W.E. Moerner is really the father of single-molecule spectroscopy. It’s not surprising that a prize for single molecules went to him. His early work laid the foundation for single-molecule photophysics that made PALM-type super-resolution possible.
Also, most people don’t realize that almost all the early cryogenic single-molecule imaging resolved molecules that were closer than the diffraction limit. At temperatures near absolute zero, the spectral linewidths get super narrow. This means that any one laser wavelength excites only a fraction of the dyes in a crystal; dyes in different parts of the solid experience slightly different nano environments, and their spectral properties are different. This is called inhomogeneous broadening. By tuning the wavelength of a dye laser, Moerner and others were able to excite different dyes at different times, all within one diffraction-limited laser spot. That was routinely done, and many of the early single-molecule images were actually plots of intensity, with distance on one axis (moving the laser spot) and wavelength on the other (changing the laser color).
Fluorescence excitation spectra for pentacene in p-terphenyl at 1.5 K measured with a tunable dye laser of line width ∼3 MHz. The laser detuning frequency is referenced to the line center at 592.321 nm. (a) Broad scan of the inhomogeneously broadened line; all the sharp features are repeatable structure. (b) Expansion of 2 GHz spectral range showing several single molecules. (c) Low-power scan of a single molecule at 592.407 nm showing the lifetime-limited width of 7.8 MHz and a Lorentzian fit. [From: Moerner, W. E. J. Phys. Chem. B 2002, 106, 910– 927.]
[From: Ambrose, W. P. and Moerner, W. E. Nature 1991, 349, 225– 227]
Eric Betzig contributed to single-molecule spectroscopy early on, imaging single molecules at room temperature with near-field super-resolution microscopy (Betzig 1993) and proposing an early variant of PALM super-resolution imaging back in the 1990s (Betzig 1995). (His proposal was realized at cryogenic temperatures by van Oijen in 1998.) After that, he left science and worked at his father’s tool factory.
When Betzig heard about the development of GFPs that could be easily photoswitched on and off, he realized that these could be applied to his super-resolution concept he proposed a decade earlier (Betzig 1995). So he built a super-resolution microscope in his friend’s living room and published the first PALM paper in 2006. It should be noted that Xiaowei Zhuang and Sam Hess each independently published similar super-resolution methods in 2006 (Betzig 2006; Hess 2006; Rust 2006).
[From: Betzig 1995]
Stefan Hell has a very interesting story. After proposing STED microscopy in the 1990s (Hell 1994), he worked for years with little funding and almost no support or recognition. A decade later he got his STED microscope producing super-resolution images and now he’s a huge force in the field.
It goes without saying that there were many others who contributed to the field of super-resolution and single-molecule imaging (Yanagida, Webb, Zhuang, Hess, Gustafsson, Lippincott-Schwartz, Zare, Vale, Orrit, Rigler, Xie, Cremer, Baer…) and many people will probably be disappointed. But is hard to argue that these three were not deserving and I congratulate them!
Also, Ash at Curious Wavefunction has a great summary. See my post from 2006 on super-resolution methods. And my single-molecule timeline (please excuse any omissions: it is impossible to include everyone!). And remember when the Simpsons predicted W.E. to win?
And full disclosure: W.E. was my PhD advisor. :)
Ambrose, W. P. and Moerner, W. E. Nature 1991, 349, 225– 227.
Betzig E and Chichester RJ (1993) Single molecules observed by near-field scanning optical microscopy. Science 262:1422-1425.
Hell SW and Wichman J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion-microscopy. Opt. Lett. 19:780-782.
Hess, S. T., Girirajan, T. P. K. and Mason, M. D. Biophys. J. 2006, 91, 4258–4272
Rust, M. J., Bates, M. and Zhuang, X. Nat. Methods 2006, 3, 793– 795
van Oijen AM, Kohler J, Schmidt J, Muller M and Brakenhoff GJ (1998) 3-Dimensional super-resolution by spectrally selective imaging. Chem. Phys. Lett. 292:183–187.