|
|
|
Low-temperature strategies for immunocytochemistry: choice or compromise J.N.Skepper, Multi-Imaging Centre, Department of Anatomy, University of Cambridge.. A bewildering number of choices of immunogold based labelling techniques confront electron microscopists. Some have been developed for use on heat or chemically cured resins, some for low temperature embedding strategies and others for use with unembedded, thawed cryo-sections. It is painfully obvious that methods need to be tailored to meet individual requirements. Antigens differ vastly in their response to fixation, dehydration and embedding. There are invariably compromises to be made between the contrasting requirements for the retention of antigenicity and the preservation of ultrastructure. I hope to demonstrate that by asking appropriate questions to the generators of antibodies ad undertaking simple pilot studies the choice of technique can be dramatically simplified. Quantitative immunoelectron microscopy reveals 2,6 sialytransferase is concentrated in the central cisternae of rat hepatocyte Golgi apparatus John Lucocq and Caroline Lovelock, Department of Anatomy and Physiology, University of Dundee. The Golgi apparatus is a membrane bound organelle involved in synthesis of N-linked oligosaccharides which are trimmed and then lengthened by a series of sugar transferases adding N-acetylglucosamine, galactose and sialic acid in sequence. We previously published qualitative work which localized Galb1,4GlcNAc 2,6 sialyltransferase of rat hepatocytes to trans cisternae and Trans Golgi Network. We now report the use of combined stereological and immunoelectron microscopical techniques for mapping the Golgi stack composition and distribution of sialyltransferase protein in rat hepatocytes. The Golgi stack showed substantial variation in composition consisting of 1,2,3,4,or 5 cisternae with an average of 2.5 cisternae. Sialyltransferase labelling was mainly located in the central cisternae of the Golgi stacks irrespective of whether the stacks were orientated in a cis/trans direction using morphological criteria. Only 20% of the total sialyltransferase labelling was present in the trans-most cisterna and 2% in the Trans Golgi Network. The low labelling in the trans-most cisterna was essentially due to the presence of a sialyltransferase negative cisterna. These data emphasize the importance of quantitation in obtaining a representative picture of Golgi enzyme distribution in three dimensions. They suggest that central cisternae, rather than the trans-most cisterna and Trans Golgi Network, function in sialation along the secretory pathway of rat hepatocytes. Confocal Microscopy with high light budget T. Wilson, R. Juskaitis and M.A.A. Neil The key elements in the optical system of a confocal microscope are the point source and point detector. The use of these has the effect that only light which has arisen from the focal region is detected efficiently since the point pinhole detector serves to attenuate greatly light from out of focus regions of the object. However the use of a single pinhole source and detector means that only one object point is imaged and so it is necessary to introduce scanning to image a finite region of the object. Further drawbacks to this single point source/point detector approach are that the image is not usually obtained in real time and also incandescent light sources are not usually sufficiently bright and hence lasers are usually used. We will describe a new form of confocal microscope in which there is no scan and traditional white light sources may be used and the image is formed in real time at T.V. rate. In order to be able to replace the laser source with an incandescent white light source it is necessary to increase the light budget of the system. One approach would be to build many confocal systems in parallel. This is the approach adopted in the tandem scanning microscope which employs an aperture disc containing many pinholes. However since it is necessary to avoid cross-talk between neighbouring 'confocal systems' it is necessary to space the pinhole apertures far apart. In practice this means that only about 1% of the available light is used for imaging and that the aperture disc must be rotated (scanned) in order to 'fill in' the gaps between the points in the object samples by the neighbouring confocal systems. However these systems do reduce real time confocal images with non laser sources. Our approach is to increase the light budget further and to obviate the need for scanning by placing the pinhole apertures as close together as possible. This will result in efficient use of the available light but a method of preventing cross-talk between neighbouring confocal systems must be found. we achieve this by using an aperture mask which is programmable in the sense that the transmissivity of the individual pixels may be changed in time. We use aperture correlation techniques to eliminate cross talk. The gene for the green fluorescent protein (GFP) from the jellyfish Aequorea victoria was cloned in 1992 and since then GFP has become one of the most important marker proteins available. GFP has been expressed from a potato virus X (PVX) vector and used to pinpoint areas of infection and to track the progress of the virus as it moves throughout the plant. Constructs of PVX has been prepared in which the GFP is produced either free in the cytoplasm of infected cells or fused to the viral coat protein. In the later case, virus particles become fluorescent and naturally occurring aggregates of particles are easily visible within cells. Since GFP is also visible under UV illumination, it is now possible to determine the patterns of viral spread at a whole-plant level. Confocal laser scanning microscopy (CSLM) has been used to detect the GFP-labelled virus at both high magnifications, across tissues. By dual imaging GFP with more traditional fluorescent markers, such as Texas Red, it is possible to relate the spread of PVX to the underlying vascular system of the plant. The main advantages of CSLM are that live tissue can be studied non-invasively and in real time, allowing dynamic changes in the tissue to be monitored. Finally, at a subcellular level, electron microscopy can be used to detect the presence of either virus or GFP using antibody labelling. Gold labelled thin sections of tissues can then be related to light micrographs, or used to add subcellular detail to lower magnification images obtained from the confocal microscope. By using a variety of imaging techniques to detect GFP, it has become possible to gain a broader understanding of the movement of viruses, such as PVX, within their host plants. Differences between ESEM and Low Vacuum SEM illustrated with application examples. Dirk van der Wal, Philips Electron Optics, Eindhoven Both ESEM and Low Vacuum SEM allow a gas in the specimen inspection chamber. The principle functionality of the gas is charge neutralisation, which allows imaging and analysis of non-conductive specimen without applying conductive coating layer. Differences between Low Vacuum SEM and ESEM are not well known, and can be summarised as follows:
The above differences and their implications for SEM imaging will be illustrated with application examples. Staining resin sections: why infiltration is (and should be) incomplete R. W. Horobin, Division of Neuroscience & Biomedical Systems, Institute of Biomedical & Life Sciences, University of Glasgow. For effective staining of resin sections, infiltration must be incomplete. This is so as the resin, whether cross-linked and hydrophobic as used for TEM or water-miscible as used in LM, will exclude and limit staining. In fact incomplete infiltration is routine, as shown by:
What structures are poorly infiltrated, and hence easy to stain?
Material neither hydrophilic nor dense is usually well infiltrated, and so harder to stain. these rules of thumb apply to all types of resins. They are understandable in terms of viscous, rather lipophilic media not easily penetrating impermeable and hydrated structures. Influences of incomplete infiltration on staining
Practical problems: case examples
Efficient and accurate 3D measurements in microscopy - unbiased stereology Vyvyan Howard & Matt Reed,Fetal and Infant Toxico-Pathology, University of Liverpool. The past decade has seen the appearance and maturation of a whole technology for the precise and accurate measurement of volumes, surfaces, lengths and numbers of features in 3D space from histological sections. There has been a radical change of approach compared to previous methods in that no assumptions about feature shape, orientation or spatial distribution are required. The accuracy of the estimates relies solely upon the design of the sampling and the mathematically proven rigour of the estimators, which means that they are guaranteed to work without the need for validation studies (in contradistinction to the model-based approach). The designs of methods for estimating volumes (Cavalieri principle) and the total number of cells in an organ (Disector principle) will be presented and examples from developmental pathology presented. Modern design-based stereological methods are now gaining widespread acceptance in neuroscience and in quantitative toxico-pathology. Drug licensing authorities such as the US FDA are beginning to ask for total cell counts in drugs that may affect neurological development. These methods in biology are still performed manually with the use of interactive computer programs, complete and reliable segmentation being very difficult to achieve in low contrast images. However if segmentation is achievable, as is often the case in materials science then completely automatic software for making stereological measurements is also available (Kinetic Immaging Ltd). A Royal Microscopical Society handbook on the subject will be published in January 1988. Minimal preparation procedures for SEM of botanical specimens. Stephan Helfer, Royal Botanic Gardens, Edinburgh Preparation procedures lead to artifacts. In many cases these artifacts are useful and desirable, as in the preparation of pollen grains using acetolysis, or in the removal of surface waxes and dust particles from herbarium specimens using CPD and involving solvents like ethanol or acetone. However, for some delicate specimens, such as powdery mildew conidia, minimal preparation procedures are required to preserve the morphological arrangement of structures. These procedures consist of no pre-treatment or of sputter coating only. Minimal procedures have themselves limitations, and can only be used for short times of exposure of specimens to the conditions in conventional SEMs. Data and examples of successful and less successful specimen preparation are shown. Alternative techniques such as ESEM are discussed. |