Elucidating cell structure in 3-D by automated electron microscopy

The fine structural detail within cells has traditionally been examined using either scanning EM (SEM) or transmission EM (TEM). TEM is performed through a single thin section of the structure of interest and therefore often just gives a 'snapshot' 2D image. SEM gives a 3D-like image but only images the surface of the structure and not the detail therein. With TEM images, researchers often question what is happening above and below the single section and, to answer this, serial-sectioning is required. This is very time-consuming and technically difficult - the orientation of each section cannot be controlled and, therefore, 3-D-reconstruction of the digital images from these multiple serial sections requires frequent re-alignment that takes many hours. Even the most skilled technician will lose some sections resulting in incomplete series. Remarkably, these problems can be overcome by the 3View system, an automated sectioning and image capture system incorporated into a high resolution SEM. Computer-controlled, multiple sections and images can be made with one sample and 3-D-reconstructions performed without the need for realignment. This results in the best features of TEM and SEM being combined to show how cells are organized - both internally and in relation to their neighbouring cells and environment - at unprecedented levels of spatial information. Our immediate research projects using this equipment will cover a wide range of subjects - how do cells of the heart develop structures to beat every second? how do mitochondria - the 'batteries of the cell' - re-model themselves under different conditions? how can stem cells transform themselves into different cell types? how does the brain makes split-second judgments and more. Completely new understanding of these subjects will arise from being able to produce high resolution 3-D images of the structures of interest with this technology.

We request the purchase of an advanced scanning electron microscope (SEM) incorporating the Gatan 3View2.XP system to enable high-resolution 3-D-reconstruction of cell and tissue components. A high-precision ultramicrotome is operated within the SEM and allows the automated acquisition of 3-D ultrastructure by sequentially imaging the serially sectioned resin block face. Serial block face imaging and 3-D volume EM is particularly suited to studying the spatial arrangement of cells and their organelles and will enable a step-change in our ability to reconstruct intracellular and intercellular structural arrangements that underpin fundamental biological processes.
The theme common to all the projects is the need for 3-D imaging at an x-y resolution, and a z-axis depth, far higher than can be achieved with confocal microscopy. Only with EM resolution, and 3-D volume analysis, will the ultrastructure of cellular compartments and their interactions with the surrounding environment be fully explored. 11 Newcastle University research groups investigating mammalian and non-mammalian cell biology, supported by Research Council (BBSRC, MRC) and Charity (Wellcome Trust, BHF) funding, will be the first to benefit from in-house access to this equipment for topics including: developmental and age-dependent remodelling of striated muscle, mitochondrial reorganization during cell division and ageing, CNS neuronal cell polarity and plasticity, synaptic formation in the visual cortex, primary ciliary formation in developing and diseased kidney, and mechanisms of stem cell commitment.

The 3View system is step-changing technology and is already revolutionising how we look at cellular ultrastructure in terms of learning about biological function. The immediate impact of this technology will be on the researchers whose projects are outlined in this proposal. During the following months this impact will extend to other internal and external researchers as seminars, workshops and demonstrations are held to highlight the potential of the equipment. Access to this technology will open up new applications to these researchers and allow then to study their structures of interest in a way that has previously been either difficult or impossible. Significantly, the automated nature of the data acquisition, its scale and the time efficiency all means that these opportunities for discovery are available to everyone with the sample and question - not just the dedicated EM researcher who is prepared to spend months painstakingly doing a single manual 3D reconstruction. However, the acquisition of the 3View system and the ability to explore cellular ultrastructure in three dimensions will have an impact beyond the academic environment. There will also be a commercial impact - one of the co-applicants (C Rind) is working with industry to design collision sensors based on the locust visual system for use on cars. In the long-term we envisage further commercial involvement particularly in the area of bioengineering.

More immediate impact will come with the opportunities for public engagement. The EM facility regularly hosts groups of school children who learn about EM as part of the A-level curriculum. Illustrating to them 3D-reconstructions, often in step-by-step movie format, rather than 2D 'snapshots', will truly open up their learning experience about the spatial features of intracellular structures and cell-to-cell arrangements in ways not previously feasible.