This is an interdisciplinary Project between applied physicists, and structural and cardiovascular biologists. This collaboration will develop and implement the methodology required for imaging the elemental composition of cell membranes and organelles at 2nm resolution and protein structure at 1nm resolution. Major objectives to be achieved through these methods include: mapping functionally distinct intracellular Ca2+-storage sites in vascular smooth muscle and endothelial cells; quantitatively localizing the concentration of Ca-bound to cardiac intercalated discs, gap junctions and to non-specialized regions of the plasma membrane and determining the chemical (sulfur containing basement membrane and determining the chemical (sulfur containing basement membrane or phosphorus containing phospholipid) components associated with bound Ca. A 200kV field emission gun (FEG) equipped electron microscope, available for the first time, will be used for X-ray mapping and scanning transmission energy filtered electron microscopy (STEFEM) required for compositional imaging of biological cryosections. Quantitative methods of signal collection and data analysis will be developed for biological electron energy loss spectroscopy (EELS). Tissues will be prepared by rapid freezing and cryoultramicrotomy. The quantitative localization of Ca2+-bound to cardiac membranes is expected to provide information about the Ca2+-dependence of normal and abnormal cardiac conduction. The compositional imaging methods to be developed will provide a unique and general approach to a broad range of physiological and pathological biomedical problems, and represent a major significant advance in analytical electron microscopy. The 200kV microscope will also be used for spot scan imaging of Ca2+-ATPase (Project 1), the problems of intracellular Ca2+-storage and Ca2+-binding sites on cardiac membranes are intimately related to the aims of Project 3, and this Project (5) will also provide electron microscopic support to Project 2.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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