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SUMMARY Some problems of the quantitative analysis of diffusible elements in cryosections are reviewed. The two prevalent methods for obtaining concentrations from X‐ray data, one based on characteristic radiation alone and the other on continuum‐normalization, are recapitulated. Both methods seem suitable at cellular level while the latter seems preferable at finer spatial resolution. Recourse to both methods together is desirable in the analysis of frozen‐hydrated sections especially when there is no peripheral standard. Selective local contamination is a particular hazard in the analysis of chlorine. In the case of sodium, physical parameters set restrictive limits to the minimum concentration measurable by ‘energy‐dispersive’ X‐ray spectrometry (about 20 mm kg−1) and to the spatial resolution attainable by diffractive X‐ray spectrometry (∼0·2 μm). One obvious danger to meaningful quantitative analysis is inadvertent redistribution of diffusible elements during the moments preceding the freeze‐quenching of a tiny piece of tissue. Data are presented to show that concentration changes due to simple evaporation are a real hazard prior to the quenching of sub‐millimetre size samples.

The principle of the microprobe analysis of chemical elements is illustrated in Fig. i. Some kind of radiation is directed on to the specimen, generating signals characteristic of the elements present. Local analysis in situ is achieved in one of two ways. Most often the impinging beam is finely focused so that the signal at any moment comes only from a selected microregion. Alternatively, in some instruments, the impinging beam floods a larger region but the emergent signals characteristic of a particular element may be selected and focused to give an elemental ‘map’ or ‘image’.

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Mature nematocysts of the sea anemones Rhodactis rhodostoma and Anthopleura elegantissima contain a fluid that has a high concentration of solutes and is extraordinarily rich in calcium (ca. 500-0 mmol/kg wet weight); this contrasts with the surrounding cytoplasm which is rich in potassium but poor in calcium. The undischarged capsule is surrounded by a membrane that probably acts as a selective permeability barrier between the cytoplasm and the nematocyst fluid. During discharge the nematocyst moves to the surface of the nematocyte and comes into contact with the external sea water medium. Calcium, which may be bound to proteins in the undischarged state, is rapidly lost from the fluid; at the same time, sea water enters the capsule. In vitro experiments have already shown that calcium loss increases the osmotic pressure of the capsular fluid, causing an influx of water from the external medium; this influx appears to increase the hydrostatic pressure inside the capsule to the point that the thread everts explosively.Nematocysts are stinging organelles found in cnidarians; each one consists ofa closed capsule which contains an inverted harpoon-like thread that can be discharged explosively. Although the structural events during eversion of the thread have been described in detail (1), little is known about the mechanism of discharge (2-5). Recently, Lubbock and Amos (6) found that discharge could be initiated in vitro by the removal of bound calcium from the fluid inside the nematocyst; calcium removal increased the osmotic pressure of the nematocyst fluid, thus producing an influx of water from the external medium and an increase in the internal hydrostatic pressure of the capsule. It was notable that alterations in the permeability of the capsule wall to water did not seem to be involved in controlling discharge in vitro. At that time, the relevance of the in vitro experiments to nematocyst discharge in vivo was not clearly established because data were not available on the ionic changes occurring under natural conditions.In the present study, we examined, by x-ray microanalysis of frozen sections, the elemental composition of both excited and unexcited nematocytes in the sea anemones Rhodactis rhodostoma and Anthopleura elegantissima. Our results enable us to present a generalized model of the events that take place during excitation. MATERIALS AND METHODSMesenterial filaments from R. rhodostoma were placed in synthetic seawater (Tropic Marin Neu; specific gravity, 1.021) containing 16.8% (wt/wt) dextran (250,000 daltors; Sigma, clinical grade). To quench freeze, filaments with droplets of dextran/ sea water were placed on the truncated conical tips of copper pins and rapidly immersed in a well-stirred slush of (monochlorodifluoromethane, m.p. -181'C) (7-9). In certain cases, filaments mounted on copper pins were electrically stimulated <3 sec before instant quenching in order to induce nematocyst discharge. Acrorhagial tissue was obtained from A. elegantissima by removing inflated acrorhagi with ...

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