Cryo transmission X-ray imaging of the malaria parasite,P. falciparum

Cryo transmission X-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin.