Unit

UNIT 2.20 Comparative and Practical Aspects of Localization-Based Super-Resolution Imaging

  1. Gary S. Laevsky,
  2. Christopher B. O'Connell

Published Online: 1 JAN 2013

DOI: 10.1002/0471142956.cy0220s63

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Laevsky, G. S. and O'Connell, C. B. 2013. Comparative and Practical Aspects of Localization-Based Super-Resolution Imaging. Current Protocols in Cytometry. 63:2.20:2.20.1–2.20.11.

Author Information

  1. Nikon Instruments, Melville, New York

Publication History

  1. Published Online: 1 JAN 2013
  2. Published Print: JAN 2013

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. Curr. Protoc. Cytom. 63:2.20.1-2.20.11. © 2013 by John Wiley & Sons, Inc.

Keywords:

  • super-resolution microscopy;
  • STORM;
  • PALM;
  • tandem dyes