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Tracing Malaria’s Pathways

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Membrane fluctuations offer clues to cells’ health

Gary Boas,

For the past five or six years, a team at MIT in Cambridge has been probing the structural, biochemical and mechanical changes induced by the malaria parasite Plasmodium falciparum. These changes include the formation of vacuoles around parasites as they grow in their host red blood cells, decreases in cell volume and the emergence of nanoscale “knobs” on the surface of the cell membranes.

In the Sept. 16 issue of PNAS, a study by the group reported additional changes and how they might be influenced by pathological status. Researchers have long suspected that membranes fluctuate because cells are active and, furthermore, that the amount of vibration can be an indicator of health or disease. But the amount of vibration is so small that you can’t see it with a regular microscope, explained Subra Suresh and Michael Feld, the principal investigators of the study.

The investigators used two noninvasive optical techniques, diffraction phase microscopy and tomographic phase microscopy, to acquire three-dimensional maps of refractive index and membrane fluctuations in P. falciparum-invaded human red blood cells. Understanding these changes could help advance our knowledge of the possible mechanistic pathways in the pathogenesis of malaria.

Figure 1. Shown here are three-dimensional maps of the refractive index and nanoscale cell membrane fluctuations at different maturation stages of the malaria-inducing parasite Plasmodium falciparum, acquired using the noninvasive optical techniques tomographic phase microscopy and diffraction phase microscopy. Color has been added to enhance visualization. Images courtesy of Technology Review.

This work builds directly upon research previously conducted in the lab. “In the last few years,” Suresh said, “we’ve been able to measure, with extreme precision, the way in which the parasite interacts with hemoglobin to stiffen the cell, and we’ve been able to measure, systematically, the extent of stiffening over the course of maturation inside the cell.” Stiffness is also related to membrane vibrations and, in the present study, the investigators wanted to show how vibrations affect stiffness.

Focus on fever

Suresh and colleagues focused on the periodic episodes of high fever that are found with P. falciparum malaria. Previous studies have shown that both survival rate and deformability of red blood cells infected by the parasite are influenced by the presence of fever and that deformability is more pronounced at fever temperatures (41 °C) than at physiological temperatures (37 °C).

Figure 2. The investigators reconstructed three-dimensional images of infected human red blood cells at different stages of parasite development, based on the 3-D maps of the refractive index in the cells. Healthy cells exhibited a characteristic biconcave shape (top cell). During the early stage of parasite maturation (middle), the parasitophorus vacuole is shown as a yellow region. In a later stage, parasitized red blood cells are subjected to severe morphological changes (bottom). The blue regions inside the cell indicate parasite-produced hemozoin, a crystallized form of digested hemoglobin.

The in vitro experiments on which these studies are based do not provide a full picture, however. The changes in deformability may not be reversible and, therefore, the effects of fever might remain even after normal physiological temperature is restored. Suresh and colleagues looked into this possibility by measuring the deformability of infected cells at physiological temperature after exposing them to fever temperatures, simulating the conditions of a fever episode. They compared these measurements with those static measurements obtained at physiological and fever temperatures.

The diffraction phase microscopy measurements used an Olympus inverted microscope outfitted with a 40×, 0.65-NA objective, which provided a diffraction-limited transverse resolution of 400 nm. A Melles Griot line-tunable argon-ion laser (457 to 514 nm) served as the illumination source. A Princeton Instruments electron-multiplying CCD imaged the interferogram. Because it relies on the principle of laser intereferometry in a common path geometry, diffraction phase microscopy could give them full-field quantitative images of the red blood cells with high optical path length stability.

They mapped the three-dimensional distribution of refractive index using tomographic phase microscopy and imaged the sample-induced optical phase shift with a phase-shifting heterodyne interferometer, recording the images by varying the illumination angle. They measured interferograms using a custom-built microscope and a CMOS camera.

The optical techniques used in the study offer a variety of advantages over other methods that are more widely used for such applications, including micropipette aspiration, optical tweezers, laminar shear flow and magnetic twisting cytometry. Tomographic phase microscopy can provide measurements of the hemoglobin content in single P. falciparum-invaded red blood cells and, furthermore, because the refractive index is an intrinsic optical property, the technique does not demand any special sample preparation.

Diffraction phase microscopy offers the ability to measure fluctuations at the nanoscale level without any direct contact with the cell, avoiding the possibility of damaging it. Also, the optical configuration used with the method makes it relatively easy for researchers to regulate the temperature of the sample without affecting other parts of the instrument. Finally, because measurements are acquired within seconds after identifying a cell, researchers can experiment on large numbers of samples using a range of controlled test conditions within acceptable time spans.

The measurements revealed a direct correlation between the amplitude of membrane fluctuations and parasite development stage, with the distribution of fluctuations growing sharper in later stages. The researchers further noted a 53 percent increase in fluctuations from the physiological to the fever state, which represents only a 7.5 percent increase in absolute temperature. They concluded that equilibrium thermodynamics alone cannot explain the increase in fluctuations and speculated as to what might account for this significant change.

Irreversible changes

They also noted that exposure to fever temperatures resulted in irreversible changes to the deformability of P. falciparum-invaded red blood cells. This information could not have been gleaned from measurements at constant temperatures, they said.

Thus, they demonstrated that the techniques used in the PNAS study offer a means to draw out the relationships between cell membrane fluctuations and pathological conditions associated with human disease, offering quantitative information about these relationships that might not be available otherwise.

The techniques could help to advance understanding of diseases besides malaria. “We have shown that we can get 3-D maps of what goes on inside the cell,” Suresh said, “and this can open up very interesting future studies – for example, in people with sickle cell disease.” Studies have demonstrated that, in patients with the sickle cell trait, the cells interact very differently with P. falciparum-invaded red blood cells than they do with healthy ones. The mechanisms of these interactions have not been well studied, and the tools described here could help to shed light on these mechanisms.

Dec 2008
biochemicalBiophotonicscellsmembranesMicroscopynanoscaleResearch & Technology

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