ABSTRACT
Fluorescence microscopy, a special form of light microscopy, is subject to the limitations of diffraction, resulting in a resolution limit of about 200 nm in the visible light range. Single-molecule localization microscopy (SMLM) is an imaging technique that circumvents this limit and can produce super-resolved images with near-molecular spatial resolution. This significant gain in resolution is achieved by spatially and temporally separating the emission events of individual fluorophores [1, 2]. One way to achieve this separation is to use photoswitchable fluorophores and to adjust the fraction of active fluorophores to a low density, resulting in separate blinking of individual fluorophores. Examples of such fluorophores are photoactivatable or photoconvertible fluorescent proteins, as used in (fluorescence) photoactivated localization microscopy ((F)PALM) [3, 4], or photoswitchable organic fluorophores, as used in (direct) stochastic optical reconstruction microscopy ((d)STORM) [5, 6]. Fluorescence emission from individual fluorophores is first detected as a point spread function (PSF), and subsequently its centroid is determined, for example, by approximation with a Gaussian function. The precision with which the center of the PSF can be determined scales inversely with the square root of the number of detected photons originating from the fluorophore [7]. Finally, a super-resolved image is reconstructed from the collected coordinates of a sufficiently large number of individual fluorophores. The resulting list of coordinates can then be used for post-processing of the imaging data (Figure 8.1).
