Identification of microstructure and localization of ions in unfixed deep frozen muscle sarcomeres

Cold thin sections (1400 Å) of rabbit psoas muscle deep frozen in vivo were examined at -150°C in the scanning electron microscope using a special cold stage designed for vacuum compatability of hydrated tissue and transfer system to preclude frosting. Identification and localization of ions in the scan beam path is achieved by detection of their fluorescent X rays. Myofibrils and bands generally corresponding to A, I and Z of the sarcomeres are identified. Myofilaments are not seen; rather myofibrils appear to be composed of bundles of previously undescribed longitudinal elements, 500 to 1000 Å thick. In comparison to standard electronmicrographs Z bands appear relatively broader and are traversed by the longitudinal elements. Independent experiments verify that freezing per se does not alter basic architecture down to the myofilament level. Comparison of frozen sections of tissue previously fixed with frozen unfixed tissue reveals marked difference in electron density of the optical image without striking changes in general structure. Fluorescent X ray analysis of the spatial location of constituent ions clearly identifies all basic elements heavier than Na. Quantitative analysis is in progress.