Correlative light and electron microscopy : strategies and applications

Abstract

Correlative light and electron microscopy (CLEM) refers to the observation of the same structures or ultrastructures with both light microscopy (LM) and electron microscopy (EM). LM provides an overview of the studied material, and enables the quick localization of structures that are fluorescently labeled, while EM allows high resolution imaging of structures. The thesis describes several technical developments that allow CLEM. In the first chapters, methods are described for room temperature CLEM. This includes tissue labeling with fluorescent dyes in ultrathin resin sections, and fluorescence imaging before further preparation for EM and electron tomography. The methods are applied to tissue biopsies, and to the study of regulated exocytosis of Weibel-Palade bodies. In the following chapters, CLEM developments are extended with CLEM of vitreous samples, i.e. cryo-CLEM. Vitrification allows easy maintenance of fluorescent probes, and it allows correlation with cryo-EM, a technique that preserves biological structures to atomic resolution. The thesis describes the design of a microscope stage for cryo-fluorescence microscopy. Following cryo-FM, samples are studied directly by cryo-EM and cryo-electron tomography. The technique is used to study mitochondrial ultrastructure, and the role of actin in the exocytosis of pancreatic zymogen granules.