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 ...
We have developed diffraction phase microscopy as a new technique for quantitative phase imaging of biological structures. The method combines the principles of common path interferometry and single-shot phase imaging and is characterized by subnanometer path-length stability and millisecond-scale acquisition time. The potential of the technique for quantifying nanoscale motions in live cells is demonstrated by experiments on red blood cells.
We present spatial light interference microscopy (SLIM) as a new optical microscopy technique, capable of measuring nanoscale structures and dynamics in live cells via interferometry. SLIM combines two classic ideas in light imaging: Zernike’s phase contrast microscopy, which renders high contrast intensity images of transparent specimens, and Gabor’s holography, where the phase information from the object is recorded. Thus, SLIM reveals the intrinsic contrast of cell structures and, in addition, renders quantitative optical path-length maps across the sample. The resulting topographic accuracy is comparable to that of atomic force microscopy, while the acquisition speed is 1,000 times higher. We illustrate the novel insight into cell dynamics via SLIM by experiments on primary cell cultures from the rat brain. SLIM is implemented as an add-on module to an existing phase contrast microscope, which may prove instrumental in impacting the light microscopy field at a large scale.
Using novel interferometric quantitative phase microscopy methods, we demonstrate that the surface integral of the optical phase associated with live cells is invariant to cell water content. Thus, we provide an entirely noninvasive method to measure the nonaqueous content or "dry mass" of living cells. Given the extremely high stability of the interferometric microscope and the femtogram sensitivity of the method to changes in cellular dry mass, this new technique is not only ideal for quantifying cell growth but also reveals spatially resolved cellular and subcellular dynamics of living cells over many decades in a temporal scale. Specifically, we present quantitative histograms of individual cell mass characterizing the hypertrophic effect of high glucose in a mesangial cell model. In addition, we show that in an epithelial cell model observed for long periods of time, the mean squared displacement data reveal specific information about cellular and subcellular dynamics at various characteristic length and time scales. Overall, this study shows that interferometeric quantitative phase microscopy represents a noninvasive optical assay for monitoring cell growth, characterizing cellular motility, and investigating the subcellular motions of living cells. phase microscopy; interferometric microscopy; cell growth PHASE-CONTRAST (PC) and differential interference contrast (DIC) microscopy have been used extensively to study live cells without the need for exogenous contrast agents (32). The tremendous success of these methods is due to the fact that the optical phase shift through a given sample contains information about the refractive index (n) variations that directly result from structural features within the sample. Refractive index n can therefore be regarded as a powerful endogenous contrast agent for cellular structure (6). However, as the relationship between the irradiance and phase of the image field is generally nonlinear (30, 39), both PC and DIC are qualitative in nature and limited to morphological observations without specific structural data.Quantitative phase microscopy has received substantial interest in recent years, as quantifying optical phase shifts associated with cells provides structural and dynamical information at the nanometer scale without the need for any cell preparation or the use of exogenous contrast or labels. Existing methods for biological quantitative phase measurements can be divided into single-point and full-field techniques. Several point measurement techniques have been used for investigating the local structure and dynamics of live cells (1,7,10,14,29,36,37). In contrast, full-field phase measurement techniques provide simultaneous information from a large region of the sample, which offers the additional benefit of studying both the temporal and spatial behavior of the sample (2, 5, 9, 13, 18 -20, 40, 41).Over the past several years, our laboratory has developed new full-field phase imaging techniques that are suitable for spatially resolved investigation of live cell...
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