PROFILE Profile of Eric Betzig, Stefan Hell, and W. E. Moerner, 2014 Nobel Laureates in Chemistry Jennifer Lippincott-Schwartz1 Eunice Kennedy Shriver National Institute of Child Health and Human Development, National it could be used to quench all fluorescence Institutes of Health, Bethesda, MD 20892 except that in a nanometer-sized volume. The shrinking could be accomplished, Hell reasoned, if one laser was used to make a clus- Scientists once believed that the laws of the field of superresolution imaging, which ter of dye molecules fluoresce and a second physics would prevent them from peering has allowed the visualization of nanometric- beam, of a different wavelength, was used to switch off some of those fluorophores. By into the structures of the cell. The story of the level structures inside cells using visible light scanning across a fluorescent sample in this winners of the 2014 Nobel Prize in Chemis- for the first time. manner, it would then be possible to obtain try is about how three imaginative scientists Stefan Hell began seeking ways to over- a superresolution image, bringing fine detail pioneered ways to work around these sup- come Abbe’s limit as a graduate student at to diffraction-limited fuzzy blobs of fluores- posed limits, transforming microscopy into theUniversityofHeidelberginthelate1980s. cence. In 1994, Hell published his theory of nanoscopy. His first idea for improving resolution was to this new imaging method, which he called In 1873, German physicist Ernst Abbe use two opposing lenses with interfering light stimulated emission depletion (STED) (2). theorized that the laws of physics dictate that paths both focused to the same geometrical The next challenge Hell faced was to show visible light cannot distinguish between location. When he joined the group of Ernst that STED worked. Fortunately, the Max objects closer to each other than around Stelzer at the European Molecular Biology Planck Institute for Biological Chemistry in 200 nm (about half the wavelength of visible Laboratory in Heidelberg, Germany, he used Gottingen, Germany, was willing to let him light), which inevitably appear as one blob. this approach to devise the 4Pi microscope try, despite many scientists’ skepticism about Visual resolution above this level, known as (1), which improved resolution three to seven the possibility of breaking the diffraction ’ z Abbe s diffraction limit, is good enough to times, but only along the axis. Determined limit. Assembling a team of physicists, Hell reveal the organelles inside cells but not to to find another way to achieve superresolu- worked tirelessly to construct a STED micro- see their detailed structures. The 2014 chem- tion, Hell moved to a fluorescence microcopy scope. In 1999, he wrote up his success, istry laureates, Stefan Hell, Eric Betzig, and laboratory at the University of Turku in imaging an Escherichia coli bacterium at a res- W. E. Moerner, together defied these restric- Finland. There, Hell found the clue he olution never before achieved in an optical tions by conceptualizing and developing needed. Reading about stimulated emis- microscope. Both Nature and Science promptly a suite of tools. Their work helped found sion in a quantum optics book, he realized 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 im- prove STED (4), which is now used world- wide 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 ob- tain subdiffraction-limit resolution. He shined light through a subwavelength aperture (pro- ducing an evanescent wave limited both axially and laterally to within 20 nm) and scanned it across a surface, detecting unprec- edented detail. The technique, called near- field scanning optical microscopy (NSOM) (5), transcended Abbe’s limit by an order of

Author contributions: J.L.-S. wrote the paper. 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 The author declares no conflict of interest. Sweden, Queen Silvia, Stefan W. Hell, and Anna Kathrin Hell. Copyright © Nobel Media AB 2014. Photo: Niklas Elmehed. 1Email: [email protected].

2630–2632 | PNAS | March 3, 2015 | vol. 112 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1500784112 Downloaded by guest on October 1, 2021 PROFILE reactivated, it once again fluoresced at 488 nm (9). The variant was thus optically controllable. Within a few years, research- ers began developing a palette of fluores- cent 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 imple- ment his 10-year-old idea for overcoming Abbe’s limit using single molecules. He con- tacted one of the groups specializing in these proteins (my own) to test whether his ideawouldholdupinpracticeintheirlabora- tory. Although George Patterson in our group had developed a photoactivatable GFP that al- lowed 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 Fig. 2. In the case of photoactivable localization microscopy, a weak pulse of UV light is used to activate a very small Mike Davidson contributing key probes and 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 Harald Hess helping to build the novel appa- proteins are superimposed to generate an image with a resolution many times greater than the diffraction limit. Il- ratus, photoactivable localization microscopy lustration: © Johan Jarnestad/The Royal Swedish Academy of Sciences. (PALM) emerged (12). In PALM, a weak pulse of UV light is used to activate a very small subset of photo- magnitude in all dimensions and therefore provided the seed for Betzig’s approach activatable molecules in a dense sample. initially sparked widespread interest. How- for breaking Abbe’s diffraction limit. He Most of these switched-on molecules will ever, because its resolution decreased with reasoned that a dense ensemble of mole- be positioned at a distance from each distance between the aperture and the sam- cules could be imaged at superresolution other greater than Abbe’s diffraction limit ple, NSOM was limited to the study of if individual molecules had isolatable of 0.2 μm, allowing their position to be surfaces, making its biological applica- optical properties that allowed their pre- precisely registered. Once their fluores- bility limited. Betzig therefore decided to cise positions to be determined through ’ ’ cence dies out, a new subgroup of proteins search for more tools to overcome Abbe s Gaussian fitting of the molecule s emitted can be activated and their positions regis- diffraction limit. The answer came from photons. The positions of the individual tered. After this has been repeated thou- the unexpected source of single molecule molecules could then be superimposed to sands of times, the positions of the proteins visualization. obtain a single superresolution image of can be superimposed to generate an image The foundation for this approach was laid the entire ensemble. In 1995, Betzig pub- with a resolution many times greater than Optics by W. E. Moerner, who was the first to lished this simple, elegant idea in the diffraction limit. Letters measure light absorption from a single fluo- (8), but he realized there were prac- Perhaps indicative of science’stendency rophore in a dense medium (6). Moerner was tical problems in implementation because to make significant advances once new working on optical storage devices at the there still needed to be molecules whose exploratory tools are available—in this case IBM research center in San Jose, CA, exam- emissions could be sufficiently controlled. photoconvertible probes—two other re- ining the fundamental limits for recording As the field of single molecule imaging search groups independently demonstrated digital information at different laser wave- matured, a solution to this problem emerged. a similar approach to PALM at nearly the lengths using fluorophores. Moerner decided W. E. Moerner relocated to the University same time, naming their variations sto- to look for spectral features that represented of California in San Diego to study bio- chastic optical reconstruction microscopy information from a single fluorophore in- logical systems at the single molecule level. (STORM) (13) and fluorescence photoac- stead of ensembles of fluorophores. When There, he obtained GFP variants from tivation localization microscopy (FPALM) he succeeded in observing a single fluoro- Roger Tsien, who later won the Nobel (14). Other similar uses of single molecule- phore in 1989, it was a pivotal moment. Prize in Chemistry in 2008 for developing based superresolution imaging have con- Not only did it reinforce the idea that sto- GFP technology. Using one such variant, tinued to emerge (15), so it is now possible chastic modes of molecule action could be Moerner observed something strange. Like not only to visualize the spatio-dynamics studied, but it paved the way for a host of other GFPs, after being excited with light of cellular structures subdiffractively but single-molecule techniques in spectroscopy of 488 nm, this variant fluoresced and also to track individual molecules within and microscopy. then faded. However, unlike many GFPs, dense populations and define receptor Inspired by Moerner’s achievement, Betzig which are unable to fluoresce again, this stochiometry. succeeded in using NSOM to detect fluo- variant could be brought back to life using All these superresolution ideas originat- rescence from a single molecule (7). This light of 405 nm. When the protein was ing in the last two decades are now playing

Lippincott-Schwartz PNAS | March 3, 2015 | vol. 112 | no. 9 | 2631 Downloaded by guest on October 1, 2021 a vital role throughout the world of biolog- tool. Because of their perseverance and Abbe’s diffraction-limited images. The chal- ical research. Hell, Betzig, and Moerner de- creativity, commercial turn-key superreso- lenge now is to understand and interpret the serve recognition for their foundational lution microscopes are now available, end- cellular processes we see unfolding at nano- contributions to this essential new research ing the days of interpreting fuzzy blobs from meter scales.

1 Hell SW, Stelzer EH, Lindek S, Cremer C (1994) Confocal 6 Moerner WE, Kador L (1989) Optical detection and 11 Shcherbakova DM, Sengupta P, Lippincott-Schwartz J, microscopy with an increased detection aperture: type-B 4Pi confocal spectroscopy of single molecules in a solid. Phys Rev Lett 62(21): Verkhusha VV (2014) Photocontrollable fluorescent proteins for microscopy. Opt Lett 19(3):222. 2535–2538. superresolution imaging. Annu Rev Biophys 43:303–329. 2 Hell SW, Wichmann J (1994) Breaking the diffraction resolution 7 Betzig E, Chichester RJ (1993) Single molecules observed by 12 Betzig E, et al. (2006) Imaging intracellular fluorescent proteins limit by stimulated emission: stimulated-emission-depletion near-field scanning optical microscopy. Science 262(5138): at nanometer resolution. Science 313(5793):1642–1645. fluorescence microscopy. Opt Lett 19(11):780–782. 1422–1425. 13 Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging 3 Klar TA, Jakobs S, Dyba M, Egner A, Hell SW (2000) Fluorescence 8 Betzig E (1995) Proposed method for molecular optical imaging. by stochastic optical reconstruction microscopy (STORM). Nat microscopy with diffraction resolution barrier broken by stimulated Opt Lett 20(3):237–239. Methods 3(10):793–795. emission. Proc Natl Acad Sci USA 97(15):8206–8210. 9 Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off 14 Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution 4 Hell SW (2003) Toward fluorescence nanoscopy. Nat Biotechnol blinking and switching behaviour of single molecules of green imaging by fluorescence photoactivation localization microscopy. 21(11):1347–1355. fluorescent protein. Nature 388(6640):355–358. Biophys J 91(11):4258–4272. 5 Betzig E, Trautman JK (1992) Near-field optics: Microscopy, 10 Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable 15 Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) spectroscopy, and surface modification beyond the diffraction limit. GFP for selective photolabeling of proteins and cells. Science Superresolution imaging using single-molecule localization. Annu Rev Science 257(5067):189–195. 297(5588):1873–1877. Phys Chem 61:345–367.

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