Development of quantitative microanalysis techniques and their application to selected biological systems
The objective of the research that I have undertaken was to develop two quantitative techniques used in analytical electron microscopy, energy dispersive X-ray microanalysis (EDX) and electron energy loss spectroscopy (EELS) and to apply those techniques to a selected biological system. More specifically the aim was to develop a technique for quantifying, at high spatial resolution, trace concentrations of Al in mineralised bone. The Al was highly localised and therefore it was important to select the microanalytical technique with the required spatial resolution (m) compatible with the highest sensitivity. In practice, the choice was relatively simple because the signals from the trace concentrations could not be detected using EELS due to the combined effects of plural inelastic scattering in the sample, and a low signal to background ratio. The former problem resulted from the difficulty in preparing sufficiently thin samples, and the latter is an inherent problem of EEL spectroscopy. Therefore, EDX was chosen for the mineralised bone study. As is always the case, artefacts are introduced into the spectral data by the measurement system itself and it was necessary to first quantify these artefacts before attempting to process the EDX spectra. Standards of accurately known composition are often used to quantify the characteristic signals extracted from EDX spectra. Unfortunately, suitable standards were not available for the elements of interest. Therefore, a standardless quantitation procedure was used. Since the accuracy of the method was dependent upon the accuracy of the parameterised characteristic cross-sections, some time was spent determining the optimal parameterisation. Significant problems exist when attempting to extract the Al and Mg signals from the EDX mineralised bone spectra. Essentially these are due to the extensive overlap between the major P peak and the vanishingly small Al (and Mg) signals of interest. In addition it is difficult to model the bremsstrahlung background (for characteristic signal extraction) at the Al and Mg X-ray energies because of the combined effects of absorption in the detector and specimen, and incomplete charge collection in the detector. However, a technique was developed which greatly improves the accuracy of the characteristic signal extraction over existing methods. A new method of EEL spectral processing was developed in which the characteristic signals are separated from the background counts and quantified in a single process. This `single-stage' technique appears to have some advantages over the standard `extrapolation' method of spectral processing: e.g. when dealing with adjacent edge signals or small signals on relatively large backgrounds. A detailed investigation was made into all the problems associated with the application of EELS to the mineralised bone study of interest here; i.e. the difficulties of EEL mineralised bone specimen preparation and the very low Al signal/background ratio which is predicted, even if suitable mineralised bone samples could have been prepared. Finally, a brief investigation was made into the physiological implications of the mineralised bone atomic ratios obtained in the EDX study.