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

Curr Protoc Cytom. 2013 Jan:Chapter 2:2.20.1-2.20.11. doi: 10.1002/0471142956.cy0220s63.

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.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / pharmacology
  • Humans
  • Image Cytometry / instrumentation
  • Image Cytometry / methods
  • Image Enhancement* / instrumentation
  • Image Enhancement* / methods
  • Image Processing, Computer-Assisted / instrumentation
  • Image Processing, Computer-Assisted / methods
  • Microscopy / instrumentation*
  • Microscopy / methods*
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Microtubules / chemistry
  • Microtubules / ultrastructure
  • Mitochondria / chemistry
  • Mitochondria / ultrastructure
  • Professional Practice
  • Staining and Labeling / methods

Substances

  • Fluorescent Dyes