Diffraction‐Unlimited Fluorescence Microscopy of Living Biological Samples Using pcSOFI

Sam Duwé1, Benjamien Moeyaert1, Peter Dedecker1

1 Department of Chemistry, University of Leuven, Heverlee, Belgium
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140025
Online Posting Date:  March, 2015
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The complex microscopic nature of many live biological processes is often obscured by the diffraction limit of light, requiring diffraction‐unlimited fluorescence microscopy to resolve them. Because of the vast range of different processes that can be studied, sub‐diffraction imaging should work efficiently under many different conditions. Photochromic stochastic optical fluctuation imaging (pcSOFI) is a recent addition to the field of diffraction‐unlimited fluorescence microscopy. This robust and versatile method employs a statistical analysis of random fluctuations in the emission of single labels, in this case reversibly switchable fluorescent proteins (RSFPs), to retrieve super‐resolution information. Added to the resolution enhancement, pcSOFI also offers contrast enhancement and background reduction in a practical and convenient way. Here, we describe the necessary steps to obtain diffraction‐unlimited images, including multicolor and three‐dimensional imaging, and highlight the advantages of pcSOFI together with the circumstances under which pcSOFI can be favorably applied. © 2015 by John Wiley & Sons, Inc.

Keywords: super‐resolution microscopy; reversible photoswitchable fluorescent proteins; pcSOFI; 3D imaging; fluorescence imaging

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1:

  • Plasmid DNA encoding the target protein or localization signal fused to a RSFP
  • HeLa cells (ATCC # CCL‐2)
  • Growth medium: phenol red‐free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) glutaMAX and 0.1% (v/v) gentamicin (store at 4°C)
  • Ultrapure H 2O, sterile
  • 2 M calcium chloride (CaCl 2), sterile (store indefinitely at 4°C)
  • 2× HEPES buffered saline (2× HBS), pH 7.05 to 7.06 (see recipe)
  • Hanks’ balanced salt solution (HBSS), pH 7.4 (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • 4% (w/v) paraformaldehyde in PBS (optional; store aliquots at −20°C for ≤14 days)
  • 35‐mm glass‐bottom culture dishes (MatTek)
  • Wide‐field fluorescence microscope
  • pcSOFI analysis software
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Literature Cited

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Key References
   Dertinger et al., 2013. See above.
  A review detailing the mathematics and assumptions that form the base of SOFI and pcSOFI, together with practical tips on when to consider (pc)SOFI or other techniques and how SOFI can be used on multiple platforms.
   Cremer and Masters , 2013. See above.
  An extensive review detailing the historical development and achievements of super‐resolution microscopy methods including applications in the biosciences.
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