(Left) Viewing a mitochondrion using conventional diffraction-limited microscopy offers a resolution (200 nanometers) barely sufficient to visualize the mitochondrial internal membranes. (Right) Viewing the same mitochondrion by imaging sparsely activated fluorescent molecules one at a time—using PALM—provides much better resolution (20 nanometers), producing a detailed picture of the mitochondrion’s internal membranes.

More than a century ago, German physicist Ernst Abbe discovered a fundamental limitation on how sharply a conventional light microscope can focus on extremely small objects.

Because of the way light moves in the realm of the infinitesimal, no matter how powerful the microscope, it cannot distinguish particles closer than about 200 nanometers (one-quarter the diameter of a typical bacterium) as separate objects.

The problem is that the wave nature of light becomes more apparent in the microscopic world. Particles inside cells distort light waves in much the same way that raindrops falling into a pond form concentric ripples. With so many structures packing cells, high-magnification microscope images become torrents of overlapping light patterns.

As a graduate student in the 1980s, Eric Betzig wasn't satisfied living within Abbe's constraints, so he developed an imaging technique called near-field microscopy to circumvent them. Honing the method at Bell Labs, Betzig achieved unprecedented resolutions of 30-50 nanometers.

But even at that level, images weren't sharp enough to meet what Betzig calls the First Holy Grail of optical microscopy: imaging individual protein molecules within cells. He and his Bell Labs colleague Harald Hess realized that, by discerning individual proteins, cell biologists could keep more intimate tabs on what those proteins were doing and how they interacted.

Last year, the two inventors—both unemployed at the time—developed an elegant procedure out of an idea they had begun exploring together in the 1990s. Instead of having a muddle of fluorescently labeled proteins glowing at once, sloshing light waves everywhere, they found a way to turn on just a few molecules at a time. That way, the concentric waves surrounding each molecule wouldn't overwhelm the image.

Images: From Science 2006 Sep15; 313(5793):1642-5. Epub 2006 Aug 10. Reprinted with permission from AAAS.

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