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Techniques

Energy dispersive X-ray microanalysis

Energy dispersive X-ray spectroscopy (EDXS) allows the measurement of elements on an inter- and intracellular level. This technique is based on the interaction of a primary electron beam with specimen elements resulting in X-ray emissions that are measured by crystal spectrometers (wavelength-dispersive X-ray spectroscopy, WDXS) or a solid-state detector (energy-dispersive X-ray spectroscopy, EDXS). Advantage of the wavelength-dispersive system is a higher sensitivity compared to the energy-dispersive system. However, EDXS is more often used, since it allows the parallel analysis of different elements at one specific measurement point.

This figure shows a typical X-ray microanalytical spectrum of polyphosphates, a common phosphate storage molecule in microorganisms, algae and fungi. Besides phosphate, high contents of magnesium and potassium can be found in these granules.

For measurements with a high spatial resolution it is generally necessary to analyse sections. For that reason, a preparation procedure must be used, that guarantees a sufficient structural preservation but prevents dislocations and losses of elements. The commonly used preparation technique in electron microscopy with a chemical fixation and chemical dehydration can not be used, because changes of the in-vivo elemental distribution can not be excluded. This means, that alternative techniques, such as cryofixation followed by freeze-drying or freeze substitution must be used.

Besides the possibility to analyse specific points in a sample, also measurements of a larger sample area can be carried out. In these so called mappings, the whole sample area is scanned by the primary electron beam and at each specific point an X-ray microanalytical spectra is analysed.

The left side of the figure shows a mapping of the fungal sheath of an ectomycorrhizal association, in which electron dense granules can be seen. These granules are clearly associated with phosphate (center) and with potassium (right). This indicates, that these granules are polyphosphates.

References:

Hall, J.L. & Hawes, C. 1991. Electron microscopy of plant cells. Academic Press, London.
Robinson, D.G., Ehlers, U., Herken, R., Herrmann, B., Mayer, F. & Schürmann, F.-W. 1985. Präparationsmethodik in der Elektronenmikroskopie. Springer Verlag, Berlin.