Milestone 21: Light Microscopy at the Limit

Milestones

MILESTONE 21 Light microscopy at the limit

theoretically and then experimentally, photoactivatable or photoswitchable that a stimulated-emission-depletion fluorophores and high-accuracy single- (STED) microscope can break the molecule localization to break the diffraction barrier. To do this, the diffraction barrier. Although the details excitation focal spot is shrunk to a differ, all three methods are based on very small size by depleting the fluoro- a single principle: a sparse subset of phores at its rim through stimulated fluorophores is switched on at any one emission with a doughnut-shaped time, and each molecule is localized STED beam of red-shifted light. The with high accuracy. These fluorophores tiny spot is then scanned over the are then switched off and the process sample to generate a sub-diffraction is repeated with another subset until image. In the first experimental enough information has been collected Stimulated-emission-depletion image recorded from a living neuron of an organotypical implementation of STED, which to generate a sub-diffraction image. hippocampal slice, showing dendritic spines in included imaging of live yeast and The hope for super-resolution unprecedented detail. The neuron is expressing yellow fluorescent protein. Image courtesy of K. bacteria, resolution was improved microscopy is that visualizing cells Willig, V. Nägerl, N. Urban, T. Bonhoeffer & both axially and laterally and reached at this unprecedented scale will yield S. W. Hell, MPI Biophysical Chemistry, Göttingen, Germany about 100 nm. The resolution has unprecedented insights. Although since been improved further, allowing the application of these methods the technique to be used to show that to biological questions has begun, For most of the twentieth century, the synaptic vesicle-associated protein the full realization of their potential it was held that far-field light synaptotagmin remains clustered on is still to come. Their extension to microscopy could not resolve objects the neuronal plasma membrane after imaging in all three dimensions and closer than 150–200 nanometres exocytosis. within living cells — capabilities that (see Milestone 3). This is because While STED was being demon- are developing rapidly — will be a light diffracts as it passes through strated, Mats Gustafsson was develop- crucial part of this process. different media, so that the light ing an approach called structured Natalie de Souza, Associate Editor, emanating from a point is detected as illumination microscopy (SIM). In Nature Methods

emerging from a larger volume. Light SIM, the excitation light is structured PRIMARY REFERENCES Klar, T. A., Jakobs, S., microscopy was therefore said to be in a controlled pattern, which renders Dyba, M., Egner, A. & Hell, S. W. Fluorescence ‘diffraction-limited’. normally unresolvable information microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Yet many cellular processes take accessible after image processing. Sci. USA 97, 8206–8210 (2000) | Betzig, E. et al. place within these diffraction-limited Although still diffraction-limited, SIM Imaging intracellular fluorescent proteins at distances, at length scales of tens to improved lateral resolution by about nanometer resolution. Science 313, 1642–1645 (2006) | Rust, M. J., Bates, M. & Zhuang, X. Sub- hundreds of nanometres (nm). To be twofold and produced clearer images diffraction-limit imaging by stochastic optical able to visualize these processes, there- of biological structures. The technique reconstruction microscopy (STORM). Nature Methods 3, 793–796 (2006) fore, microscopy with substantially has since been extended to three FURTHER READING Hell, S. & Stelzer, E. H. K. improved resolution was needed. It dimensions using 3D structured light; Fundamental improvement of resolution with a became necessary to ‘break’ the dif- moreover, it enables the diffraction 4Pi-confocal fluorescence microscope using two-photon excitation. Opt. Commun. 93, fraction barrier. barrier to be broken, using nonlinear 277–282 (1992) | Hell, S. W. & Wichmann, J. After early work that achieved processes. Breaking the diffraction resolution limit by the maximum theoretical resolu- Finally, by exploiting the known stimulated emission: stimulated-emission- depletion fluorescence microscopy. Opt. Lett. 19, tion — including the standing-wave ability to localize single molecules with 780–782 (1994) | Gustafsson, M. G. L. Surpassing illumination reported by Fred Lanni nanometre precision, several single- the lateral resolution limit by a factor of two using structured illumination microscopy. and Jans Taylor and the two-photon molecule approaches to achieving sub- J. Microsc. 198, 82–87 (2000) | Gordon, M. P., Ha, T. 4Pi microscope of Stefan Hell and diffraction resolution were developed. & Selvin, P. R. Single-molecule high-resolution Ernst Stelzer — Hell showed, first The pioneers of these methods — Eric imaging with photobleaching. Proc. Natl Acad. Sci. USA 101, 6462–6465 (2004) | Willig, K. I., Betzig and Harald Hess for photoacti- Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. vated localization microscopy (PALM), STED microscopy reveals that synaptotagmin Xiaowei Zhuang for stochastic optical remains clustered after synaptic vesicle It became necessary to exocytosis. Nature 440, 935–939 (2006) | reconstruction microscopy (STORM) Hess, S. T., Girirajan, T. P. K. & Mason, M. D. ‘break’ the diffraction and Samuel Hess for fluorescence pho- Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. barrier. toactivation localization microscopy Biophys. J. 91, 4258–4272 (2006) (FPALM) — used the combination of