In-depth biological analysis of alteration in Plasmodium knowlesi-infected red blood cells using a noninvasive optical imaging technique

Abstract

Background Imaging techniques are commonly used to understand disease mechanisms and their biological features in the microenvironment of the cell. Many studies have added to our understanding of the biology of the malaria parasite Plasmodium knowlesi from functional in vitro and imaging analysis using serial block-face scanning electron microscopy (SEM). However, sample fixation and metal coating during SEM analysis can alter the parasite membrane. Methods In this study, we used noninvasive diffraction optical tomography (DOT), also known as holotomography, to explore the morphological, biochemical, and mechanical alterations of each stage of P. knowlesi-infected red blood cells (RBCs). Each stage of the parasite was synchronized using Nycodenz and magnetic-activated cell sorting (MACS) for P. knowlesi and P. falciparum, respectively. Holotomography was applied to measure individual three-dimensional refractive index tomograms without metal coating, fixation, or additional dye agent. Results Distinct profiles were found on the surface area and hemoglobin content of the two parasites. The surface area of P. knowlesi-infected RBCs showed significant expansion, while P. falciparum-infected RBCs did not show any changes compared to uninfected RBCs. In terms of hemoglobin consumption, P. falciparum tended to consume hemoglobin more than P. knowlesi. The observed profile of P. knowlesi-infected RBCs generally showed similar results to other studies, proving that this technique is unbiased. Conclusions The observed profile of the surface area and hemoglobin content of malaria infected-RBCs can potentially be used as a diagnostic parameter to distinguish P. knowlesi and P. falciparum infection. In addition, we showed that holotomography could be used to study each Plasmodium species in greater depth, supporting strategies for the development of diagnostic and treatment strategies for malaria. Graphical Abstract

Quantitative Measurements of Red Blood Cell Indices Using Spectroscopic Differential Phase-Contrast Microscopy

“…Optical diffraction tomography (ODT) has also been applied to map out the three-dimensional RI information on RBCs. − For instance, Ryu et al combined a microfluidic device and ODT to achieve the actuation- and dilution-free formation of sparse RBC distribution in the measurement volume and applied the ODT method to measure the RI and 3D volume maps. These methods can reliably decouple the RI and the thickness of RBCs but require multiple measurements with various incident angles, and the cells should be stationary during the measurement.…”