HIGH PRESSURE FREEZING SYSTEM FOR ELECTRON MICROSCOPY

Emory University, Atlanta GA

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Abstract

Morphological and cytochemical data obtained from electron microscopy (E.M.) provide biomedical researchers with information on the nature of molecular components within cells and tissues. The high pressure freezing (HPF) method makes possible the cryofixation of bulk tissue specimens to a depth of 200mum for scanning and transmission E.M. without chemical fixatives or cryoprotectants. A HPF system for the Emory community would centralize a facility, within a specially engineered cryogenic E.M. laboratory located in the Integrated Microscopy & Microanalytical Facility (IM&MF). Administered through the Department of Chemistry, the IM&MF would route specimens within Chemistry and to research groups in Neurology, Yerkes Research Center (YRC) and at ACRI. The IM&MF will house, maintain, and operate the HPF system. Four groups will utilize cryofixed samples in ongoing NIH funded biomedical research involving neural and vascular tissues and giant vesicle systems. A 10-year NIH merit study of biological membranes employ the Giant Vesicle model (GV, 1-100 mum in diameter). GV posses a phospholipid bilayer that approximates the curvature of, and compartmentalize a volume similar to, plasma membranes of living cells. GV membranes are destroyed by ice crystal formation when plunged frozen. The Menger group would employ the HPF for GV vitrification and imaging of frozen, hydrated specimens by cro-HRSEM and cryo-TEM. The requested HPF system would provided quality cryofixation of vascular and neural tissue for cryo-ultramicrotomy and immuno-cytochemistry studies for 3 other NIH funded groups. (1) The Robinson group will localize extracellular matrix proteins (collagen, fibronectin, and factor VIII-related antigens) and cell surface integrins within vascular walls after endovascular radiation therapy used during obstructive coronary artery disease treatments. (2) The most abundant excitatory and inhibitory neurotransmitters in the mammalian central nervous system are glutamate and GABA. The Smith group is elucidating the subsynaptic localization of various types of ionotropic and metabotropic GABA and glutamate receptors on neurons of the basal ganglia. (3) The neuropathology of Huntington's disease is selective for degeneration of specific neuronal populations in the basal ganglia and cerebral cortex. Expansion of CAG repeats in gene 15 of chromosome 4 codes for a mutant form of the protein huntington, which associates with other proteins forming a cytotoxin in Huntington brain tissue. The Hersch group is localizing huntington within specific intraneuronal components and involved in Fragile X syndrome research.