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José Ramón Isasi Allica,,, Professor of Chemistry Physics of the School of Sciences of the University of Navarra

Nanoscopic superresolution

Thu, 09 Oct 2014 14:21:00 +0000 Published in Navarra Newspaper

"It is impossible to discern two elements closer to each other than half the wavelength of the light used." This is the limit of optical microscopy, defined already at the end of the 19th century by the German physicist Abbe, and which prevents us from obtaining detailed images of the internal Structures of cells and microorganisms because of the diffraction of light. And what do we do if we want to "see" something smaller? In the first half of the 20th century, the electron microscope was developed: the wavelength of an electron is much smaller than that of visible light. Problem solved, if it were not for its limitations when analyzing living specimens.

A fluorescence microscope detects the visible light emitted by sample, rather than that reflected or absorbed in a traditional optical microscope. This technique has the advantage that it can be used on living cells and allows a high contrast between cells. It is possible, for example, to mark a molecule that we are interested in investigating by "attaching" a fluorescent label to it. In fact, fluorescence microscopy is used, for example, at Clínica Universidad de Navarra for brain tumor surgery.

What about the theoretical barrier of resolution? By itself, this technique would not be sufficient. An additional step is needed. More like a leap. Or two types of leaps, since, as is often the case in these awards, two very different methods for overcoming the barrier have been awarded on this occasion. In the first, developed by Stefan Hell, (called STED or "stimulated emission depletion"), the sample receives two beams of light: the first one illuminates the sample, which would be blurred, as expected, if it were not for the second beam, which "turns it off" before the light leaves it. Well, not quite, because the intensity of this second "quenching" beam is minimal on the point being focused, which will be the only thing we see. The second methodology is based on the possibility of measuring the fluorescence emitted by individual molecules, demonstrated by W.E. Moerner in the late 1980s. Shortly thereafter, Eric Betzig was able to apply this idea by developing photoactivated localization microscopy (PALM). With very weak illumination, only a few molecules are illuminated, quite far apart, so that they can be localized very precisely, as they do not interfere with each other. If this image capture is repeated a few times (by illuminating a few more molecules randomly distributed on the sample), the only thing left to do is to superimpose the captured photographs to obtain a high-resolution image.

This year, the Nobel Prize for Physics has awarded a more efficient and therefore more "ecological" light (LEDs) and the Nobel Prize for Chemistry a new way to illuminate the beauty hidden in the tiny.