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The custom built microscopy and CMOS camera (FASTCAM 1024 PCI, Photron, Inc.) were used to measure interferograms.
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Phase image at each step of angle corresponds to angular projection of refractive index at the illumination angle. It takes about 10 seconds to scan the entire angular range. The angle of illumination ranges from -60 to 60 degree and angular step is 0.2 degree. Phase images are recorded by varying the directions of illumination. In TPM, the sample-induced optical phase shift is imaged using a phase-shifting heterodyne interferometer. Tomographic Phase Microscopy: Tomographic phase microscopy (TPM) is a technique that can map the 3-D distribution of refractive index in live cells and tissues. The DPM optical path-length stability is 2.4 mrad, which corresponds to a membrane displacement of 3.3 nm. We employed tomographic phase microscopy (TPM) to retrieve 3-D refractive index for all the stages of Pf-RBCs and healthy RBCs. The refractive index contrast between the RBC and the surrounding PBS is mainly contributed to the Hb, which is optically homogeneous in cytosol. The instantaneous cell thickness map is obtained as, with the quantitative phase image measured by DPM. DPM employs the principle of laser interferometry in a common path geometry and thus provides full-field quantitative phase images of RBCs with unprecedented optical path-length stability. EMCCD (Photonmax 512B, Princeton Instruments, Inc.) was used to image interferogram. With the additional relay optics used outside the microscope, the overall magnification of the system was approximately 200´. The microscope was equipped with a 40´ objective (0.65 NA), which facilitates a diffraction-limited transverse resolution of 400 nm. Diffraction Phase Microscopy: An Ar++ laser (l=514 nm) was used as illumination source for an inverted microscope (IX71, Olympus). These changes lead to sequestration in microvasculature in the later stages of parasite development, which is linked to vital organ dysfunction in severe malaria. Two major mechanical modifications are loss of cell deformability and increased adherence of Pf-RBC membrane to vascular endothelium and other RBCs. Hemozoin appears as brown crystals in the vacuole of parasite in later maturation stages of Pf-RBCs. From the biochemical standpoint, a considerable amount of hemoglobin (Hb) is digested by parasites during development and converted into insoluble polymerized forms, known as hemozoin. Major structural changes include the growing of vacuole of parasites inside host RBCs, loss of cell volume, and the appearance of small, nano-scale protrusions on the membrane surface. falciparum causes structural, biochemical, and mechanical changes to host RBCs. During the intra-erythrocytic development, P. These properties can be used to systemically study the patho-physiology of human malaria disease. falciparum infected red blood cells ( Pf-RBCs). falciparum): three dimensional distributions of refractive index and the membrane fluctuations in P. We present, for the first time, two intrinsic indicators that quantitatively and non-invasively elucidate the consequences on cell biomechanics of the malaria parasite Plasmodium falciparum ( P. falciparum Infected Human Red Blood Cells Intrinsic Indicators of Pathological States: Refractive Index Maps and Membrane Fluctuations in P.