Summary

Electron energy-loss spectroscopical (EELS) data and those acquired by electron probe X-ray microanalysis (XRMA) add chemical information to morphological structures in electron microscopical images. Recent instrumental developments lead to the need for a comparison of their various microanalytical potentials in relation to biological materials, especially their possibilities for quantitative image analysis. The aim of this chapter is to describe the present state of the art in electron energy-loss image analysis and to compare the results with those from previous studies on images of the same material with electron probe X-ray microanalysis. The acquisition of digitised electron spectroscopic images (ESI) from ultrathin sectioned cells and tissues allows the simultaneous morphometric and chemical analysis of such material. A recurring problem in the analysis is the segmentation of the images. It is shown that segmentation can be achieved by thresholding, using the first derivative of the grey-value frequency histograms constructed from such images. This method applies to electron energy-loss images as well as to images acquired by X-ray microanalysis. The influence of the image contrast and of the image averaging procedure during acquisition on the morphometrical parameters and perimeter is indicated. It is demonstrated that quantification of element-related spectra and net-intensity images is possible. One of the advantages of digitised images, to show the co-localisation of several elements within a single organelle, is illustrated. An example of element concentration determination using co-embedded Bio-standards present in the same section as the ‘unknown’ is given.