Subcellular Stucture in Glial Networks by TEM

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The aim of this project is to study ultrastructural features at the supramolecular scale in glial cells in the zones where mitochondria and endoplasmic reticulum adjoin. Interactions between these two types of organelle are associated with calcium signaling, through which glial cells communicate with each other during central nervous system development and synaptic activity. To preserve glial networks in their physiological state, high-pressure freezing was used. Cultured cells were grown on sapphire discs where they could be assessed by optical microscopy. At the appropriate time, the discs were high-pressure frozen at liquid nitrogen temperature to preserve ultrastructure. Specimens were then freeze-substituted with osmium fixative before being embedded in plastic, sectioned and stained. Experimental conditions are being investigated that are suitable for structural studies. The aim is to image interaction zones of the glial cells in three dimensions by using electron tomography in a 300 kV energy-filtering transmission electron microscope.