SUMMARY The quantitative X‐ray microanalysis of ultrathin biological sections is exemplified by a recent study of the distribution of calcium in mineralizing cartilage and bone. The determination of calcium mass‐fraction in one microarea of a vesicle within a section of rabbit epiphyseal plate cartilage is presented in detail in order to display all steps of the processing of the data. Mass fractions are obtained from an equation which is approximate but which is adequately accurate for most cases of interest, specifically for ultrathin biological sections where the analysed area consists predominantly of organic matrix. We shall examine the physical assumptions behind the computation, and the extent to which these assumptions have been verified in the analytical microscope. The purpose of this paper is to describe in detail the scheme of data collection and processing, which has now been in use for some years in the Cavendish Laboratory, for the measurement of local elemental mass fractions in thin biological specimens in instruments like the EMMA. After an outline of the physical theory we shall describe the handling of the data by means of working through an example of an actual measurement, and finally we shall add some comments, mainly precautions and reservations.

Summary Conventional preparative procedures for the examination of tissues in the electron microscope involve the use of fixatives, dehydration in alcohol or acetone, embedding in plastics and staining. Such procedures remove soluble components and are therefore often unsuitable for chemical analysis of naturally occurring electrolytes. Ultrathin frozen sections of unfixed, unembedded biological tissue can be cut onto dry glass knives, freeze‐dried and viewed in the electron microscope without staining. Morphological detail is sufficient to identify cell types and ultrastructure. X‐ray microanalysis in the analytical electron microscope (EMMA‐4) has shown that highly soluble electrolytes can be detected and that intracellular compartments are retained.

Observations made during the freezing and thawing of mouse and human oocytes and mouse embryos with the cryomicroscope suggest that physical factors as well as physicochemical factors may play a role in the development of lethal damage upon thawing. The point of contact with the approaching ice front may predispose that area to the appearance of future cytoplasmic blebbing. The ice front distorts the oocyte and this distortion remains during its subsequent thermal history and is unrelated to desiccation distortion. Ice initiates the formation of both intra- and extracellular gas bubbles which are apparent upon thawing; with the progression of the thawing process they can be seen to grow in volume. Growth of these bubbles can give rise to expanding vesicles which can totally destroy an embryo. The consequences of these physical factors for the successful cryopreservation of oocytes, embryos, tissues and organs are discussed.

SYNOPSISA new technique of autoradiography is described for studying the incorporation of soluble isotopes and for work on unextracted materials. No solutions that might leach out soluble compounds are used until after the exposure is complete. Frozen sections are cut in a cryostat which is kept in the darkroom and mounted directly onto cover-slips coated with Kodak AR.10 stripping film with the emulsion upwards. Melting of the sections is avoided by exposing at -20 to -30°C. The autoradiographs are given a quick fixation in 5% acetic acid in alcohol immediately prior to development. The method is simple and effective and the section is in close and secure contact with the emulsion 80 giving very good resolution.