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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.

A previous study demonstrated that connecting the human body to the earth during sleep (earthing) normalizes the daily cortisol rhythm and improves sleep. A variety of other benefits were reported, including reductions in pain and inflammation. Subsequent studies have confirmed these earlier findings and documented virtually immediate physiologic and clinical effects of grounding or earthing the body. It is well established, though not widely known, that the surface of the earth possesses a limitless and continuously renewed supply of free or mobile electrons as a consequence of a global atmospheric electron circuit. Wearing shoes with insulating soles and/or sleeping in beds that are isolated from the electrical ground plane of the earth have disconnected most people from the earth's electrical rhythms and free electrons. The most reasonable hypothesis to explain the beneficial effects of earthing is that a direct earth connection enables both diurnal electrical rhythms and free electrons to flow from the earth to the body. It is proposed that the earth's diurnal electrical rhythms set the biological clocks for hormones that regulate sleep and activity. It is also suggested that free electrons from the earth neutralize the positively charged free radicals that are the hallmark of chronic inflammation. A relationship between cortisol and inflammation was established in the pioneering work of H. Selye published in the 1950s. Current biomedical research has led to an inflammation hypothesis that is establishing chronic inflammation as the culprit behind almost every modern chronic illness. The research summarized here and in subsequent reports provides a basis for a number of earthing technologies that restore and maintain natural electrical contact between the human body and the earth throughout the day and night in situations where going barefoot on the earth is impractical. It is proposed that free or mobile electrons from the earth can resolve chronic inflammation by serving as natural antioxidants.