Comparative and practical aspects of localization-based super-resolution imaging

Super-resolution microscopy overcomes diffraction to generate images with superior resolution compared to conventional light microscopy. Localization-based super-resolution methods result in up to ten-fold improvement in resolution by determining the positions of fluorescent molecules with sub-pixel accuracy. This process critically depends on controlled emission at the level of individual fluorophores so that fluorescence is non-overlapping, allowing for accurate centroid determination of diffraction-limited spots by Gaussian fitting of the pixel intensities. The intrinsic photoswitching behavior of many fluorophores provides a convenient way to achieve emitter isolation. Here, we describe methods for label preparation and staining of cellular structures to obtain high-quality images using localization super resolution. We also compare labeling strategies and dye characteristics relevant to all localization-based techniques, such as STORM and PALM.