Quantitative analysis of astrocytic processes

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1) Three dimensional analysis of astrocytic processes by means of the computer tomography (CT) technique and high voltage electron microscopy : Thick sections (2-3 m thick) of protoplasmic astrocytes hippocampus and cerebral cortex of rats were prepared from Golgi stained and plastic embedded materials. Series of tilted high voltage electron micrographs were taken by tilting the specimen stage from -60 to +60 with 2 intervals. Three dimensional morphometrical analysis of these tilted images were performed by means of computer tomography technique. We used an ONIX REALENGIN II computer for image analysis. We succeeded in three dimensional imaging of strocytic processes. The research of estimating the surface area and volume of the astrocytic processes are now in progress.2) Astrocytes in various brain areas were stained immunocytochemically. Using an anatibody against glial protein, S 100, and observed under the light microscope. Images were similar to those stained with the Golgi impregnation technique. CT analysis of astrocytic processes was performed using the immunostained specimens and high voltage electron microscopy.It was found that the contrast of the immunostained material was not high enough for the high voltage electron microscope CT analysis. We are trying to find out other contrasting techniques for high voltage electron microscopy.3) We made a special objective lens to get a parallel electron beam at the specimen position, and to make possible in positioning the objective aperture on the back focal plane of the objective lens. As a result the contrast of the specimen improved very much, and further improvements in our research can be expected.