It was not until recently that several novel fluorescence microscopy approaches completely shattered the diffraction limit of image resolution in the far field. In general, all of these approaches generate diffraction-unlimited images by using the physical properties of fluorescent probes to distinguish emissions from two nearby molecules within a diffraction-limited region. These super-resolution approaches can be divided into two primary classes. The first category is ensemble imaging approaches that use patterned illumination to spatially modulate the fluorescence behavior of molecules within a diffraction-limited region, such that not all of them emit simultaneously, thereby achieving subdiffraction limit resolution. This category includes stimulated emission depletion (STED) microscopy (Hell and Wichmann, 1994,Klar and Hell, 1999) and the related RESOLFT technology (Hofmann et al., 2005), as well as saturated structured illumination microscopy (SSIM) (Gustafsson, 2005,Heintzmann et al., 2002). The second category takes advantages of single-molecule imaging, using photoswitching or other mechanisms to stochastically activate individual molecules within the diffraction-limited region at different times. Images with subdiffraction limit resolution are then reconstructed from the measured positions of individual fluorophores. This second class has been termed stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006), photoactivated localization microscopy (PALM) (Betzig et al., 2006), and fluorescence photoactivation localization microscopy (FPALM) (Hess et al., 2006).
Although these two categories of methods use different approaches to accomplish subdiffraction resolution, these techniques also share important commonalities. In both cases, a physical or chemical property of the fluorophore is used to maintain neighboring molecules in different states (i.e., on and off), enabling them to be resolved from each other (Hell, 2007).