“Ingenious.” That is how the Royal Swedish Academy of Sciences described the work of Eric Betzig of the Howard Hughes Medical Institute, Virginia, USA; Stefan W. Hell of the Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; and W. E. Moerner from Stanford University, USA, who have shared the 2014 Nobel Prize in Chemistry. The prize was awarded for the development of fluorescence techniques with nanometre 'super-resolution' that have enabled scientists to observe chemistry occurring in unprecedented detail. These techniques sidestep a fundamental physical law — first described by Ernst Abbe in the late nineteenth century — that limits standard optical microscopy to a resolution of roughly 200 nm.
Working separately, Moerner (pictured left) and Betzig (pictured middle) laid the foundations for 'single-molecule spectroscopy'. Moerner, as well as being the first person to measure the absorption spectrum of a single fluorophore molecule in 1989, discovered a controllable variant of green fluorescent protein (GFP) in 1997. This GFP could be switched on with light of a specific wavelength and would intermittently fluoresce for a period of time before switching off — it could then be reactivated by a second wavelength of light. Using similar proteins, Betzig reported the technique of 'photo-activated localization microscopy' (PALM) in 2006. This method uses a weak laser to switch on the fluorescence of a fraction of the fluorophores in a sample, which are almost always farther apart than the 200 nm diffraction limit. They are imaged before the fluorescence is turned off, and when the process is repeated a different set of fluorophores light up. This process is carried out many times and a composite of the images gives a high-resolution picture of the positions of the fluorophores.
Hell (pictured right) developed a technique known as stimulated emission depletion (STED) microscopy. It works by using a laser to stimulate fluorescence in a sample of labelled molecules and a second laser beam that prevents fluorescence from a doughnut-shaped region around the point of interest. Only the fluorescence from the central area is detected. The microscope can then be swept across the sample, nanometre-by-nanometre, until a large picture is built up. Hell first published the concept in 1994, but it was not demonstrated experimentally until 2000.
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Armstrong, G. No limit. Nature Chem 6, 1027 (2014). https://doi.org/10.1038/nchem.2125
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DOI: https://doi.org/10.1038/nchem.2125