Abstract
Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.
Highlights
Malaria is one of the most deadly infectious diseases worldwide, responsible for 247 million cases and 881 thousand deaths (85% of deaths occurring in children under 5 years old) annually in more than 100 countries
As the milling is made within the microscope chamber, it is possible to control the milling thickness and the number of slices so that the sampled volume obtained remains mainly limited by the quantity of material and the time available
As the erythrocyte has no intracellular organelles or trafficking machinery, Plasmodium constructs an entirely novel trafficking system for protein and antigen transport. This secretory system has been extensively studied in P. falciparum, where the Maurer’s clefts and a tubovesicular network were characterized three-dimensionally
Summary
Malaria is one of the most deadly infectious diseases worldwide, responsible for 247 million cases and 881 thousand deaths (85% of deaths occurring in children under 5 years old) annually in more than 100 countries. Because the mature erythrocyte has no intracellular organelles and none of the trafficking machinery used by most eukaryotic cells to transfer proteins to their correct destinations, the parasite needs to set up a completely novel system for trafficking of proteins and antigens to the surface of red blood cells [3,4] This system includes insertion of membrane profiles in the cytoplasm of the erythrocyte, such as membrane clefts, named Maurer’s clefts in P. falciparum [5] or membrane clefts in other species such as the human parasite P. vivax [6] and rodent malaria parasite P. berghei [7], and a tubovesicular network, a term that has been introduced for a membranous network formed by projections of the parasitophorous vacuole membrane (PVM), described in P. falciparum [8], P. vivax [6] and P. berghei [7]. An association between these membranous structures with alterations on the surface of infected erythrocyte has been demonstrated by Bhattacharjee et al [9]
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