ChemPhysChem

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Editor: Greta Heydenrych; Editorial Board Chairs: Christian Amatore, Michael Grätzel, Michel Orrit

Online ISSN: 1439-7641

Associated Title(s): Advanced Materials, ChemBioChem, ChemCatChem, ChemElectroChem, ChemSusChem, Small

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October 08, 2014

2014 Nobel Prize in Chemistry: Breaking the Barrier

2014 Nobel Prize in Chemistry: Breaking the BarrierThe 2014 Nobel Prize in Chemistry goes to Stefan W. Hell (Max Planck Institute for Biophysical Chemistry and German Cancer Research Center, Germany), W. E. Moerner (Stanford University, USA ), and Eric Betzig (Janelia Research Campus, Howard Hughes Medical Institute, USA) "for the development of super-resolved fluorescence microscopy”. With the help of fluorescent molecules, the three scientists were able to overcome the diffraction barrier –a presumed limitation stipulating that an optical microscope could never yield a resolution better than 200 nanometers. This diffraction barrier, proposed by the physicist Ernst Abbe in 1873, was considered unbreakable and had not been questioned for more than a century. Hell, Moerner and Betzig found brilliant ways to bypass this limitation. Their groundbreaking work has brought optical microscopy into the nanodimension.

"The most important issue about the development of optical nanoscopy was that it showed that a physical limit that over years was thought to impede the applicability of far-field optical microscopy –one of the most important tools for live-cell investigations– can be overcome," says Professor Christian Eggeling (University of Oxford, UK), who has worked closely with Stefan Hell during the past years and is co-author of several of his highly cited publications. "This was based on the insight by Stefan Hell in the 1990s that nearby sample molecules are no longer discerned just by the phenomenon of focusing light, but by prompting them to briefly assume at least two different states (e.g. an on and an off state)."

Based on this principle, Hell developed a method called stimulated emission depletion (STED) microscopy, in which two laser beams are utilized; one stimulates fluorescent molecules to glow while the other one cancels out all the fluorescence except for that in a nanometer-sized volume. Scanning over the sample, nanometer for nanometer, yields an image with a resolution better than Abbe's stipulated limit. "I realized that silencing a fluorophore by stimulated emission or keeping it in a metastable dark state would be very powerful for separating fluorophores at sub-diffraction length scales," Hell told ChemPhysChem recently.

W. E. Moerner and Eric Betzig, working separately, laid the foundation for another method: single-molecule microscopy. This technique relies on the possibility to turn the fluorescence of individual molecules on and off. The same area is imaged repeatedly, but only a few molecules are allowed to glow each time. The images are then superimposed, yielding a dense super-image resolved at the nanolevel.

"These three researchers have laid the foundations for the field of nanoscopy or super-resolution microscopy," says Suliana Manley, Professor of Physics at the Ecole Polytechnique Federale de Lausanne, EPFL, in Switzerland. "Moerner was the first to image single molecules in ambient conditions, a remarkable feat at the time; Hell performed the seminal development of STED, and was incredibly persistent, continuously improving the method and keeping the field alive with his breakthroughs; and Betzig made brilliant connections to go from spectral separation of emitters in near-field microscopy to temporal separation of fluorophores in far-field photoactivated localization microscopy (PALM)." Manley explains that the different methods, STED, PALM and structured illumination microscopy (SIM), compete in a very positive way. "All three have pushed to enable multicolor, live, 3D imaging, and historically the breakthroughs have come in bursts," she says.

"One of the challenges in biological imaging is that the sizes of cellular structures are inherently mismatched with the diffraction limit of light," says Dr. James Fitzpatrick (Salk Institute of Biological Studies, USA), a well-known researcher in the field of super-resolution imaging. "Take for example macromolecular structures such as nuclear pore complexes, the gates that control the flow of molecules between the nucleus and the rest of the cell. Their true size is on the order of 100 nm, yet distinguishing them from other cellular components can be challenging because they appear larger in traditional fluorescence images as a result of optical diffraction. Super-resolution imaging, using either localization or point-spread function engineering approaches overcome these limitations by allowing the visualization of such structures at higher fidelity than was previously thought possible."

Christian Eggeling agrees that super-resolution imaging techniques are crucial to biological research. "Tools such as STED nanoscopy have been implemented on turn-key instrumentation and have found widespread inset into open facilities of biological institutes, where the applications involve all biological and biomedical areas such as neurobiology, immunology, cancer research or plasma membrane organization and thus functionality of cellular receptors," he says. But there's still a lot of work to do, says James Fitzpatrick: "Pushing the envelope in terms of spatial resolution has been an incredible leap forward, allowing us to access and visualize the world within our cells at length scales previously unattainable. But continued research is required to couple these advances with the ability to visualize changes over shorter and shorter periods of time. Greater temporal resolution will be critical to understanding how structural changes resulting from disease, injury or aging compromise the function of living biological systems," he adds.

Image: Nobel Medal (© ® The Nobel Foundation. Photo: Lovisa Engblom). Further information at www.nobelprize.org.

-Kira Welter

This article was also published on the news site of ChemElectroChem.

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