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Apoptosis - when discretion is better than valour David J. Harrison, Department of Pathology, University Medical School, Teviot Place, Edinburgh, EH8 9AG Apoptosis describes a morphological series of events which culminate in fragmentation of the cell and its engulfment by adjacent or incoming phagocytes. The discrete series of events that make up apoptosis implies both an energy dependent and genetically regulated process. It is for this reason that programmed cell death which occurs in many situations during embryological development often is effected by the process of apoptosis. In pathophysiological terms apoptosis is critically important in the response of cells to injury. Cells which sustain severe injury have a number of options. First, they may endeavour to repair and to recover structure and function. The risk of this approach is that repair may not be adequate and particularly if this refers to DNA there may be opportunity to acquire mutations which ultimately lead to cancer. Second, the cell may resist the toxic or damaging stimulus and try valiantly to preserve the integrity of cell membranes at all costs. The problem with this approach is that if the cell's defences are overwhelmed there is a catastrophic loss of membrane integrity with spillage of cell contents and resultant inflammation. This is the process known as necrosis. Third, the cell may have a series of measures and thresholds which will determine at certain levels of injury that future viability is compromised. In these situations, the cell may "opt" to die by a process of apoptosis. Because apoptolic bodies are membrane bound there is no inflammation, as cell contents are not released. In this situation, it is clear to see that apoptosis may have an important function in determining the response to many different injuries, but particularly relevant may be its role in the prevention of tumour formation.
En bloc optical sectioning of resin-embedded specimen using a confocal laser scanning microscope Ian M. Roberts, SCRI, Invergowrie, Dundee, Scotland This paper describes the use of the phenomenon of glutaraldehyde-induced autofluorescence and the optical sectioning facility of the CLSM to obtain images which allow accurate determinations of the depths of structural features within resin blocks down to c.200µm and, when required, to create 3-D images of such features. Preliminary examinations were usually performed on whole specimens embedded in Araldite or in LR White resins with a block face size of c.3x2mm and with the specimen mounted in a microtome chuck. It was often convenient to pre-trim several blocks at the one time for subsequent examination, and in these instances, the specimens were held upright on glass slides using Blu-Tack adhesive putty to allow both transmitted light and laser illumination. Specimens could be examined using low power objectives, or at higher magnifications through oil mounted directly on the block faces. Blocks which had been irrradiated by the CLSM were compared with blocks which had not been treated in this way. The structural appearance of cell contents and organelles remained unchanged, as did the more detailed features of structures such as plasmodesmata, nuclear pores, viral inclusions and chloroplast grana. When comparisons were made between tissues with or without Safranin O staining, the fluorescence was intense compared with autofluorescence, but no other differences were observed and the IGL of virus particles and viral coat protein in the cytoplasm and in plasmodesmata remained the same The method is simple and reliable, and removes most of the uncertainty normally experienced when looking at specific structures for serial section studies.
Cathodoluminescence microscopy Adrian Finch, Department of Environmental Sciences, University of Hertfordshire, Hatfield, HERTS AL10 9AB. Cathodoluminescence (CL) is the emission of light (generally close to or in the visible range) from materials when bombarded by electrons. The nature and spatial variation in the CL emission is an important indicator of the electronic structure of materials, and its variability across macroscopic crystals. CL has been particularly important in the Earth Sciences, where it has been adopted as a tool in the study of rocks. 'Flood-gun'-type CL equipment is relatively cheap to buy, and analysis of rock thin sections can be completed in a few minutes. Highly photogenic views of CL zoning in minerals often result. Applications have included processes of diagenesis in carbonates, since subtle variations in pore fluid chemistry in carbonates rocks give rise to clear zonation in the CL emission. More recently, CL has also been applied to igneous and metamorphic rocks and has been particularly informative about the interaction of rocks with mineralising fluids. Zonation in the CL emissions of feldspars change in response to fluid interaction and thereby can provide information about not only the volumes of rocks that have experienced fluid interaction, but also the mechanisms by which fluids pass through the crust. Petrological interpretation of CL textures is parallelled by studies attempting to determine the causes of luminescence. Luminescence in carbonates is controlled by Mn2+-activation giving an emission in the yellow region, set against quenching processes that result from the presence of Fe2+. However, CL of many minerals is poorly understood, particularly in complex natural solid solutions such as feldspar. In these cases, CL relates to defect concentrations and states of order. CL zoning therefore provides a means of visualising defect distributions and variations in state of or order in crystals.
Blackcurrant fruit development in three dimensions by NMR microscopy and complementary techniques. S. M. Glidewell1,
B. Williamson1 G. H. Duncan1 J. A. Chudek2,1-
Scottish Crop Research institute, lnvergowrie, Dundee. The development of fruits of blackcurrant (Ribes nigrum L.) cv. Ben Alder from flower to maturity was studied non invasively by NMR microscopy, using attached and detached fruits, and the images were compared with those from low temperature scanning electron microscopy (LTSEM) and conventional resin histology. NMR microscopy allows non-invasive imaging of entire live tissue in 3 dimensions, illustrated in this presentation by animations of serial slices of the same specimen in mutually orthogonal directions and rotations of maximum intensity and surface-rendered projections to allow visualisation of complex structures such as the vascular system and the growth of the arillus. The NMR signal intensity is a function, not only of mobile proton concentration, but of relaxation rate, thus allowing the production of a variety of contrast patterns without the use of stains by selection of appropriate imaging protocols, to reveal different aspects of structure. However, it is only by the use of more established microscopical techniques that the origin of the NMR images may be fully understood. This is illustrated by the appearance of the vascular tissues in the pericarp and placentae as small bright cores surrounded by dark regions, within a matrix of moderate signal intensity. Conventional microscopical studies showed that the bright core discernible by NMR imaging encompassed an entire vascular bundle whereas the darker surrounding region represented small parenchyma cells with pronounced intercellular gas spaces. Other regions of the pericarp which included extremely large parenchyma cells, however, had few intercellular spaces and consequently gave rise to brighter regions of the image.
High resolution cryotransfer techniques for both SEM and TEM Judith Brock, Oxford Instruments, Eynsham, Oxford The advantages of low temperature techniques are well known, in that they stabilise the hydration of a sample and reduce beam heating effects. With the improvements in EM column design, high-resolution imaging has become a routine tool in most labs. High-resolution cryo TEM is now used to study molecular information from both isolated macromolecules, single particles and assemblies such as viruses. The sample is first vitrified in ice, then transferred to the microscope using a TEM cryotransfer system such as CT3500. Imaging then takes place in low dose mode to reduce beam damage. Cryo transfer protects the sample from contamination by the use of retractable cooled blades while the holder is protected from frosting by a nitrogen purged sleeve. The ultra lightweight materials used in the holder design allow a guaranteed resolution in excess of 0.34 nm. SEM resolution can now exceed 5nm. As a result SEM cryotransfer systems have also been improved to give sub 10 nm resolution. To achieve such resolutions high vacuum, high resolution coating and vibration free imaging conditions are key. Addressing all these issues, Oxford Instruments has recently launched ALTO 2500, a new high-resolution cryo system to replace the successful CT1 5OOHF. This new system combines user friendly electronics with a versatile high vacuum preparation chamber. Now there is a significant overlap in the level of performance of cryo transfer for both SEM and TEM. Oxford Instruments remains committed to continue pushing the limits.
John Lucocq, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH In mitotic and okadaic acid treated mammalian cells the Golgi apparatus shrinks to form aggregates of remnant tubular structures lacking cisternae. Most likely, shrinkage results from membrane loss driven by arrested endoplasmic reticulum (ER) to Golgi transport combined with continued recycling from post-ER compartments. To better understand membrane dynamics in the ER Goigi region during mitosis and okadaic acid treatment, we have used quantitative immunoEM and biochemical techniques to locate the site of ER-Goigi transport arrest and also assess the degree of protein recycling from the ER-related intermediate compartment and Golgi stack. Our results indicate that under these conditions, Golgi stack residents do not recycle to the ER. However ERGIC53, a protein of the intermediate compartment, recycles and accumulates in the ER. Accumulation of ERGIC53 and other forward directed proteins in the ER is most likely a result of restricted COPII coated transport vesicle formation at the ER membrane. Thus an inhibition of transport vesicle budding at the ER membrane is a key event in driving the structural changes to the secretory pathway of mitotic animal cells.
Microscopy in studies of plant surface, structure and function *C.E. Jeffree, †S. P. Hoad and ‡J. Grace, *University of Edinburgh, Institute of Cell and Molecular Biology, †Scottish Agricultural College, Crop Systems Department, Bush. ‡University of Edinburgh, Institute of Ecology and Resource Management The properties of the cuticular membrane, the plant's superficial permeability barrier for water, solutes and gas exchange, are known primarily from measurements made on intact, astomatous cuticles of substantial area, isolated from pristine leaves. Conceptually, the cuticle is therefore often treated as a homogeneous layer covering the plant surface. However, throughout the life of leaves, cuticles are subject to continuous chemical and physical degradation by insects, wind action and pollution. The characteristics of this damage, its consequences for plant performance, and its implications for the responses of plants to wind and pollutant stress have been difficult to observe directly. Observations from Low-temperature scanning electron microscopy of leaf surfaces collected into a cryo-specimen storage system in the field, coupled with a variety of other simple imaging techniques, have enabled the detection of a spectrum of microscopic injuries to the cuticle which breach cuticular integrity in a spatially heterogeneous manner, and which account for responses of plants to wind and pollution damage that are otherwise difficult to explain.
Visualisation of PrP protein in uninfected and scrapie infected spleen Diane Ritchie, Karen L Brown and Moira Bruce, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh, EH9 3JF Scrapie is a member of the Transmissible Spongiform Encephalopathies (TSEs), a neurodegenerative disease which occurs naturally in sheep and goats. TSEs are characterised by a number of pathological changes, including the accumulation of a protease resistant protein (PrP) in the central nervous system (CNS). Although the major pathology associated with this disease is confined to the CNS the peripheral lymphoid system is important in the pathogenesis of the disease in both natural and experimental models. Within the peripheral lymphoid system, PrP protein is associated with follicular dendritic cells (FDCS) in both normal and scrapie infected mice. FDCs are believed to play an important role in the replication of the scrapie agent outside the CNS. To examine in more detail the association between PrP protein and FDCs we have challenged mice with the ME7 strain of scrapie and a control group with an uninfected brain homogenate. Animals from both groups were perfused at various timepoints following infection, spleens removed and vibratome sections examined using immunohistochemistry. Fluorescent labelled markers were used to establish the relationship between PrP and FDCs following infection. immunohistochemistry. The development of these protocols and results will be presented at the meeting in November.
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