The 2014 Nobel Prize in Chemistry recognizes the efforts that resulted in super-resolution fluorescence microscopy. New work by the honorees demonstrates the benefits of their contributions to life sciences.
On October 8, 2014, the winners of the 2014 Nobel Prize in Chemistry were honored for their work to overcome the resolution limit of standard optical microscopy, which Ernst Abbe famously stated in 1873 was 0.2 μm, or 200 nm (the shortest wavelength of visible light).
|Left to right: Betzig, Hell, and Moerner. (Photos courtesy of Matt Staley/HHMI; Bernd Schuller, Max-Planck-Institut; and LA Cicero/Stanford University)|
The honorees—Eric Betzig, Ph.D., a group leader at Howard Hughes Medical Institute's Janelia Research Campus (Ashburn, VA); Stefan W. Hell, Prof. Dr. Dr. h. c. mult., a director with the Max Planck Institute for Biophysical Chemistry (Göttingen, Germany); and William E. Moerner, Ph.D., Harry S. Mosher Professor in Chemistry and Professor, by courtesy, of Applied Physics at Stanford University—worked out different approaches to achieve super-resolved fluorescence microscopy, which has enabled nanoscale imaging of living tissue (see Fig. 1) and innumerable benefits for life sciences and for society.
|FIGURE 1. A comparison of standard confocal microscopy with super-resolved fluorescence in the form of stimulated emission depletion (STED). Here, HeLa cells are shown stained with NUP153-Alexa 532 (green), Clathrin-TMR (red), and Actin-Alexa 488 (white).|
"This year's prize is about how the optical microscope became a nanoscope," said Staffan Normark, Permanent Secretary of the Royal Swedish Academy of Sciences, in announcing the academy's decision. Sven Lidin, Chair of the Nobel Committee for Chemistry 2014, said that with super-resolution microscopy, "guesswork has turned into hard facts, and obscurity has turned into clarity."
The prize recognizes two separate principles:
The first enables stimulated emission depletion (STED) microscopy, which Hell developed in 2000 (see Fig. 2).
|FIGURE 2. STED uses two laser beams: one makes fluorescent molecules glow, while the other cancels out all fluorescence except for that in a nanometer-sized volume. The image is generated by scanning the specimen nanometer by nanometer.|
One laser beam excites fluorescent molecules, while another cancels out all 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 limit (see Fig. 3).
|FIGURE 3. Super-resolution optical microscopy enables imaging of such living viruses, proteins, small molecules, and other nanoscale structures. Electron microscopy images at even higher resolutions, but cannot image live tissue.|
The second, single-molecule imaging, was pioneered separately by Betzig and Moerner, using the concept of switching molecular fluorescence on and off. The same area is scanned multiple times, and each time the glow of a few molecules is captured. Stacking these images produces a super-resolution image (see Fig. 4).
|FIGURE 4. In single-molecule microscopy, fluorescence of individual molecules is switched on and off while the area is imaged multiple times. Stacking the images produces the super-resolved result.|
The Academy highlighted work that the researchers have pursued with the help of the imaging advance that they developed: the ability to follow individual proteins in fertilized eggs as they divide into embryos (Betzig); the understanding of how molecules create synapses between nerve cells in the brain (Hell); and the ability to track proteins involved in Huntington's disease as they aggregate (Moerner).
In fact, Moerner's most recent publication demonstrates use of super-resolution fluorescence to understand the mechanism underlying the progression of Huntington's disease and to designing therapeutics for the disease, as well as for aggregates implicated in Alzheimer's and Parkinson's diseases.1
And Hell, in delivering the plenary talk at LASER World of Photonics trade fair and World of Photonics Congress (May 13-16, 2013, Munich, Germany), said that nonlinear microscopy "is not the best line of thinking" to achieve nanoscale resolution. In fact, he presented the RESOLFT (REversible Saturable OpticaL Fluorescence Transitions) approach, which uses on-off switching of various states to enable imaging at molecular and even atomic scales.
A new imaging platform developed by Betzig and colleagues, announced on October 24, 2014, offers another leap forward (see "Laureate's reprise shows real-time subcellular activity in 3D"). Lattice light-sheet microscopy's ability to collect high-resolution images rapidly and minimize damage to cells means it can image the three-dimensional activity of molecules, cells, and embryos in fine detail over longer periods than was previously possible.
1. W. C. Duim, Y. Jiang, K. Shen, J. Frydman, and W. E. Moerner, ACS Chem. Biol., doi:abs/10.1021/cb500335w (2014).