X-ray microanalysis has become increasingly used to study the elemental composition of biological specimens and has provided valuable information on the identity and occurrence of diffusible and bound ions, inorganic deposits and elements that are structural components of macromolecules. The attractions of X-ray microanalysis to the potential investigator in terms of spatial resolution and range of elements that can be detected have already been mentioned in Section A. The analytical sensitivity in terms of minimal detectable mass (10−19 g) also presents considerable opportunity to the analyst, though the value for sensitivity on a mass fraction basis (normally no better than 0.1 % with energy dispersive systems) poses major limitations.

The diversity of applications can be considered both in terms of general areas of study and type of specimen examined.

Applications of X-ray microanalysis to different subject areas of biology

X-ray microanalysis has been used in a variety of disciplines within biology, as exemplified by the range of chapters in this section of the book. These areas include microbiology, plant biology, animal biology (vertebrate and invertebrate tissues, cultured animal cells), medicine and environmental biology. A computer survey of the relative numbers of papers in these different areas (Table D1) indicates a clear preponderance in the spheres of animal biology and medicine.

The role of X-ray microanalysis in human physiology and medical research has recently been reviewed by Shelburne et al. (1989), and is important in the study of aspects such as the activity of Ca in cellular processes, ion transport in epithelia and the possible importance of cations in mitogenesis and oncogenesis.

The technique of X-ray microanalysis has existed for considerably longer than is, perhaps, generally realised, having been invented in 1951 and first applied to biological material at the beginning of the 1960s (Hall, 1986). Much very useful pioneering work was carried out using wavelength-dispersive detectors. However, there can be no doubt that the introduction of the energy-dispersive detector based on the Si(Li) detector crystal, with its capability of multi-element analysis, and ease of operation led to the technique becoming more widely accepted. For the detection of the majority of elements the ease of use of the EDS detector offsets its lower resolution, and both the continued improvements in the Si(Li) detectors and the introduction of germanium detectors, will help to improve the sensitivity of the technique.

Biological specimens are many and varied with their own special problems for quantification, and perhaps the second most important development in X-ray microanalysis for biologists has been the commercial availability of software for carrying out quantitative routines. The procedure most commonly used for quantification is the continuum normalisation method of Hall (Hall, 1971). With the advent of the microcomputer in the early 1980s the first software for applying the Hall technique to biological specimens became available, and now the importance of quantification of spectra from biological specimens is recognised with all major detector manufacturers offering this option with their software.

Electron probe X-ray microanalysis (EPXRMA) is only one of a large number of techniques that are available for investigating the chemical composition of cells and tissues. These techniques use a wide variety of physical and chemical principles, and each has its own advantages and disadvantages (see Table B1). Some are non-microscopical and, at best, give information only at a rather coarse level of spatial resolution, although their chemical sensitivity may be very high. Among the microscopical techniques, many should be regarded as highly specialised, in the sense that the equipment is not easily available, and in some cases is not produced commercially. Other methods, such as histochemistry and autoradiography, may be regarded as routine, but do not, on the other hand, yield the same type of information as XRMA and other techniques. Indeed, while all the techniques listed in Table B1 can be used to study the chemical composition of cells or tissues, it seems that they are complementary rather than competing. For example, histochemical procedures can identify compounds, such as enzymes, which are not generally accessible by the other methods. As well as autoradiography, techniques such as SIMS (Thellier, Ripoll & Berry, 1991) and LAMMA, which can identify isotopes, are well adapted for the study of dynamic processes using tracers. Of the techniques considered in the following two chapters, particle-induced X-ray emission (PIXE) is most similar to EPXRMA, yet nevertheless shows important differences in specimen penetration, resolution and sensitivity.

Specimen preparation is the cornerstone of successful biological X-ray microanalysis. A damaged or compromised specimen cannot be ‘recovered’ by sophisticated analytical hardware or software, or even by a skilled operator. But what constitutes a ‘good specimen’ or an ‘acceptable degree of damage’? Is there such a thing as ‘the ideal preparative procedure’? Answers to these fundamentally important questions are explicitly or implicitly offered in the chapters in this section of the book. However, two general observations should be made at this juncture.

First, there is no single ideal preparative procedure that meets all the requirements of the biological electron probe X-ray microanalyst (even if we confine our considerations to thin specimens of soft tissues). The starting point must be the nature of the biological problems that are to be tackled. Once this has been clearly defined then a suitable preparation procedure may be adopted, but even then the question (and thus the analytical objectives) may need to be modified in the light of the anticipated level of preparative damage. Measuring electrolyte concentrations in a small cohort of distinctive cells lying some distance beneath the surface of a complex tissue is a good example of a ‘real microanalytical problem’, as distinct from the analysis of well-chosen model systems. Electrolyte analysis requires cryofixation to limit redistribution, but the quality of freezing will not be high in deep cells. So the prospect of analysing compartments finer than ‘nucleus’ and ‘cytoplasm’ in such preparations may be unrealistic.