CLEM methods for studying primary cilia

FP Macaluso, GS Perumal, J Kolstrup… - Cilia: Methods and …, 2016 - Springer
FP Macaluso, GS Perumal, J Kolstrup, P Satir
Cilia: Methods and Protocols, 2016Springer
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 …
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.
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