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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

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