Superresolution imaging using single-molecule localization encompasses a number of techniques including PALM, STORM, and FPALM. The field is rapidly emerging in popularity due to the dramatic improvement in spatial resolution by over an order of magnitude (approximately 10 to 20 nanometers) to enable biological processes to be described at the molecular scale. A vast number of probes have been identified for use with these techniques, which have applications in three-dimensional and multicolor imaging, tracking of dynamic interactions, and other imaging motifs in living or fixed cells.
Betzig, E., Patterson, G. H., Sougrat, R., Lindwasser, O. W., Olenych, S., Bonifacino, J. S., Davidson, M. W., Lippincott-Schwartz, J., and Hess, H. F.
Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642-1645 (2006). The original paper on photoactivated localization microscopy (PALM), a technique that has been licensed by ZEISS. Included is a description of the theoretical basis for the methodology, as well as several examples of applications using optical highlighter fluorescent proteins in fixed cells.
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3: 793-795 (2006). Dr. Zhuang and co-workers introduce a single-molecule localization technique termed STORM based on photoswitching of carbocyanine dyes. Using short DNA molecules labeled with Cy3 and Cy5, the authors were able to achieve an imaging resolution of 20 nanometers.
Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophysical Journal 91: 4258-4272 (2006). In a technique similar to PALM and STORM, Dr. Hess and collaborators perform single-molecule imaging of photoactivatable GFP (PA-GFP) with precision localization in a method known as FPALM. The researchers were able to achieve nanometer resolution using the fluorescent protein deposited on glass slides or a sapphire wafer.
Superresolution imaging using single-molecule localization. Annual Review of Physical Chemistry 61: 345-367 (2010). A review of concepts and techniques necessary for single-molecule localization superresolution microscopy. The authors discuss the conceptual basis, probes, spatial density requirements, localization accuracy, live-cell imaging, three-dimensional imaging, and multicolor assays.
Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317: 1749-1753 (2007). In a unique demonstration of multicolor imaging, the authors introduce a family of photoswitchable fluorescent probes for use with STORM. Each probe consists of a reporter that can be cycled between bright and dark states in the presence of an activator molecule.
Shroff, H., Galbraith, C. G., Galbraith, J. A., White, H., Gillette, J., Olenych, S., Davidson, M. W. and Betzig, E.
Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proceedings of the National Academy of Sciences (USA) 104: 20308-20313 (2007). The first demonstration of two-color imaging with PALM using tandem dimer Eos and Dronpa fluorescent proteins. The authors examined apparent co-localization of the fluorescent probes when fused to different focal adhesion proteins.
Beyond Rayleigh's criterion: A resolution measure with application to single-molecule microscopy. Proceedings of the National Academy of Sciences (USA) 103: 4457-4462 (2006). The authors examine the resolution problem in widefield microscopy by adopting a stochastic framework and present a resolution measure that overcomes the limitations of Rayleigh's criterion. The measure predicts that resolution can be improved by increasing the number of detected photons.
Lemmer, P., Gunkel, M., Baddeley, D., Kaufmann, R., Urich, A., Weiland, Y., Reymann, J., Muller, P., Hausmann, M. and Cremer, C.
SPDM: Light microscopy with single-molecule resolution at the nanoscale. Applied Physics B 93: 1-12 (2008). Using a technique termed spectral precision distance microscopy or spectral position determination microscopy (SPDM), the authors exploit the novel spectral signature offered by reversible photoswitching of fluorescent proteins.
Ultrahigh-resolution colocalization of spectrally separable point-like fluorescent probes. Methods 25: 87-102 (2001). Published before the first single-molecule localization (PALM and STORM) superresolution reports, the investigators demonstrate that 10-nanometer resolution can be achieved using spectrally distinct fluorescent probes.
Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proceedings of the National Academy of Sciences (USA) 103: 18911-18916 (2006). A clever demonstration of superresolution microscopy using diffusible fluorophores for imaging that accumulates points by collisional flux. The technique is based on targeting the surface of objects by fluorescent probes diffusing in the solution.