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Immunocytochemistry of Endomembrane Dynamics in Plant Cells. Chris Hawes, School of Biological & Molecular Sciences, Oxford Brookes University, Oxford, OX3 0BP. Although the components of the secretory pathway of plant cells have been well documented, little is known about the dynamics of the organelles involved and the mechanisms behind transport of secretory products through the system. We have been using a combination of immunocytochemical location of endomembrane compartments by light and electron microscopy combined with use of the drug brefeldin A, a well characterised inhibitor of secretion in mammalian cells. Brefeldin A treatment of roots induces a rapid rearrangement of the Golgi apparatus followed by a trans-driven vesiculation of the Golgi stacks into vesicular aggregates of "BFA compartments in which secretory products accumulate. These have been proved with the Golgi marker antibody JIM84 and various antibodies to cell surface components using whole cells and confocal microscopy for immunofluorescence and low temperature embedding in LR White for immunogold labelling. The deconstruction of the Golgi is reversible both by removal of, and by prolonged incubation in the drug. No evidence was found of any redistribution of Golgi membrane into the endoplasmic reticulum. Recent work in collaboration with the SCRI using virally expressed jelly fish green fluorescent protein and confocal microscopy to image the endomembrane system has shown the brefeldin A also inhibits transport from ER to the Golgi in tobacco cells and also induces a major rearrangement in the organisation of the ER. References: SATIAT-JEUNEMAITRE, B., COLE, L., BOURETT, T., HOWARD, C. & HAWES, C. (1996). Brefeldin A effects in plant and fungal cells: Something new about vesicle trafficking? Journal of Microscopy. 181, 162-177. HAWES, C. & SATIAT-JEUNEMAITRE, B. (1996). Stacks of questions: how does the plant Golgi work? Trends in Plant Science. November '96. BOEVINK, P., SANTA CRUZ, S., HAWES, C., HARRIS, N. & OPARKA, K.J. (1996). Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells. The Plant Journal. In press. A simple immunogold silver staining (IGSS) technique for light microscopy of resin sections Ian M. Roberts, Scottish Crop Research Institute, Dundee, Scotland One major advantage of immunogold silver staining (IGSS) for light microscopy is that, compared to immunogold labelling (IGL) for electron microscopy, much larger areas of sections can be screened for the presence of a particular antigen. A simple method for such comparisons is to cut 8-10 resin sections (0.5-1.0 um thick) immediately after cutting ultrathin sections, and collect them on c. 5x20mm glass strips cut from cover slips. After careful drying, the strips and sections are placed in 1.5 ml Eppendorf tubes for IGL; two strips can be processed in each tube by placing them in back-to-back. IGL is done as standard using 250-300 ul volumes of reagents (which can be kept and reused), and the strips are washed by thorough agitation in a beaker of distilled water followed by standing in 2-3 ml of water for 10 minutes. After draining and drying, the strips are inverted on to 2-3 mm beads of "Blu-tack" adhesive putty on a glass slide to form a ramp with the sections closest to the slide, and a 25 ul drop of the sliver staining reagent is carefully pipetted under the strips where it is held by capillary forces. The sections are incubated for 5-15 min in a small, light-tight chamber then the reaction is stopped by immersing the slide and strips in a beaker of water. The strips are removed and individually washed with distilled water, drained, dried and mounted on slides for examination. The technique is simple and reliable and can be used to confirm IGL studies on ultrathin sections by electron microscopy. Principle and benefits of Energy Filter Transmission Electron Microscopy Richard Bauer, LEO Elekronenmikroskopie GmbH, D-73457 Oberkochen With the integration of an Imaging Energy Filter into a Transmission Electron Microscope (TEM) column, a new TEM was created which comprises improved imaging capabilities and outstanding analytical properties. This new type of TEM is called Energy Filtering Transmission Electron Microscope (EFTEM). In a TEM an image is formed by scattering contrast, diffraction contrast and phase contrast. All electrons which are passing the objective aperture are used for imaging. Inelastic scattered electrons which are also caused by scattering of beam electrons in a specimen cannot be influenced by a TEM. Due to their small scattering angle, they are always part of imaging electrons. However, inelastic scattered electrons loose energy and have other properties than elastically scattered, or unscattered electrons. On one hand they blur the image and reduce the image contrast, on the other hand inelastic scattered electrons carry important structure specific, element information and chemical information and cannot be used. Energy filtering enables the selection of electrons according to their energy and bandwidth. EFTEM permits elimination or reduction of inelastic scattered electrons for high contrast imaging and diffraction of thin and thick samples. A new imaging mode which is only available in EFTEM's enables imaging of selected inelastic scattered electrons. This imaging mode offers structure sensitive or element sensitive contrast. Another potential of this method is elemental mapping or chemical imaging with highest lateral resolution. Another benefit of energy filtering is the fact that an imaging energy filter has excellent properties as an energy spectrometer to record Electron Energy Loss Spectra (EELS). EELS is a sensitive qualitative and quantitative method to analyse the chemical composition of thin specimen. Due to high energy resolution, elemental information as well as molecular information can be detected. In principle, an energy filter does not limit the Microscope for other applications. An EFTEM equipped with a scanning attachment and an EDS system is a superior and universal tool for microscopy and analysis of thin, thick and bulk samples. Johnson, A.D., Nicholson, W.A.P.*, Elder, H.Y. & McGuigan, J.A.S.+ ,Institute of Biomedical and Life Sciences and *Department of Physics and Astronomy, University of Glasgow, Scotland, and +Department of Physiology, University of Bern, Switzerland In a microanalytical project to determine the quantitative distribution of Mg in myocardial cells, good morphological resolution and fidelity of elemental location are required of the subcellular compartments, especially that of SR.Freeze-dried cryosections yield higher resolution, greater image contrast and better signal to noise ratio of X-ray spectra than do frozen hydrated or resin embedded sections. However, freeze-dried sections are hygroscopic and prone to ultra-structural degradation and elemental translocation during atmospheric exposure. Freeze-drying for structural and elemental retention should be performed under precisely controlled conditions and over a longer timescale than previously considered (Edelmann, 1994). Most commercially available freeze-driers and transfer systems are inadequate. We describe a new system comprising a gas tight cartridge in which cryosections are transferred through every stage of the preparation process. An integral component of this process is a high performance freeze drier (Elder et al.,1986), modified to accommodate the gas tight cartridge and to allow for the remote operation of the cartridge lid. Once dried, the sections are transferred in the cartridge, now at ambient temperature, into the chamber of an Emscope TB500 coating unit, also allowing remote operation of the cartridge lid. The final transfer of the sections is into a dry-gas flow TEM side entry stage, for analysis. The system excludes water vapour at all stages and is combined with a freeze drier that allows great flexibility of drying protocol. The Authors gratefully acknowledge support from the British Heart Foundation. Edelmann, L. (1994). Optimal freeze-drying of cryosections and bulk specimens for X-ray microanalysis. Scan. Microsc., Suppl. 8, 67-81. Scanning Microscopy International, Chicago (AMF O'Hare), 1160666 U.S.A. Elder, H.Y., Biddlecombe, W.H., Tetley, L., Wilson, S.M. & Jenkinson, D. McE. (1986). Construction of Low Temperature Freeze Driers. E.M.S.A. Bulletin, 16, 111-113 Variable Pressure Scanning Electron Microscopy - Principles and Applications Peter Clark, LEO Electron microscopy Ltd, Clifton Road, Cambridge CB1 3QH The concept of high pressure electron microscopy has been around for many years, but has recently come to the fore with the development of the Variable Pressure Scanning Electron Microscope, the VP SEM. This breed of instrument has a dual personality, a familiar conventional SEM in part, that can be easily and rapidly transformed into a flexible instrument, overcoming many of the limitations of its other half. The reaction of many, when confronted by the variable pressure concept, for the first time, is to remark "Err... Yes... Nice... but what can it do for me". The scope of this presentation is to help those people to answer that all encompassing question themselves. The first part of the equation is to look at some of the limitations of the conventional analytical SEM that were the driving force for the development of the VP SEM. From there we will look at the VP SEM in it physical form, examining how the concept has been put into reality, and how the vacuum control system of the instrument works in both conventional mode and VP mode. Fundamental to the VP SEM is the interaction of the electron beam with both the specimen, and the "atmosphere" within the sample chamber, we will look at positive and negative effects of this interaction on the imaging and analysis of the sample. Finally, we will look at the imaging and analytical applications and techniques that are developing through use of the VP SEM, and explore application areas that may benefit from use of a VP SEM in the future. Visualising neurons using Golgi impregnation and confocal imaging of intracellular dyes. Brown D, Halliday WG* and Fraser J. NPU, Institute for Animal Health, Edinburgh. EH9 3JF, * CVL, Lasswade Laboratory, Edinburgh EH26 0SA The single section Golgi impregnation technique rapidly stains single neurons 70 um vibratome sections from paraformaldehyde/glutaraldehyde perfused brains. A modification of this technique was used to compare the morphology of individual pyramidal cell neurons in the hippocampus of normal brain injected and scrapie infected mice. These single neurons were observed for differences in shape and numbers of dendrites and also for numbers of dendritic spines. Using this technique spine loss from scrapie infected neurons was quantified. The limitations of this technique are that it only stains a few neurons at random, and the entire dendritic tree cannot be seen. To overcome these problems, a fluorescent dye, in 200 um vibratome sections from brains perfused with 4% paraformaldehyde. The sections were imaged using a Biorad 600 confocal laser scanning microscope. This gave a 3D picture of these neurons, providing much more information for analysis, especially the depth of the neuron and dendrite and spine numbers. Image Archiving Dr Andrew Yarwood, JEOL (UK) Ltd, Silver Court, Watchmead, Welwyn Garden City, Herts. AL7 1LT. The current trend towards the use of digital imaging in scientific laboratories is gathering pace as the quality of the results improves. Electron microscopes have used digital imaging output for some time. Improved digital imaging resolution, quality and output now means that many electron microscopists are now seriously considering converting traditional wet chemistry darkrooms into digital "darkrooms". This lecture will outline the methods used to digitise, archive and output electron microscope images. The advantages and limitations of digital imaging will be discussed, with particular reference to the problems encountered when handling large image files. Reference will be made to products currently available to the electron microscopists, including products which can update existing electron microscopes to high resolution digital image acquisition systems. Examples of digital output from a selection of electron microscopes and printers will be shown. A novel image analysis and data visualisation method for the study of biological structures. Steve Campbell, Department of Obstetrics and Gynaecology, University of Glasgow, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER Many researchers have avoided the use of image analysis when describing staining patterns in tissue sections and have instead used qualitative scoring systems. This is partly due to the inherent complexity of many biological structures and the need to use a variety of coloured stains to provide useful information for the observer. We have sought to make the task of interactive image analysis technically easier. We have carried out interactive image analysis and simple thresholding on tissue sections of human and cow uterus in which carbohydrates were localised with a lectin. These sections were neither counterstained to reveal the cell nuclei nor dehydrated at the end of the staining procedure.Sections were mounted in aqueous medium and epithelial glands were examined by manipulating the condenser settings or by using Nomarski optics. The outlines of the glands were traced manually and a variety of measures of gland staining used. In some circumstances it may also be relevant to know how the features that are being analysed vary over the area of the tissue section. This particularly applies to situations where spatial gradients exist. We have therefore devised a way of graphically displaying the data in combination with the microscope image by a technique that preserves spatial information. In order to examine gradients of carbohydrates within the lining of the cow uterus we have used the technique of texture mapping to project the image on to the surface of a thin 3-D slab. Microscope images treated in this way can be therefore be viewed and rotated almost like any other flat solid object seen in 3-D perspective. The data was represented graphically as cones emerging from the section where the height of the cones was proportional to the size of the parameter displayed. The data therefore emerges like mountain peaks from the section representing the spatial distribution of the image analysis measurements The combined image of section and data cones was then examined at a variety of tilts and rotational angles so that the data distribution could be better understood. |