The position of a single isolated fluorescent emitter, although its image appears as a diffraction-limited spot, can be precisely determined by finding the centroid of its image. The precision of this localization process is given approximately by
, where s
is the standard deviation of the PSF and N
is the number of photons detected [25
]. This concept has been used to track small particles with nanometer-scale accuracy [26
]. Recently it has been shown that, even when the emitter is a single fluorescent dye, its position can be determined with an accuracy as high as ~1 nm [28
]. Nanoscale precision in single molecule localization does not, however, translate directly into image resolution. When multiple fluorophores are positioned close together such that they are separated by a distance less than the PSF width, their images overlap and this prevents accurate localization of each of the fluorophores. In order to distinguish the fluorescence signal from nearby fluorescent emitters, several approaches have been used, based on differences in emission wavelength [29
], the sequential photobleaching of each fluorophore [32
], or stochastic blinking [34
]. These methods have obtained high accuracy localization for several closely spaced emitters, but are difficult to extend to densities higher than 2 - 5 fluorophores per diffraction-limited area. A fluorescently labeled biological sample, by contrast, may be labeled with hundreds or thousands of fluorophores per diffraction-limited region.
Super-resolution imaging based on fluorophore localization takes advantage of the properties of photo-switchable fluorescent molecules, which can be switched between a non-fluorescent (dark) state and a fluorescent (bright) state by exposure to light of particular wavelengths. The imaging process consists of many cycles during which fluorophores are activated, imaged, and deactivated (). During each cycle, the density of activated molecules is kept low by using a weak activation light intensity, such that the images of individual fluorophores do not typically overlap and therefore can be localized with high precision. This process is repeated until a sufficient number of localizations have been recorded, and a high resolution image is constructed from the measured positions of the fluorophores. The resolution of the final image is not limited by diffraction, but by the precision of each localization. This concept, independently developed by three research groups, has been given the names Stochastic Optical Reconstruction Microscopy (STORM) [22
], Photoactivated Localization Microscopy (PALM) [23
], or Fluorescence Photo-activation Localization Microscopy (FPALM) [24
Figure 1 Super-resolution imaging by high-precision localization of photo-switchable fluorophores. (A - D) The imaging concept. (A) Schematic of a cell in which the structure of interest (grey filaments in this case) are labeled with photo-switchable fluorophores (more ...)
The initial results obtained with this method yielded images with extraordinary spatial resolution and employed a number of different fluorescent probes. Using a reversibly switchable synthetic dye pair, Cy3 - Cy5 [35
], Rust et al.
achieved an experimental localization precision of 8 nm (standard deviation) for each switchable probe, corresponding to an image resolution of ~ 18 nm in terms of the limit of resolvability for two adjacent probes (the FWHM of the localization probability distribution) [22
]. DNA and DNA-protein complexes were imaged in vitro
using these probes and fluorophores separated by ~ 40 nanometers were clearly resolved. Betzig et al.
demonstrated imaging of fixed cell samples expressing target proteins fused with the photo-switchable fluorescent proteins Kaede [36
] and EosFP [37
]. A variety of targets were labeled and the super-resolution images revealed cellular structures with sizes well below the diffraction limit [23
]. Hess et al.
imaged photo-activatable Green Fluorescent Protein (PA-GFP) [38
] on a surface, achieving a spatial resolution of several tens of nanometers [24
]. Collectively, these results demonstrated a resolution improvement of an order of magnitude over conventional imaging, while requiring no specialized setup other than a standard fluorescence microscope, low power continuous wave (CW) lasers, and a sensitive CCD camera.
A number of photo-switchable proteins and organic dyes have been applied to these techniques (summarized in ), and variations in the image acquisition process have been demonstrated. In addition to those mentioned above, other photo-switchable fluorescent proteins have been used for super-resolution imaging including KikGR [39
], Dronpa [40
] and its mutants Dronpa-2, Dronpa-3, and rsFastLime [41
], photo-switchable CFP2 [43
], and other photo-switchable organic dyes including caged rhodamine [23
], caged fluorescein [44
], and a newly developed photochromic rhodamine compound [45
]. Increased imaging speeds were demonstrated by using asynchronous activation and matching the camera frame rate with the switching kinetics of the fluorophore (this approach has been termed PALM with independently running acquisition, or PALMIRA) [46
], and also by stroboscopic illumination [47
]. In certain cases, the dynamic binding or translational motions of fluorescent molecules have also been used to produce high-resolution images of cellular structures without requiring the use of photo-switchable probes [48
Spectral properties of photo-switchable organic dyes and fluorescent proteins, including the fluorescence excitation and emission wavelengths of the pre- and post-activation states, and the wavelength used for activation of the fluorophore