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STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells

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Super-Resolution Microscopy

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

Abstract

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

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Acknowledgments

This study was supported by the Wellcome Trust Foundation and by the National Institutes of Health (GM83257 to A.S.). R.S.E. was supported by an Advanced Postdoc. Mobility fellowship from the Swiss National Science Foundation.

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Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06520, USA

    Roman S. Erdmann & Derek Toomre

  2. Department of Chemistry, Yale University, New Haven, CT, 06520, USA

    Roman S. Erdmann & Alanna Schepartz

  3. Department of Molecular, Cellular, and Developmental Biology, Yale University, P.O. Box 208107, New Haven, CT, 06520, USA

    Alanna Schepartz

Authors
  1. Roman S. Erdmann
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  2. Derek Toomre
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  3. Alanna Schepartz
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Corresponding author

Correspondence to Alanna Schepartz .

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Editors and Affiliations

  1. High-Content Analysis of the Cell (HiCell) and Advanced Biological Screening Facility, BioQuant, Heidelberg University, Heidelberg, Germany

    Holger Erfle

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Erdmann, R.S., Toomre, D., Schepartz, A. (2017). STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7265-4_6

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  • DOI: https://doi.org/10.1007/978-1-4939-7265-4_6

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7264-7

  • Online ISBN: 978-1-4939-7265-4

  • eBook Packages: Springer Protocols

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