title: High data output method for 3-D correlative light-electron microscopy using ultrathin cryosections.
abstract: Investigation of intracellular dynamics requires a detailed description of the molecular topography and ultrastructural morphology of the cell, for example, the position of a protein in relation to a given compartment of the cell and the morphology of the compartment. Standard fluorescence light microscopy (FLM) localizes proteins in living or fixed cells with a resolution of few hundreds of nanometers, but the unlabeled cellular context is partially missing. Electron microscopy (EM) techniques, such as immuno-EM, reveal protein topology with a few tens of nanometer resolution and retain the cellular context. However, EM analysis shows shortcomings compared to FLM, such as, lower statistical output, applicability only to fixed cells, and higher technical difficulties. To bridge the gap between fluorescent cell imaging and EM, several laboratories have developed methods for correlative light-electron microscopy (CLEM). In CLEM, a limited number of fluorescently labeled cell compartments are first imaged by light microscopy and then visualized and analyzed by EM. Recently, two different CLEM approaches using the EM cryo-immunogold method have been developed to extend the analysis to a high number of regions of interest and to correlate the topology of specific antigens. In this chapter, we describe one of these methods, the High Data Output CLEM (HDO-CLEM) approach. The major benefits of HDO-CLEM are the possibility to (1) correlate several hundreds of events at the same time, (2) perform three-dimensional (3D) correlation, (3) immunolabel both endogenous and recombinantly tagged proteins at the same time, and (4) combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometric analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D FLM reconstruction and defined preliminary conditions for a hybrid light/electron microscopy morphometry approach.
authors.affiliation: MicroscoBio Research Center, Department of Experimental Medicine (DIMES), University of Genoa, Genoa, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Cortese
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Vicidomini
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Gagliani
authors.forename: Patrizia
authors.initials: P
authors.lastname: Boccacci
authors.forename: Alberto
authors.initials: A
authors.lastname: Diaspro
authors.forename: Carlo
authors.initials: C
authors.lastname: Tacchetti
mesh_terms: D016616:Cryoultramicrotomy / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008854:Microscopy, Electron / Q000379:methods*; D016253:Microscopy, Immunoelectron