Contribution of cryofixation and freeze-substitution to analytical microscopy: a study of Tritrichomonas foetus hydrogenosomes.

The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. It is found in several trichomonad species, protists living in oxygen-poor environments, as well as certain free-living ciliates, rumen ciliates, and some fungi. We performed a comparative microanalytical study (energy dispersive X-ray analysis and electron spectroscopic imaging) of different fixation methods for electron microscopy analyzing hydrogenosomes of the bovine parasite Tritrichomonas foetus. The study included the elemental composition and the mapping of calcium, phosphorus, and oxygen. A preparation of T. foetus cells, based on cryoimmobilization by high-pressure freezing and freeze-substitution, was compared to a second preparation based on chemical fixation followed by dehydration and routine processing. The ultrastructural preservation achieved by the cryotechnique was far superior to the chemical fixation, since it allowed the successful cryoimmobilization of intracellular ion contents. The detection of several cations (Al, Mg, Co, Ca, Fe) by X-ray microanalysis inside the peripheral vesicle of the hydrogenosome was only possible in cryofixed cells. The presence of aluminum and cobalt ion in the hydrogenosomal vesicle was established for the first time. Electron-spectroscopic images of calcium showed that this element, in addition to the vesicle compartment, is present in the hydrogenosome's membrane in varying concentrations, which might reflect changes in the physiology of this organelle.