissue via Field Emission scanning electron microscopy in back scattered electron (BSE) imaging mode

The central issues of our current research are the cellular mechanisms that contribute to the function and modification of individual central synapses. For over 20 years we have utilised high-resolution transmission electron microscopy (TEM) and 3-dimensional (3-D) reconstructions of neural ultrastructure in the hippocampus of the central nervous system, mainly of rodents, following use a several learning and plasticity paradigms. The TEM methods used have advanced considerably in recent years with application of computer controlled stage positioning, while the use of film to capture images has been supplanted via application of high resolution digital cameras on electron microscopes. This has enabled production of digital images where the resolution is at least the equal of film derived prints and thus has facilitated rapid acquisition of a much greater number of images. However, while advances in computing power have enabled more rapid processing of images, the process of making 3-D reconstructions remains a very time intensive process with little automation of the processes being feasible. Alternative approaches in the ability to study connections between synapses and spines have been suggested by optical advances in at light microscope level but because of resolution limitations these do not permit with any precision, measurements of the contacts between nerve cells - synapses. Scanning electron microscopes (SEMs) are much more suited to automation than TEMs and can easily work with much larger samples. It has already been shown that field emission (FE)-SEMs working in back scattered electron (BSE) imaging mode with conventionally prepared and stained TEM sections can produce excellent resolution and contrast images, comparable to TEM images. Our strategy will therefore be to develop a novel method to make montages, and reconstructions of a large area of serial sections of mouse or rat hippocampal tissue. Our study will be a collaborative effort using state of the art facilities at JEOL UK (SEM and TEM manufacturers). The key of the new method to make such a gigantic montage is the automatic image acquisition system with very accurate stage control of the SEM (in position terms). Software would automatically acquire multiple images from each of a number of sections. Images from each section can be montaged to form single, large montages of each section, which can then be combined into a large-scale 3-D reconstruction. We believe that this methodology can then be applied to our regular studies of synaptic and dendritic plasticity.

The aim of the proposed research is to enhance automation of production of high resolution 3-D reconstructions of brain tissue via Field Emission scanning electron microscopy (FE-SEM) in back scattered electron (BSE) imaging mode. We will produce reconstructions of similar quality to those of our existing 3-D methods using our JEOL 1400 electron microscope of 3-D reconstruction of brain samples. Brain tissue will be fixed as per our standard fixation perfusion protocols of rats but a higher than normal concentration of osmium tetroxide will be used (4%) to improve back scatter signal in the FE-SEM. Ultrathin Serial sections of rat brain tissue at ~ 50 -70 nm in thickness will be collected on carbon coated slot grids (2mm x1mm) and attached to a 13mm aluminium stub using a double sided carbon sticky tab. A collaboration with JEOL UK will enable the stub to be placed in the chamber of a state of the art FE-SEM at JEOL UK headquarters at Welwyn Garden City. For imaging with an FE-SEM the surface of the stub is coated with carbon to avoid specimen charging. The FE-SEM to be used will be a JSM-7001F equipped with a high precision stage. The BSE imaging is employed to obtain contrast of fine intracellular structures. A YAG detector is used for BSE detection. Image acquisition will take place using an automatic process with automatic correction for astigmatism and brightness/contrast. Analyses of data and 3-D reconstructions. The images collected from the SEM will be collected using JEOL 3D reconstruction software on a 64-bit PC which is available in our electron microscope suite. Reconstructions can then be made from the images which have been automatically collected from the FE-SEM system. We will analyse the images using our 3-D systems and comparison will be made of image quality, time saving with 3-D reconstructions of similar fixed brain tissue where a number of reconstructions are made and prepared using our traditional TEM 3-D methods.

Beneficiaries from this research The main beneficiaries of the body of knowledge arising from this research are expected to be firstly, the academic community, who will be able to utilise our development of methods of automation of tissue and mineral samples, for example in brain research after 3-D analyses of brain circuitry after experiential treatments. This will be of particular importance in helping to develop further ideas and research on memory formation, and how this may be relate to cognitive dysfunction, especially that associated with ageing. Secondly, data from our project could have importance for the imaging manufacturing community who seek to advance methods of automation of image collection and analyses. How benefits will be realised Staff working on this project will have developed and utilised skills, which are of very considerable importance in the imaging and analyses fields, not only in neuroscience but also in the wider imaging community medical and pharmaceutical fields, and thus be attractive to employers. The methodologies involving will employ cutting edge microscopy techniques and cellular imaging neuroscience methodology that puts us in the forefront of biomedical research. The project will be developed in 3 phases with milestones at 3, 12 and 18 months, ending with a comparison of image quality, and time saving compared with traditional 3-D reconstructions of similar fixed brain tissue where a number of reconstructions are made from ~100 TEM serial sections. This research will be innovative and internationally competitive with the best research worldwide. Ensuring engagement of beneficiaries of research Communication and track record: Plans for the engagement for users will include peer-reviewed publications in high impact journals, presentations at international meetings and public engagement and dissemination of science to the broadest possible audience, which both of the main applicants have very considerable experience as evidenced by their previous records. The beneficiaries will be engaged by presentations at meetings, publications of our results in peer-reviewed journals, reviews in more general journals, and information placed on our web sites in understandable language. Furthermore, we will communicate key findings though our public relation offices at the Open University. Prof. Stewart together with the postdoctoral research assistant (PDRA) will present the research findings at meetings and write to ensure dissemination of research findings to the media. Prof Stewart has extensive experience of talking to the media (the BBC and newspapers and magazines about his key research results), and also the lay public and is completely committed to the dissemination of science to the broadest possible audience. Collaborations and exploitation Prof Stewart has worked with JEOL UK over a number of years, our first JEOL electron microscope was purchased over 20 years ago and we have frequently helped with demonstrations of equipment and techniques, so have a long standing relationship which has been of mutual benefit, though this is the first opportunity for a larger scale development of a new research method. JEOL have made clear as in their letter of support that their main interest is in a successful outcome for the project and have not specified any other demands. However, the Open University will be interested in the IP aspect and any potential commercial exploitation should the programme go ahead. The PDRA will be responsible for initiating and running the various dissemination activities, including open source and workshops. Peer-reviewed publications arising from this grant will be registered on the Open University's open access institutional repository - Open Research Online (ORO) at http://oro.open.ac.uk. ORO is now one of the largest HEI repositories in the UK with over 850,000 visits from people in 170 countries since 2006.