Visualizing the 3D Architecture of Multiple Erythrocytes Infected with Plasmodium at Nanoscale by Focused Ion Beam-Scanning Electron Microscopy

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.

Real-time cholesterol sorting in Plasmodium falciparum-erythrocytes as revealed by 3D label-free imaging

“…A variety of microscopic observations have yielded important information on the biological processes that involve structural modifications inside cells, and recently, label-free imaging, such as Raman microscopy [20][21][22] , focused ion beam scanning electron microscopy (called FIB-SEM) 23,24 and optical diffraction tomography combined with holographic microscopy (holotomographic microscopy), have been developed. Notably, holotomographic microscopy, which includes phase contrast microscopy, was developed to combine holographic and tomographic techniques based on measuring the refractive index (RI) of a sample [25][26][27][28] .…”

3D Imaging of the Early Embryonic Chicken Heart with Focused Ion Beam Scanning Electron Microscopy

“…While relatively new to biological applications, the use of FIB-SEM is not limited to embryonic or cardiac tissue; recent studies have used it to visualize the microstructure of the brain (Knott et al, 2011; Sonomura et al, 2013; Blazquez-Llorca et al, 2013), the optic nerve (Schertel et al, 2013), and red blood cells (LCS Medeiros et al, 2012). FIB-SEM has also recently been used to image lipid droplets in adult heart ventricles (Wang et al, 2013).…”

Life without a Host Cell: What is Cryptosporidium ?

“…The ability to visualize the structure of individual stages, and attachment and encapsulation with a host cell, in three dimensions at high resolution [37,38] will better reveal the true nature of Cryptosporidium's connection to the host cell and will improve our understanding of the interaction between host-and Cryptosporidium-derived structures. Such 3D imaging is beginning to receive wide attention in the study of parasite invasion and behavior [39][40][41][42][43][44][45].…”