UNIT 2.20 Comparative and Practical Aspects of Localization-Based Super-Resolution Imaging
Published Online: 1 JAN 2013
Copyright © 2013 by John Wiley & Sons, Inc.
Lab Protocol Title
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. 63:2.20.1–2.20.11.
- Published Online: 1 JAN 2013
- Published Print: JAN 2013
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.
- super-resolution microscopy;
- tandem dyes