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Defocusing improves microscopic analysis of red blood cells

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Gary Boas

Several years ago, Oscar N. Mesquita, a researcher with the Federal University of Minas Gerais in Belo Horizonte, Brazil, was measuring the movement of macrophages. At one point, he looked into the microscope before focusing and noticed that some of the otherwise transparent parts of the macrophages were visible. Not knowing exactly what he was seeing, he searched the optical theory literature for some reference to “defocused microscope.” Unfortunately, he found that — although it was a known phenomenon — the problem had not been solved.

Mesquita and his colleagues at the university set out to solve it themselves. As reported in a 2003 issue of Physical Review E, they concluded that the image of an otherwise transparent object that they observed in a defocused microscope was, in fact, that of the local curvature of the object — and that the curvature acts as a small lens that converges or diverges light, yielding dark or light contrast images.

A defocusing microscopy technique provides images of transparent objects based on their local curvature. Shown here are images of a red blood cell when the focal plane position is below (top), in the center of (center) and above the cell (bottom). Note the contrast inversion below and above the cell.

This “defocusing microscopy” technique is easier to use than the typical methods employed to image transparent (or phase) objects, they stated. And, because it provides quantitative information about the shape and fluctuations of transparent objects with curvature, it is well-suited to imaging and characterizing biological samples.

Not everyone agreed, though. A reporter writing about the study for Physical Review Focus interviewed several investigators who were not involved with the study, including Erich Sackmann of the Technical University of Munich in Germany. “He was very skeptical about our technique,” Mesquita recalled, “saying that [it] was a low-resolution technique and that we could not obtain … the bending modulus of red blood cell membranes.”

The researchers took up the challenge and began studying red blood cells, to demonstrate that the defocusing technique could provide reliable information about the cells’ optical and mechanical properties.

They used a Nikon inverted microscope outfitted with a 1003, 1.4-NA objective, and a CCD camera made by Dage-MTI of Michigan City Inc. in Indiana to capture images of the cells. Because defocusing must be tightly controlled, they coupled a piezoelectric transducer translator to the microscope’s stage, enabling control of the Z-motion.

The scientists described their work in the March 27 issue of Applied Physics Letters, reporting results that were “far better than we anticipated,” Mesquita said. They showed that they could, in a single run, obtain the refractive index, shape profile and bending modulus of red blood cells.

Therefore, they demonstrated that the technique can be applied to acquire quantitative information about the shape and optical and mechanical properties of red blood cells. Because some hemolytic disorders can alter these properties, it might even serve as a diagnostic tool. Beyond this, Mesquita said, “we solved the standing problem of interpreting and understanding red blood cell images obtained with a standard light microscope operating in bright field.”

The scientists plan to continue studying red blood cells with this technique, exploring the phase transitions that induce shape changes in the cells by investigating alterations in the optical and mechanical properties that are induced by changes in the concentration of the medium. In addition, they will work with biologists to determine how these properties change when the cells are infected by various malaria parasites.

Applied Physics Letters, March 27, 2006, 133901.

Jun 2006

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