Stage-Dependent Topographical and Optical Properties of Plasmodium Falciparum-Infected Red Blood Cells

Abstract

Efficient malaria treatment is a major healthcare challenge. Addressing this challenge requires in-depth understanding of malaria parasite maturation during the intraerythrocytic cycle. Exploring the structural and functional changes of the parasite through the intraerythrocytic stages and their impact on red blood cells (RBCs) is a cornerstone of antimalarial drug development. In order to precisely trace such changes, we performed a thorough imaging study of RBCs infected by Plasmodium falciparum, by using atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF) supplemented with bright field microscopy for stage assignment. This multifaceted imaging approach allows to reveal structure–function relations via correlations of the parasite maturation with morphological and fluorescence properties of the stages. We established diagnostic patterns characteristic to the parasite stages based on the topographical profile of infected RBCs, which show close correlation with their fluorescence (TIRF) map. Furthermore, we found that hemozoin crystals exhibit a strong optical contrast, possibly due to the quenching of fluorescence. The topographical and optical features provide a tool for locating the hemozoin crystals within the RBCs and following their growth.

Reducing data dimension boosts neural network-based stage-specific malaria detection

“…The fluorescence microscopy images were obtained as in Ref. 16 by using an Olympus IX81 inverted microscope, operated at a wavelength of 405 nm. Throughout the experiments, we used an Olympus TIRF objective (UApo N, 100x, 1.49NA).…”

“…To measure the morphology of RBCs on dried smears, we used an MFP-3D AFM (Asylum Research, Oxford Instruments) as in 16 , which was operated in AC mode at a scanning speed of 0.25 Hz. Each scan was performed on an area of 90 x 90 µm with a resolution of 512 x 512 pixels.…”

“…During maturation, the parasites change the topography [11][12][13][14] and the optical properties of the host RBCs 15 . In fact, it is well-established that malaria-infected RBCs can be characterised by their morphological properties, that can also be correlated with their characteristic fluorescence patterns 16,17 . These new insights and the widespread use of microscopy, triggered new approaches for the automatised recognition of malaria infected RBCs in blood smears, which can lead to a breakthrough in malaria diagnosis.…”

“…This also gives the hint that malaria diagnosis can benefit from the advantages of NNs in analysing images recorded by microscopy methods other than light microscopy. In fact, recently there is an increasing number of techniques successfully applied for high-resolution imaging of malaria infected RBCs, including topographic imaging by atomic force microscopy (AFM) 13,14,16 and infrared nano-imaging 12 .…”