Super‐Resolution Microscopy: A Comparative Treatment

James M. Kasuboski1, Yury J. Sigal1, Matthew S. Joens1, Bjorn F. Lillemeier2, James A.J. Fitzpatrick1

1 Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, California, 2 Nomis Center for Immunobiology and Microbial Pathogenesis, Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.17
DOI:  10.1002/0471142956.cy0217s62
Online Posting Date:  October, 2012
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One of the fundamental limitations of optical microscopy is that of diffraction, or in essence, how small a beam of light can be focused by using an optical lens system. This constraint, or barrier if you will, was theoretically described by Ernst Abbe in 1873 and is roughly equal to half the wavelength of light used to probe the system. Many structures, particularly those within cells, are much smaller than this limit and thus are difficult to visualize. Over the last two decades, a new field of super‐resolution imaging has been created and been developed into a broad range of techniques that allow routine imaging beyond the far‐field diffraction limit of light. In this unit we outline the basic principles of the various super‐resolution imaging modalities, paying particular attention to the technical considerations for biological imaging. Furthermore, we discuss their various applications in the imaging of both fixed and live biological samples. Curr. Protoc. Cytom. 62:2.17.1‐2.17.24. © 2012 by John Wiley & Sons, Inc.

Keywords: super‐resolution microscopy; PALM (photoactivation localization microscopy); STORM (stochastic optical reconstruction microscopy); SIM (structured illumination microscopy); STED (stimulated emission depletion microscopy); GSD (ground state depletion); 4Pi microscopy

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Table of Contents

  • Introduction
  • Super‐Resolution Imaging Methodologies
  • Point‐Spread Function Engineering
  • Concluding Remarks
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
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