NEWS IN FOCUS finer-resolution structure. Moerner went on to show that identical molecules could be made to fluoresce at different times, a discovery that ultimately made Betzig’s vision a reality. It was another decade before Betzig demon- strated his idea in practice. In 2006, working at the Howard Hughes Medical Institute’s Janelia Farm research campus in Ashburn, Virginia, he took a super-resolution picture of a lysosome protein dotted with green fluorescent molecules as labels. The technique can now get down to a resolution of 20 nanometres, says Markus Sauer, who studies super-resolution microscopy at the University of Würzburg, Germany. Meanwhile Stefan Hell, working at the Uni- Chemical visionaries: (from left) Stefan Hell, Eric Betzig and William Moerner. versity of Turku in Finland, had found a way around Abbe’s limit by a different technique, NOBEL PRIZE which also relies on switching fluorescent mol- ecules on and off. In 1994, he proposed using one laser to make a cluster of dye molecules flu- oresce, and a second beam, of a different wave- Insider view of length, to switch some of those fluorophores off. Hell’s trick is to use the second beam to outline the cluster illuminated by the first, so that only the molecules in a very narrow spot cells scoops Nobel fluoresce. The final image remains blurred, as light still cannot beat Abbe’s limit, but it is clear that light can have come only from the narrow Optics pioneers win chemistry prize for defying limits of central spot defined by the second beam, ena- conventional microscopes. bling researchers to pinpoint the light source. Building up a series of these tiny fluorescent spots creates a fine-resolution picture. In theory, BY RICHARD VAN NOORDEN reveal the organelles inside cells but not to see the central spot can be made as small as a few their detailed structures. Microscopes that nanometres across, but in living cells, the limit ver since the seventeenth century, when use beams of electrons, rather than light, have is around 30 nanometres, Sauer says, because pioneering microbiologist Antonie van finer resolution, but they can be used only in a it is at this stage that fluorophores are usually UNIV. L.A. CICERO/STANFORD MEDICAL INST.; HUGHES STALEY/HOWARD MATT BIOPHYSICAL CHEMISTRY; BERND SHULLER/MAX PLANCK INST. FROM LEFT: Leeuwenhoek focused light through vacuum, limiting their use to dead tissue. destroyed by the intensity of the second beam. Elenses and marvelled at the cells that swam Abbe’s limit cannot be overcome, but the “It was my view, at least, that so much phys- before his eyes, microscopes have been at the 2014 Nobel prizewinners pioneered ways to ics happened in the twentieth century that it heart of discovery. This year, the Nobel Prize in work around it using fluorophores, or fluores- was impossible there was no phenomenon Chemistry went to three scientists who defied cent molecules. Now routinely used in biologi- that would allow you to overcome the diffrac- the limits of light microscopes to reveal images cal imaging, fluorophores emit light when hit tion barrier,” Hell, who now works at the Max of molecular-scale structures in living cells. by lasers of a certain wavelength. Planck Institute for Biophysical Chemistry, The advances made by Stefan Hell, William In 1989, William Moerner, now at Stanford told the Nobel committee. Moerner and Eric Betzig in the 1990s and 2000s University in California, but then at the IBM The techniques devised by the prizewinners allow biologists to watch in real time how pro- Almaden Research Center in San Jose, detected are used by many biologists. Xiaowei Zhuang, teins are distributed and move inside cells — at the faint fluorescence of a single molecule. In a chemist at Harvard University in Cambridge, the junctions between neurons, for example, or 1997, while working at the University of Cali- Massachusetts, has invented a variation called in fertilized eggs dividing into embryos. fornia, San Diego, he found a way to control stochastic optical reconstruction microscopy, “It is really a revolution for the life sciences, the fluorescence and switch the molecules on and has used it to show how filaments of the because we can see structures that we could and off like lamps. Still, these single molecules protein actin wrap around nerve cells. “There never see before,” says Stefan Jakobs, who could be distinguished only if they were more will be many new versions of super-resolution works with super-resolution techniques at the than 200 nanometres apart. microscopes,” Hell says. ■ Max Planck Institute for Biophysical Chemis- Two years earlier, Eric Betzig, then working try in Göttingen. Or as the Nobel committee at Bell Labs in Murray Hill, New Jersey, had put it: “Microscopy has become nanoscopy.” proposed that if different molecules inside a CORRECTION & CLARIFICATION No matter how clean their lenses, optical cell could be made to glow with different col- The News story ‘Marmosets are stars of microscopes inevitably provide a blurry view ours, researchers should be able to increase the Japan’s ambitious brain project’ (Nature of the molecules inside cells, as German physi- resolution by taking a series of snapshots — 514, 151–152; 2014) misspelled Afonso cist Ernst Abbe realized in 1873. The laws of first the red molecules, then the green, then the Silva’s name. And the Toolbox story physics dictate that visible light cannot dis- blue. Any fluorophores ‘Scientific writing: the online cooperative’ tinguish between objects closer to each other NATURE.COM of the same colour would (Nature 514, 127–128; 2014) should have than around 200 nanometres (around half See a gallery of have to be more than 200 noted that although Fidus Writer does not the wavelength of visible light) — they will super-resolution nanometres apart, but the record the detailed history of every single appear as one blob. Such resolution, known microscopy images: superimposed images edit, users can save time-stamped versions. as Abbe’s diffraction limit, is good enough to go.nature.com/lgg7ep would produce a much 286 | NATURE | VOL 514 | 16 OCTOBER 2014 © 2014 Macmillan Publishers Limited. All rights reserved.