Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum

Abstract

Parasitization by malaria-inducing Plasmodium falciparum leads to structural, biochemical, and mechanical modifications to the host red blood cells (RBCs). To study these modifications, we investigate two intrinsic indicators: the refractive index and membrane fluctuations in P. falciparum-invaded human RBCs (Pf-RBCs). We report experimental connections between these intrinsic indicators and pathological states. By employing two noninvasive optical techniques, tomographic phase microscopy and diffraction phase microscopy, we extract three-dimensional maps of refractive index and nanoscale cell membrane fluctuations in isolated RBCs. Our systematic experiments cover all intraerythrocytic stages of parasite development under physiological and febrile temperatures. These findings offer potential, and sufficiently general, avenues for identifying, through cell membrane dynamics, pathological states that cause or accompany human diseases.erythrocyte ͉ febrile temperature ͉ malaria ͉ mechanical properties ͉ optical techniques D uring the intraerythrocytic development, the malaria parasite Plasmodium falciparum causes structural, biochemical, and mechanical changes to host red blood cells (RBCs). Major structural changes include the formation of parasitophorus vacuoles that surround the growing parasite in their host RBCs, loss of cell volume, and the appearance of small, nanoscale protrusions or ''knobs,'' on the membrane surface (1). From the biochemical standpoint, a considerable amount of hemoglobin (Hb) is digested by parasites during intraerythrocytic development and converted into insoluble polymerized forms of heme, known as hemozoin (2, 3). Hemozoin appears as brown crystals in the vacuole of parasite in later maturation stages of P. falciparum-invaded human RBCs (Pf-RBCs).Two major mechanical modifications are loss of RBC deformability (4-6) and increased cytoadherence of the invaded RBC membrane to vascular endothelium and other RBCs (7). These changes lead to sequestration of RBCs in microvasculature in the later stages of parasite development, which is linked to vital organ dysfunction in severe malaria. In the earlier stage, where some loss of deformability occurs, Pf-RBCs continue to circulate in the bloodstream.Membrane dynamics of RBCs can be influenced by human disease states. Fluctuations in phospholipid bilayer and attached spectrin network are known to be altered by cytoskeletal defects, stress, and actin-spectrin dissociations arising from metabolic activity linked to adenosine 5Ј-triphosphate (ATP) concentration (8-12). Proteins transported from invading organisms, such as the virulent malaria-inducing parasite P. falciparum, to specific binding sites in the spectrin network are considered to introduce significant alterations to RBC membrane dynamics and mechanical response (13,14). These changes could provide insights into possible mechanistic pathways in the pathogenesis of malaria, because the parasite alters biophysical properties of RBCs during its intraerythrocyte stage that lasts up to 48 ...

Quantifying the biophysical characteristics of Plasmodium-falciparum -parasitized red blood cells in microcirculation

“…In malaria disease, progression through the parasite development stages (ring → trophozoite → schizont) leads to a considerable stiffening of Pf-RBCs compared to healthy ones (22,23). Furthermore, in the schizont stage the RBC shape becomes near-spherical whereas in the preceding stages RBCs maintain their biconcavity.…”

“…Recent experiments (22) suggest that the volume of cytosol may be reduced threefold in the later stages of intracell parasite development compared to healthy RBCs, indicating that the parasite takes up considerable volume within a Pf-RBC. Hence, a sufficiently large parasite provides a rigid backbone within a Pf-RBC to strongly affect its adhesive dynamics.…”

Measurement of red blood cell mechanics during morphological changes

“…RBCs were separated into three groups corresponding to their shapes: DCs, ECs, and SCs. ECs can be classified into three types (type I, II, and III) according to progressive stages in formation and maturation (38,39). We have excluded type III ECs (with high speculation) and have analyzed only type I and II ECs.…”

White-light diffraction tomography of unlabelled live cells

“…In recent years, as a result of new advances in light sources, detector arrays and computing power, quantitative phase imaging (QPI), in which optical path-length delays are measured at each point in the field of view, has become a very active field of study 9 . Whether involving holographic or non-holographic methods 10-16 , QPI presents new opportunities for studying cells and tissues non-invasively, quantitatively and without the need for staining or tagging [17][18][19][20][21][22][23] . Projection tomography using laser QPI has made use of ideas from X-ray imaging and enabled three-dimensional imaging of transparent structures [24][25][26] .…”