title: Comparative and practical aspects of localization-based super-resolution imaging.
abstract: 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.
authors.affiliation: Nikon Instruments, Melville, New York.
authors.forename: Gary S
authors.initials: GS
authors.lastname: Laevsky
authors.affiliation: Nikon Instruments, Melville, New York.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: O'Connell
mesh_terms: D000818:Animals; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure; D008928:Mitochondria / Q000737:chemistry / Q000648:ultrastructure; D011364:Professional Practice; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes