Quantification for the X-ray microanalysis of cryosections

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.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

Theoretical prediction of the electron backscattering coefficient for multilayer structures

Based on existing formulae for the backscattering coefficient for coated samples, i.e. for samples consisting of one film on a massive homogeneous substrate, a theory for the backscattering coefficient for multilayer structures is derived in a clear mathematical way without using approximations. The resulting expression is simple and ready to be applied in scanning electron microscopy or other related analytical techniques.     

Calculation of concentration depth profiles in electron probe microanalysis

Based on a multiple reflection model, the concentration depth profiles of gold diffused into a silver substrate are determined. To conclude the structure of the sample from the measured intensity of radiation, an accurate correction procedure is necessary. A new model was developed for this purpose. Since the new electron probe microanalysis (EPMA) correction procedure is also valid for tilted samples, measurements with different geometries could be performed. The different angles of incidence and take-off angles allow for a better comparison of theory with experiment. One advantage of the applied multiple reflection model is that it can be extended comparatively easily with the analysis of layered specimens. Measurements of some specimens with one layer on a substrate are reported. The mean deviation of calculated thicknesses is about 10%. To calculate depth profiles, gold was evaporated with physical vapor deposition on silver foils. The thicknesses of evaporated gold films were 50 and 100 nm. Then the specimens were annealed at a temperature of approximately 400°C. Finally, the concentration depth profiles were quantified with EPMA. The shape of the profiles was derived from a simple diffusion model valid for the samples under consideration. To check the shape of the functions obtained, a comparison with measurements by secondary ion mass spectrometry was performed.     

Monte Carlo modeling of secondary x-ray fluorescence across phase boundaries in electron probe microanalysis

Secondary fluorescence induced by photoelectric absorption of x-rays generated by an electron beam can occur when the characteristic x-ray energy of material “A” exceeds the critical excitation energy of material “B.” An expression is developed to calculate secondary fluorescence across a planar boundary from a discrete source placed at any (X, Y, Z) coordinates relative to the boundary. The expression can be incorporated into a Monte Carlo electron trajectory simulation which calculates the discrete distribution of primary x-ray generation.     

Cryofixation methods for ion localization in cells by electron probe microanalysis: a review

Electron probe microanalysis data on the intracellular content and distribution of electrolyte ions depends critically on the functional state of the cells at the moment of cryofixation. Whereas tissue specimens often require special in-situ freezing techniques, isolated and cultured cells can be frozen within their environmental medium under physiologically controlled conditions. Thus, they represent a feasible system to study functional ion-related intracellular parameters such as the K/Na ratio. Specifically modified freezing devices allow the study of ion shifts related to dynamic processes in cells, for example, locomotion and exocytosis. The time resolution achieved by time-controlled cryofixation is approximately 1 ms.     

The influence of oxide surface layers on bulk electron probe microanalysis of oxygen—application to Ti-Si-O compounds

This paper discusses a generalized method to measure with the electron probe microanalyzer (EPMA) the oxygen in a material containing a surface oxide layer. The continuum background is the most difficult to measure, particularly for materials in which oxygen-free samples cannot be produced. The method depends on the preparation of either oxygen-free samples or well characterized oxygen-containing samples. Specific application of the method to the Ti-Si-O system is discussed. In addition, measurements of oxide surface-layer thickness of 3.6–8.0 nm on Ti and Ti-Si compounds were obtained using EPMA and a scanning Auger microprobe (SAM). The nature of the oxide surface layers was shown using x-ray photoelectron spectroscopy (XPS).