Optimisation of freeze substitution protocols for the examination of malaria parasite structure by volumetric electron microscopy.

Malaria is one of the deadliest infectious diseases in the world, annually responsible for over 400,000 deaths. It is caused by parasites of the genus Plasmodium, which undergo remarkable structural changes during their development within different cells across various hosts. An important approach to understand the structural basis of biochemical and physiological processes during Plasmodium infection has been the quantitative measurement of dimensional parameters obtained by different microscopy techniques. In this regard, sample preparation, particularly electron microscopy protocols that rely on room-temperature chemical fixation, has posed significant challenges, as it is known to produce artefacts such as shrinking, swelling and displacement of structures and osmolytes. In contrast, specimen immobilisation by cryofixation followed by freeze substitution minimises these artefacts and provides better sample preservation. Nevertheless, the composition of the freeze substitution medium may vary depending on the cell type, making it a critical factor for achieving optimal sample preparation. In this work, we optimised a freeze substitution protocol for the structural analysis of intraerythrocytic stages of the murine malaria models Plasmodium chabaudi and P. berghei. We tested different freeze substitution recipes, considering the biochemical composition of malaria membranes, and compared the results with those obtained through conventional chemical fixation. Overall, the results showed a significant improvement on the preservation of cell morphology and haemozoin crystals. Establishing an efficient and reproducible freeze substitution protocol for murine malaria models provides an important tool for advancing our understanding of the structural organisation of Plasmodium spp.