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

Abstract

Hydrogenosomes are organelles found in anaerobic protists such as trichomonads, certain ciliates, and some fungi, generating molecular hydrogen and ATP through pyruvate or malate oxidation. This study compared two electron microscopy preparation methods—high-pressure freezing with freeze-substitution versus conventional chemical fixation—for analyzing hydrogenosomes in the bovine parasite Tritrichomonas foetus. Energy dispersive X-ray analysis and electron spectroscopic imaging were used to map elemental composition, focusing on calcium, phosphorus, and oxygen. Cryofixation preserved intracellular ionic content far more effectively than chemical fixation, enabling detection of multiple cations (Al, Mg, Co, Ca, Fe) within the hydrogenosomal peripheral vesicle. The presence of aluminum and cobalt ions in this compartment was reported for the first time. Electron spectroscopic imaging further revealed calcium distribution not only in the vesicle but also within the hydrogenosomal membrane at variable concentrations, suggesting dynamic physiological changes in this organelle.

Mechanism

Hydrogenosomes oxidize pyruvate and malate under anaerobic conditions to produce molecular hydrogen and ATP, functioning analogously to mitochondria in oxygen-poor environments.