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.

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle . In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles . The Electron probe X-ray microanalysis also showed a highly significant (P < 0 .001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P . Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions . Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum . X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions . The presence of Sr was similarly verified in the sarcoplasmic reticulum . These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle .

The electron-probe x-ray microanalyser is normally used to study surfaces of thick specimens. A method is given here for the microanalysis of thin films. The film thickness is monitored by using the continuous x-ray spectrum. The ratio of the numbers of characteristic quanta to continuum quanta gives a measure of the concentration of an element in the film which is independent of the thickness of the film. The accuracy of the method is approximately 8% M.P.E. for films in the thickness range 0-0·5 mg cm−2.

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SUMMARY Difficulties in the quantitative X‐ray microanalysis of frozen sections may conceivably arise from ice‐crystal damage and from electron‐beam damage. X‐ray peak‐to‐continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice‐crystal formation existing prior to sublimation. The experiments in these reports are re‐interpreted here; it is argued that peak intensities may be affected by ice‐crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum. The accuracy of the peak‐to‐continuum method is affected by beam‐induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold‐stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 μm frozen‐hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, can be completed before substantial mass loss occurs. However, analysis of frozen‐hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss. Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X‐ray analysis at very high resolution.