Life and Medical Sciences
Cell & Developmental Biology
Wiley InterScience Backfile Collection 1832-2000
Natural Sciences in General
Electrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosections are cut with a LKB cryoultramicrotome at temperatures of -130 to -140°C, freeze-dried at 10-6 Torr vacuum and carbon-coated before analysis. The freeze-dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMol/kg d.w.) of Na, Mg, P, S, Cl, K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x-ray microanalysis can be successfully applied to heart physiology.
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