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New Optics Strategies Cut Through Diffraction Barrier

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Science  11 Aug 2006:
Vol. 313, Issue 5788, pp. 748a-749a
DOI: 10.1126/science.313.5788.748a

Optical microscopes gave birth to cell biology, revealing a Lilliputian world of mitochondria, chromosomes, and much more. Yet as biologists grew more adept at illuminating the cell's interior, light's physical properties stopped their progress dead in its tracks. The so-called diffraction barrier limits resolution to 200 nanometers in the case of visible light, or half the wavelength used to make an image. To see more detail, scientists had to turn to the shorter wavelengths of electron microscopes.

Now, two research teams have independently developed light microscopy techniques that resolve objects on the nanometer scale. “The diffraction barrier is not only gone in theory. It's really gone,” says physicist Stefan Hell of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, the leader of one of the groups. He and others expect the new methods to enable biologists to visualize how proteins interact with one another and the cell membrane, and to solve numerous mysteries about how cells function. “I see a whole array of applications,” says Shuming Nie, a biomolecular engineer at Emory University in Atlanta, Georgia.

One of the new techniques, described online in Science this week ( by physicists Eric Betzig, Harald Hess, and colleagues, began with a device assembled in Hess's living room while both he and Betzig were unemployed. Betzig had pioneered a technique called near-field microscopy at Bell Labs in the 1990s, but he then went to work at his father's machine tool company in Michigan. “I was going through my midlife crisis, [and] I didn't want to do microscopy,” says Betzig. Leaving the machine company in 2003, he began talking microscopy again with Hess, a longtime friend from Bell Labs.

Up close.

A high-tech microscope, assembled in a living room (above), revealed molecules (red, inset) nanometers apart inside a cell's mitochondria.


Together, the two arrived at a way to break the diffraction barrier. Using new technologies for labeling cellular machinery with light-activated fluorescent markers, they could “turn on” just one molecule at a time. Such pinpoints of light can be located much more precisely than when all are glowing at once. By slowly mapping the cell molecule by molecule, they could piece together a high-resolution picture of the whole thing.

They constructed a microscope that flashes a violet light at proteins designed to activate under such rays. By keeping the light flash brief and the light extra dim, the scientists ensured that just some molecules activate. Then, the pair zapped the molecules with a yellow light that made them glow brightly for up to a few seconds before flaring out. By repeating the process over and over again—roughly 10,000 times in all over 2 to 12 hours—the researchers could gather enough information to compile a “supermap” of the cell, distinguishing molecules just 2 to 25 nanometers apart in regions with up to 100,000 molecules per square micrometer. For example, they assembled detailed images of the Golgi apparatus and the retroviral protein Gag bound to the cell's membrane. “They are, in a sense, pushing the power of single molecules as nanoscale light sources to the limit,” says W. E. Moerner, a physical chemist at Stanford University in Palo Alto, California.

The new technique, dubbed photoactivated localization microscopy, currently has a resolution similar to that of electron microscopy. But scientists say that it has potential for even better resolution and for examining protein-protein interactions, particularly if fluorescent labels of different colors can be applied to proteins.

Hell's barrier-busting technique, which he first sketched out in 1994, takes the opposite approach from Betzig's. Instead of turning on fluorescently labeled molecules one by one, Hell turns them off, using a hollow needle of light that darkens a ring of molecules but leaves the ones in the very center glowing. In 2000, Hell tested the technique—known as stimulated emission depletion microscopy—on cells and found that it worked. Last year in Physical Review Letters, Hell and colleagues reported even better resolution in nonbiological samples. Now, in the 1 August Proceedings of the National Academy of Sciences, Hell and colleagues report imaging molecules 15 to 20 nanometers apart in dead cells.

One challenge now is to apply the new techniques to living cells, whose parts are often in rapid motion. The Betzig technique may face more hurdles because it relies on hours of snapshots before building a picture of a cell's static state. Still, says Moerner, there's hope that scientists will find ways around the roadblocks. “The ingenuity of people always surpasses what we say can be done,” he says.

Fortunately, Hess's living room won't be needed anymore. Both Hess and Betzig have been recruited to lead groups at Janelia Farm, the new Virginia campus of the Howard Hughes Medical Institute devoted to developing new research techniques.

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