to BioTechniques free email alert service to receive content updates.
A STED-y route to commercialization
Jeffrey M. Perkel, Ph.D.
BioTechniques, Vol. 50, No. 6, June 2011, pp. 357–363
Full Text (PDF)

“The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken.” With that blunt assessment, published in the Proceedings of the National Academy of Science in 2000, Stefan Hell introduced the world to a “super-resolution” microscopy technique called stimulated emission depletion, or STED.

Or rather, he reintroduced the technique. Hell had first described STED—which cracks the diffraction limit that has for so long bounded optical microscopy—in a brief two-and-a-half page mathematical treatise six years earlier. “I had a very hard time to convince people it would work,” Hell recalls. The idea of a “diffraction barrier,” a physical constraint on light microscopy resolution, was simply too well ingrained.

Following his 2000 article, though, researchers started paying attention; the publication has racked up some 310 citations, according to the ISI Web of Knowledge. It also attracted the notice of microscopy manufacturer Leica Microsystems who licensed STED technology in 2001 and began converting it into a viable commercial system. It would take some five years. Along the way, Leica's engineers, physicists, and microscopists, aided by members of Hell's own lab, would have to rethink most of the original benchtop design.

The Seed of STED

In the late 1980s Hell was a physics graduate student at the University of Heidelberg using confocal microscopy to measure features on computer microchips. “The actual thesis subject … was so technical that I started to think about more fundamental things that one could do with this outdated field of physics,” he says, referring to optical microscopy. “I thought about breaking the diffraction barrier, which seemed to me a goal worthwhile to pursue scientifically.”

The diffraction barrier (also known as the Abbe limit) limits how closely together two objects in a cell can be and still be resolved using a light microscope. A function of the wavelength of light illuminating a sample and the physical properties of the optics used to visualize it, this barrier hovers around 200–250 nm, and it explains why microscopists cannot resolve macromolecular complexes such as densely packed microtubules into individual subunits via fluorescence light microscopy.

Hell, though, had a gut feeling the barrier was less solid than imagined. He suspected it might be possible to overcome it by manipulating the “light-driven state transitions” of fluorescent dyes: in other words tinkering with the process by which the dyes absorb and release energy. He pursued the idea in the early 1990s, first at the European Molecular Biology Laboratory and then at the University of Turku in Finland, where he had a senior postdoctoral fellowship. In 1993, he had a breakthrough. “I realized that one could do it by turning off a dye by stimulated emission.”

Hell's vision was to use two superimposed laser spots, an excitation beam and a beam for molecularde-excitation, and alternate their pulses so that some of the molecules in the excitation area are prevented from fluorescing – the foundation of STED. “STED introduces a mechanism by which we keep molecules dark even though they are illuminated with excitation light,” he says.

As in standard confocal microscopy, the excitation beam excites the fluorophores in a diffraction-limited region in the sample, rasterizing across the sample and collecting fluorescence intensity spot-by-spot. First, though, the STED beam—shaped like a doughnut with a hole in the center—deactivates the dyes at the periphery of that relatively large spot by forcing them back down to their ground state without fluorescing, acting like a light-based photomask. The net result is to shrink the effective excitation region below the diffraction limit.

“The purpose of that ring is to keep molecules fluorescent at the center of the doughnut while at the periphery, where the [STED laser] intensity is strong, the molecules are shut off,” Hell explains. “So the region in which molecules are allowed to emit is made smaller in the end, and so it's possible to see much finer detail.”

  1    2    3    4