Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry

Scientists once believed that the laws of physics would prevent them from peering into the structures of the cell. The story of the winners of the 2014 Nobel Prize in Chemistry is about how three imaginative scientists pioneered ways to work around these supposed limits, transforming microscopy into nanoscopy.

Fig. 1.

The 2014 Chemistry laureates and their significant others appear with the Swedish royal family after the Nobel Banquet. From left to right: Sharon Stein Moerner, William E. Moerner, Na Ji, Eric Betzig, King Carl XVI Gustaf of Sweden, Queen Silvia, Stefan W. Hell, and Anna Kathrin Hell. Copyright © Nobel Media AB 2014. Photo: Niklas Elmehed.

Fig. 2.

In the case of photoactivable localization microscopy, a weak pulse of UV light is used to activate a very small subset of photoactivatable molecules in a dense sample. Once their fluorescence dies out, a new subgroup of proteins is activated and their positions registered. The process is repeated thousands of times, and the positions of the proteins are superimposed to generate an image with a resolution many times greater than the diffraction limit. Illustration: © Johan Jarnestad/The Royal Swedish Academy of Sciences.

In 1873, German physicist Ernst Abbe theorized that the laws of physics dictate that visible light cannot distinguish between objects closer to each other than around 200 nm (about half the wavelength of visible light), which inevitably appear as one blob. Visual resolution above this level, known as Abbe’s diffraction limit, is good enough to reveal the organelles inside cells but not to see their detailed structures. The 2014 chemistry laureates, Stefan Hell, Eric Betzig, and W. E. Moerner, together defied these restrictions by conceptualizing and developing a suite of tools. Their work helped found the field of superresolution imaging, which has allowed the visualization of nanometric-level structures inside cells using visible light for the first time.

Stefan Hell began seeking ways to overcome Abbe’s limit as a graduate student at the University of Heidelberg in the late 1980s. His first idea for improving resolution was to use two opposing lenses with interfering light paths both focused to the same geometrical location. When he joined the group of Ernst Stelzer at the European Molecular Biology Laboratory in Heidelberg, Germany, he used this approach to devise the 4Pi microscope (1), which improved resolution three to seven times, but only along the z axis. Determined to find another way to achieve superresolution, Hell moved to a fluorescence microcopy laboratory at the University of Turku in Finland. There, Hell found the clue he needed. Reading about stimulated emission in a quantum optics book, he realized it could be used to quench all fluorescence except that in a nanometer-sized volume. The shrinking could be accomplished, Hell reasoned, if one laser was used to make a cluster of dye molecules fluoresce and a second beam, of a different wavelength, was used to switch off some of those fluorophores. By scanning across a fluorescent sample in this manner, it would then be possible to obtain a superresolution image, bringing fine detail to diffraction-limited fuzzy blobs of fluorescence. In 1994, Hell published his theory of this new imaging method, which he called stimulated emission depletion (STED) (2).

The next challenge Hell faced was to show that STED worked. Fortunately, the Max Planck Institute for Biological Chemistry in Gottingen, Germany, was willing to let him try, despite many scientists’ skepticism about the possibility of breaking the diffraction limit. Assembling a team of physicists, Hell worked tirelessly to construct a STED microscope. In 1999, he wrote up his success, imaging an Escherichia coli bacterium at a resolution never before achieved in an optical microscope. Both Nature and Science promptly rejected it, arguing that the technique did not reveal any new biology and thus would be of limited interest. However, PNAS recognized STED’s potential and published the data in 2000 (3). Over the ensuing 14 years, Hell and his colleagues have continued to improve STED (4), which is now used worldwide for acquiring images of specimens at the nanometer scale.

In parallel, across the Atlantic, a young graduate student at Cornell University was also obsessed with the idea of bypassing Abbe’s diffraction limit. Eric Betzig used a different, more intuitive approach to obtain subdiffraction-limit resolution. He shined light through a subwavelength aperture (producing an evanescent wave limited both axially and laterally to within 20 nm) and scanned it across a surface, detecting unprecedented detail. The technique, called near-field scanning optical microscopy (NSOM) (5), transcended Abbe’s limit by an order of magnitude in all dimensions and therefore initially sparked widespread interest. However, because its resolution decreased with distance between the aperture and the sample, NSOM was limited to the study of surfaces, making its biological applicability limited. Betzig therefore decided to search for more tools to overcome Abbe’s diffraction limit. The answer came from the unexpected source of single molecule visualization.

The foundation for this approach was laid by W. E. Moerner, who was the first to measure light absorption from a single fluorophore in a dense medium (6). Moerner was working on optical storage devices at the IBM research center in San Jose, CA, examining the fundamental limits for recording digital information at different laser wavelengths using fluorophores. Moerner decided to look for spectral features that represented information from a single fluorophore instead of ensembles of fluorophores. When he succeeded in observing a single fluorophore in 1989, it was a pivotal moment. Not only did it reinforce the idea that stochastic modes of molecule action could be studied, but it paved the way for a host of single-molecule techniques in spectroscopy and microscopy.

Inspired by Moerner’s achievement, Betzig succeeded in using NSOM to detect fluorescence from a single molecule (7). This provided the seed for Betzig’s approach for breaking Abbe’s diffraction limit. He reasoned that a dense ensemble of molecules could be imaged at superresolution if individual molecules had isolatable optical properties that allowed their precise positions to be determined through Gaussian fitting of the molecule’s emitted photons. The positions of the individual molecules could then be superimposed to obtain a single superresolution image of the entire ensemble. In 1995, Betzig published this simple, elegant idea in Optics Letters (8), but he realized there were practical problems in implementation because there still needed to be molecules whose emissions could be sufficiently controlled.

As the field of single molecule imaging matured, a solution to this problem emerged. W. E. Moerner relocated to the University of California in San Diego to study biological systems at the single molecule level. There, he obtained GFP variants from Roger Tsien, who later won the Nobel Prize in Chemistry in 2008 for developing GFP technology. Using one such variant, Moerner observed something strange. Like other GFPs, after being excited with light of 488 nm, this variant fluoresced and then faded. However, unlike many GFPs, which are unable to fluoresce again, this variant could be brought back to life using light of 405 nm. When the protein was reactivated, it once again fluoresced at 488 nm (9). The variant was thus optically controllable. Within a few years, researchers began developing a palette of fluorescent proteins with various optical control capabilities (10, 11).

While perusing the scientific literature, Betzig happened to read about the optically controllable fluorescent proteins. He realized that this was the tool he needed to implement his 10-year-old idea for overcoming Abbe’s limit using single molecules. He contacted one of the groups specializing in these proteins (my own) to test whether his idea would hold up in practice in their laboratory. Although George Patterson in our group had developed a photoactivatable GFP that allowed switching on of discrete ensembles of proteins (10), it had not occurred to us that it could be used, like STED and NSOM, to break the Abbe diffraction limit. Once Betzig explained his concept, we enthusiastically joined his project. Within a few months, with Mike Davidson contributing key probes and Harald Hess helping to build the novel apparatus, photoactivable localization microscopy (PALM) emerged (12).

In PALM, a weak pulse of UV light is used to activate a very small subset of photoactivatable molecules in a dense sample. Most of these switched-on molecules will be positioned at a distance from each other greater than Abbe’s diffraction limit of 0.2 µm, allowing their position to be precisely registered. Once their fluorescence dies out, a new subgroup of proteins can be activated and their positions registered. After this has been repeated thousands of times, the positions of the proteins can be superimposed to generate an image with a resolution many times greater than the diffraction limit.

Perhaps indicative of science’s tendency to make significant advances once new exploratory tools are available—in this case photoconvertible probes—two other research groups independently demonstrated a similar approach to PALM at nearly the same time, naming their variations stochastic optical reconstruction microscopy (STORM) (13) and fluorescence photoactivation localization microscopy (FPALM) (14). Other similar uses of single molecule-based superresolution imaging have continued to emerge (15), so it is now possible not only to visualize the spatio-dynamics of cellular structures subdiffractively but also to track individual molecules within dense populations and define receptor stochiometry.

All these superresolution ideas originating in the last two decades are now playing a vital role throughout the world of biological research. Hell, Betzig, and Moerner deserve recognition for their foundational contributions to this essential new research tool. Because of their perseverance and creativity, commercial turn-key superresolution microscopes are now available, ending the days of interpreting fuzzy blobs from Abbe’s diffraction-limited images. The challenge now is to understand and interpret the cellular processes we see unfolding at nanometer scales.