Dissection ofPlasmodium falciparumdevelopmental stages with multiple imaging methods

Efficient malaria treatment is a global challenge, requiring in-depth view into the maturation of malaria parasites during the intraerythrocytic cycle. Exploring structural and functional variations of the parasites through the intraerythrocytic stages and their impact on red blood cell (RBCs) is a cornerstone of antimalarial drug development. In order to trace such changes in fine steps of parasite development, we performed an imaging study of RBCs infected by Plasmodium falciparum , using atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF), further supplemented with bright field microscopy for the direct assignment of the stages. This multifaceted imaging approach allows to reveal structure–functionality relations via correlations of the parasite maturation with morphological and fluorescence properties of the stages. We established identification patterns characteristic to the different parasite stages based on the height profile of infected RBCs, as obtained by AFM, which show close correlation with typical fluorescence (TIRF) maps of RBCs. Furthermore, we found that hemozoin crystals exhibit a strong optical contrast by quenching fluorescence. We demonstrate that these topographic and optical features also provide a tool to locate the hemozoin crystals within the RBCs and, in turn, to follow their growth.