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Cilia pp 193-202 | Cite as

CLEM Methods for Studying Primary Cilia

  • Frank P. Macaluso
  • Geoffrey S. Perumal
  • Johan Kolstrup
  • Peter SatirEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1454)

Abstract

CLEM (correlated light and electron microscope) imaging is a highly useful technique for examining primary cilia. With CLEM, it is possible to determine the distribution of tagged proteins along the ciliary membrane and axoneme with high precision. Scanning electron microscopy (SEM) permits measurement of ciliary length and orientation in relation to nearby cellular structures in a 3D image; in optimal cases, this can be combined with superresolution microscopy of selected ciliary components as they enter or leave the cilium. This chapter discusses CLEM methods. In the method described in detail, samples are completely processed for sequential fluorescence and SEM observation. This method is ideal for robust antibody localization and minimizes image manipulation in correlating the fluorescent and SEM images. Alternative methods prepare samples for fluorescence imaging followed by processing for SEM then observation in the SEM. This method is ideal for optimal fluorescence imaging, particularly live cell imaging.

Key words

CLEM SEM Primary cilia Axoneme MEF Superresolution 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Frank P. Macaluso
    • 1
  • Geoffrey S. Perumal
    • 1
  • Johan Kolstrup
    • 1
  • Peter Satir
    • 1
    Email author
  1. 1.Analytical Imaging Facility, Department of Anatomy and Structural BiologyAlbert Einstein College of MedicineBronxUSA

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