Moving toward the future of single-molecule-based super-resolution imaging.

S ingle-molecule fluorescence (SMF) detection was first demonstrated in 1989 by Moerner and Kador. The capability to measure the physical properties of individual molecules one at a time circumvented the need to average the properties of an ensemble, and effects such as spectral diffusion could therefore be observed. One important consequence of SMF detection was soon recognized to be the capability to measure the coordinates of spatially isolated fluorescent emitters with a certainty far better than the standard diffraction limit of light, and such localization opened the door to SMF tracking and imaging. SMF imaging provides the means to attain nanometer-scale information with a standard light microscope. Over the years, many advances have improved the resolution and applications of SMF imaging. These include improvements in sample preparation, image processing, and hardware, all of which have coalesced to aid experimenters to detect individual molecules at room temperature. A particularly important development in SMF imaging has been its application to biological imaging. This extension from immobile solid-state samples to ‘‘messier’’ biological samples relies on the fact that SMF imaging can be performed in ambient conditions and in a non-invasive, non-destructive fashion. Indeed, in SMF imaging, even live-cell samples can be investigated with only minor perturbations to the system; cells do not need to be fixed, dried, or frozen to track individual molecules within their bodies. The main physical principle of SMF imaging, that of finding the position of a single emissive molecule based on point-spread function (PSF) fitting, has remained unchanged over the years. In brief, given precise knowledge of the PSF of the microscope, the position of an isolated nanoscopic fluorophore can be localized with high accuracy. This localization accuracy can be as good as a few nm when many photons are detected from a fluorophore in a low-background sample. However, PSF fitting is limited to investigations of only one fluorophore per diffraction-limited area; more spatially dense samples cannot be processed this way. This problem can be circumvented given a distinct means to distinguish between spatially overlapping emitters. For instance, it was demonstrated early on that multiple fluorophores in a diffraction-limited spot could be resolved by spectrally selective imaging. Each single pentacene molecule in a p-terphenyl crystal has a subtly different absorbance frequency and can be differentially excited on resonance by tuning a narrow-band laser onto resonance with each molecule. Still, this could only be accomplished at low temperatures due to temperature-dependent line broadening. A second important development in the field of single-molecule imaging was therefore the realization, in 2006, that fluorophore photophysics could be used to separate fluorophores that overlap in space. By imaging a collection of single molecules over the course of an experiment and controlling the concentration via photoswitching, photoactivation, cellular dynamics, chemical control, etc., the positions of each molecule can be recorded sequentially and a superresolution image can be reconstructed from a movie (PALM, FPALM, STORM, etc.). Because, as was true for spectrally selective imaging, all these methods require some method of controlling the emission of the molecules to maintain the emitter concentration at a low level in any given imaging frame, these methods can been generally termed single-molecule active control microscopy (SMACM). With the extension of SMACM imaging to room-temperature–photophysics-based methods in 2006, SMACM imaging could be implemented on biological samples. Additionally, because these single-molecule based imaging and superresolution techniques can be implemented in relatively straightforward widefield imaging geometries with lowpower continuous-wave (CW) lasers and electron-multiplying charge coupled device (EMCCD) detectors on a standard inverted microscope, SMACM was readily extended to multicolor and three-dimensional imaging. Additionally, since 2006, further SMACM techniques have emerged that make single-molecule-based superresolution imaging possible in a wider range of settings, with a more diverse ensemble of fluorophores. These additional techniques include photoreactivation of conventional fluorescent proteins, fluoresPublished online 28 January 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21592