Hell worked out the mathematics of this idea—which he now calls “the on/off game”—in 1993 and published his proposal in 1994. He then spent the next half-decade trying to convince the scientific world (and funding agencies) that he was right. He finally got the money and lab space to implement his idea in 1998, and demonstrated the technique works in a pair of papers in 1999 and 2000 (4, 5).
The technique he invented is called STED (stimulated emission depletion) microscopy and uses two lasers to improve resolution from about 200 nm to 50 nm. The sharply focused excitation beam activates all the fluorophores in a diffraction-limited spot under the microscope. A second stimulated emission laser overlaid on the excitation beam causes fluorophores in the overlap region to stay dark. By altering the intensity of the second laser, he could raise or lower the resolution, theoretically down to the size of a single molecule. “You can break the diffraction barrier without having to break diffraction,” he says.
The result was the first so-called super-resolution technique. Alternative strategies followed a few years later, especially PALM and STORM, as well as another Hell-lab invention, RESOLFT, which derives multiple states from the cis-trans isomerization of fluorescent molecules.
Regardless of their acronyms, these techniques all rely on the same fundamental phenomenon, says Hell, the ability of fluorophores to assume two different states, making adjacent nanoscale features distinguishable.
“The on/off transition, the fact that not all the molecules residing within a 200-nm zone are capable of sending light back, this has been the essential element for overcoming the diffraction barrier,” Hell says.
Super-resolution microscopy is having “a lasting impact” on the life sciences and basic biomedical research. But the power of two-state transitions isn't yet played out. Other molecules can adopt multiple states, too, including Raman-active probes.
“I'm totally convinced that future generations will not see the light microscope just from a wave perspective, but also from a state perspective,” he says, a totally different perspective from when he started thinking about the problem in 1983.