Malaria is an infectious disease caused by the protozoan parasites-Plasmodium spp. The WHO estimated 216 million cases of the disease in 2016 and it continues to be a global burden, despite many measures undertaken to combat it. Of the species that infect humans, P. falciparum causes one of the deadliest forms of the disease. The parasites have complex life-cycle alternating between the female Anopheles mosquito and the human host, where the early phase of development is asymptomatic and is in the hepatocytes. Following this, the parasites invade red blood cells (RBCs) where they undergo cyclic asexual multiplication that results in the classic clinical symptoms of malaria. Most antimalarial agents are targeted to this stage and a systematic biological understanding of the blood stage becomes both essential and imperative. Classic electron microscopy (EM) methods have been employed to understand the ultrastructure of the parasite and infected host cells but, harsh chemical fixatives and heavy metal stains cause artefacts that can be misleading. Cryo-EM methods preclude undesirable sample preparation steps by use of instantaneous vitrification to preserve biological ultrastructure in the near native state, but pose restrictions on sample thickness. To partially overcome thickness limitation cryo-scanning transmission electron microscopy (Cryo-STEM) can be used. Here a focussed electron probe raster scans the sample and a series of images can be obtained by tilting the sample at various angles (Cryo-STEM tomography). The images are formed by detectors that collect transmitted electrons scattered at various angles. These images can be then subjected to reconstruction and image analysis protocols ultimately resulting in a 3D ultrastructural image of the native state of the parasite. We propose to use this novel technique in understanding various biological aspects of the parasite.
The present works describes encouraging data from the use of Cryo-STEM tomography to visualise and understand the ultrastructure of P. falciparum. We are in the process of standardizing the current methods and extend it towards understanding the biology of the parasite.
