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Autophagy pp 199-209 | Cite as

Correlative Light and Electron Microscopy of Autophagosomes

  • Sigurdur Gudmundsson
  • Jenny Kahlhofer
  • Nastassia Baylac
  • Katri Kallio
  • Eeva-Liisa Eskelinen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

Key words

Live-cell imaging Phagophore Autophagosome Serial sectioning Electron tomography 

Notes

Acknowledgments

Authors’ laboratory is supported by the Academy of Finland and Magnus Ehrnrooth Foundation. Live-cell imaging and confocal microscopy were carried out in the Light Microscopy Unit at the Institute of Biotechnology, University of Helsinki. We thank the Electron Microscopy Unit at the Institute of Biotechnology, University of Helsinki, for technical help in thin sectioning and for the possibility to use an electron microscope. HeLa cells stably expressing mRFP-GFP-LC3 were a kind gift from Tamotsu Yoshimori, University of Osaka, Japan, and HEK293 cells stably expressing ATG13 were a kind gift from Nicholas Ktistakis, Babraham Institute, Cambridge, UK.

References

  1. 1.
    Biazik JM, Vihinen H, Anwar T, Jokitalo E, Eskelinen EL (2015) The versatile electron microscope: an ultrastructural overview of autophagy. Methods 75:44–53.  https://doi.org/10.1016/j.ymeth.2014.11.013CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol 452:143–164CrossRefGoogle Scholar
  3. 3.
    Souslova EA, Mironova KE, Deyev SM (2017) Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing. J Biophotonics 10(3):338–352.  https://doi.org/10.1002/jbio.201600120CrossRefPubMedGoogle Scholar
  4. 4.
    Takizawa T, Powell RD, Hainfeld JF, Robinson JM (2015) FluoroNanogold: an important probe for correlative microscopy. J Chem Biol 8(4):129–142.  https://doi.org/10.1007/s12154-015-0145-1CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Killingsworth MC, Bobryshev YV (2016) Correlative light- and electron microscopy using quantum dot nanoparticles. J Vis Exp.  https://doi.org/10.3791/54307
  6. 6.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682.  https://doi.org/10.1038/nmeth.2019CrossRefGoogle Scholar
  7. 7.
    Cardona A, Saalfeld S, Schindelin J, Arganda-Carreras I, Preibisch S, Longair M et al (2012) TrakEM2 software for neural circuit reconstruction. PLoS One 7(6).  https://doi.org/10.1371/journal.pone.0038011CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sigurdur Gudmundsson
    • 1
  • Jenny Kahlhofer
    • 2
  • Nastassia Baylac
    • 1
  • Katri Kallio
    • 1
  • Eeva-Liisa Eskelinen
    • 1
  1. 1.Molecular and Integrative Biosciences Research ProgramUniversity of HelsinkiHelsinkiFinland
  2. 2.Division of Cell Biology, BiocenterMedical University of InnsbruckInnsbruckAustria

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