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Four-Channel Super-Resolution Imaging by 3-D Structured Illumination

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Part of the Methods in Molecular Biology book series (MIMB,volume 1663)


Multichannel imaging is used as a readout of relative localization of two or more components and is often the first step in investigating functional ensembles in cells. However, the localization volume of diffraction-limited light microscopy (approx. 200 nm by 500 nm) can accommodate hundred of proteins, calling for increased resolution for these types of analyses. Here, we present a protocol for 4-channel imaging using structured illumination microscopy (SIM), which increases resolution by a factor of two. We use adherent, fixed cells to identify the localization of adhesion proteins using immunofluorescence and fluorescent proteins. We discuss how labeling with the necessary brightness is achieved and how data has to be processed for colocalization analysis.

Key words

  • Cytoskeleton
  • Actin
  • Focal adhesion
  • Structured-illumination microscopy
  • Super-resolution
  • Colocalization
  • Chromatic aberration
  • Fluorescent protein

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  • DOI: 10.1007/978-1-4939-7265-4_7
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Three-dimensional structured illumination microscopy


Focal adhesion


Fluorescent protein


Green fluorescent protein


Photo activation localization microscopy


Stimulated emission depletion


Stochastic optical reconstruction microscopy


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I am grateful to Tim Mitchison and Matthias Krause for BS-C1 cell and the zyxin plasmid, respectively and Kees van der Oord and Pablo Hernandez Varas at Nikon BV for discussions on 3D-SIM technology. I want to thank all the users of the Nikon Imaging Center at the University of Heidelberg, who helped in improving our SIM routines, especially Diana Rüthnick for testing FPs and reading the manuscript. This work was supported by the CellNetworks Excellence Cluster and the SFB 873.

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Correspondence to Ulrike Engel .

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Engel, U. (2017). Four-Channel Super-Resolution Imaging by 3-D Structured Illumination. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY.

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7264-7

  • Online ISBN: 978-1-4939-7265-4

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