title: CLEM Methods for Studying Primary Cilia.
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.
authors: Analytical Imaging Facility, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Forchheimer Building, Room 602, 1300 Morris Park Ave., Bronx, NY, 10461, USA.
authors: Frank P
authors: FP
authors: Macaluso
authors: Analytical Imaging Facility, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Forchheimer Building, Room 602, 1300 Morris Park Ave., Bronx, NY, 10461, USA.
authors: Geoffrey S
authors: GS
authors: Perumal
authors: Analytical Imaging Facility, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Forchheimer Building, Room 602, 1300 Morris Park Ave., Bronx, NY, 10461, USA.
authors: Johan
authors: J
authors: Kolstrup
authors: Analytical Imaging Facility, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Forchheimer Building, Room 602, 1300 Morris Park Ave., Bronx, NY, 10461, USA. peter.satir@einstein.yu.edu.
authors: Peter
authors: P
authors: Satir
mesh_terms: D000818:Animals; D002923:Cilia / Q000378:metabolism* / Q000648:ultrastructure*; D005347:Fibroblasts; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence
keywords: Axoneme; CLEM; MEF; Primary cilia; SEM; Superresolution