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We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.

Artesunate (ART) is widely used for the treatment of malaria, but the mechanisms of its effects on parasitized red blood cells (RBCs) are not fully understood. We investigated ART's influence on the dynamic deformability of ring-stage Plasmodium falciparum infected red blood cells (iRBCs) in order to elucidate its role in cellular mechanobiology. The dynamic deformability of RBCs was measured by passing them through a microfluidic device with repeated bottleneck structures. The quasi-static deformability measurement was performed using micropipette aspiration. After ART treatment, microfluidic experiments showed 50% decrease in iRBC transit velocity whereas only small (~10%) velocity reduction was observed among uninfected RBCs (uRBCs). Micropipette aspiration also revealed ART-induced stiffening in RBC membranes. These results demonstrate, for the first time, that ART reduces the dynamic and quasi-static RBC deformability, which may subsequently influence blood circulation through the microvasculature and spleen cordal meshwork, thus adding a new aspect to artesunate's mechanism of action.

Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long-term survival of the parasite., (© 2012 Blackwell Publishing Ltd.)

Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host.

Malaria resulting from Plasmodium falciparum infection is a major cause of human suffering and mortality. Red blood cell (RBC) deformability plays a major role in the pathogenesis of malaria. Here we introduce an automated microfabricated "deformability cytometer" that measures dynamic mechanical responses of 10(3) to 10(4) individual RBCs in a cell population. Fluorescence measurements of each RBC are simultaneously acquired, resulting in a population-based correlation between biochemical properties, such as cell surface markers, and dynamic mechanical deformability. This device is especially applicable to heterogeneous cell populations. We demonstrate its ability to mechanically characterize a small number of P. falciparum-infected (ring stage) RBCs in a large population of uninfected RBCs. Furthermore, we are able to infer quantitative mechanical properties of individual RBCs from the observed dynamic behavior through a dissipative particle dynamics (DPD) model. These methods collectively provide a systematic approach to characterize the biomechanical properties of cells in a high-throughput manner., (This journal is © The Royal Society of Chemistry 2011)

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.

We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.

Parasitic infection with Plasmodium falciparum is responsible for the most severe form of human malaria in which patients suffer from periodic fever. It is well established that during intra-erythrocytic maturation of the parasite in the red blood cell (RBC), the RBC becomes significantly more cytoadhesive and less deformable; these and other biochemical factors together with human host factors such as compromised immune status are important contributors to the disease pathology. There is currently substantial interest in understanding the loss of RBC deformability due to P. falciparum infection, but few results are available concerning effects of febrile conditions or parasitization on RBC membrane rheology. Here, for the first time, we report rheology of the single, isolated RBC with and without P. falciparum merozoite invasion, spanning a range from room temperature to febrile conditions (41 degrees C), over all the stages of parasite maturation. As expected, stiffness increased with parasite maturation. Surprisingly, however, stiffness increased acutely with temperature on a scale of minutes, particularly in late trophozoite and schizont stages. This acute stiffening in late falciparum stages may contribute to fever-dependent pathological consequences in the microcirculation.

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.

We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular [ABSTRACT FROM AUTHOR]

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL-1. We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]