By means of X-ray microanalysis it is now practical to detect approximately 10(-19) g of an element in a static-probe analysis within an ultrathin section, with analytical spatial resolution in the range 20--30 nm. The main difficulties for biological microanalysis are connected not with sensitivity but with specimen preparation and beam damage. Careful cryopreparation, beginning with the quench-freezing of a small block of tissue, is essential even for determining the storage sites, or sites of binding in vivo, of physiologically active elements. In frozen-dried or frozen-hydrated sections of quench-frozen tissue, it is now possible to measure local mass fractions of diffusible as well as of bound elements.
Giant axons from the squid, Loligo pealei, were fixed in glutaraldehyde and posttixed in osmium tetroxide. Calcium chloride (5 mM/liter) was added to all aqueous solutions used for tissue processing. Electron-opaque deposits were found along the axonal plasma membranes, within mitochondria, and along the basal plasma membranes of Schwann ceils. X-ray microprobe analysis (EMMA-4) yielded signals for calcium and phosphorus when deposits were probed, whereas these elements were not detected in the axoplasm.
I N T R O D U C T I O NMany cellular processes appear to be mediated by reversible interactions of ionized calcium with the plasma membrane. We have been interested in using the electron microscope to locate membrane areas with high affinity for calcium. Addition of calcium ions to the solutions used for fixing and processing tissues for the electron microscope causes opaque deposits to be formed along the plasma membranes of a variety of ceils (I). In insect intestine, for example, deposits occur on apical membranes (microvilli) and along septate but not along gap junctions (2). Apparently, particular regions of the plasma membrane, or some structure closely associated with the membrane, can sequester enough calcium to make the region adjacent to the membrane opaque to electrons. Although we do not know precisely how the deposits form, we suspect that the presence of calcium ions during processing keeps intraceUular binding sites saturated. The deposits were not observed readily in the past because of the widespread use of osmium, which decalcifies tissues unless excess calcium is present in the fixative.To assess the significance of the deposits adjacent to the membranes, one must know their composition and how they form. The present study was undertaken to utilize the rapidly developing technique of electron-probe X-ray microanalysis (3, 4) to study the composition of the deposits. The analytical electron microscope is a transmission electron microscope fitted with X-ray spectrometers that analyze X rays generated when a micro-area of the specimen is bombarded by the electron beam. Since each element produces X rays of characteristic energy and wavelength, information is obtained about the composition of the specimen. Microprobe analysis is well suited for analyzing bound ions, since conventional fixation and embedding techniques can be used.
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