title: Combining wide-field super-resolution microscopy and electron tomography: rendering nanoscopic correlative arrays on subcellular architecture.
abstract: In this chapter, the authors outline in full detail, an uncomplicated approach that enables the combination of wide-field fluorescence super-resolution microscopy with electron tomography, thereby providing an approach that affords the best possible confidence in the structures investigated. The methodical steps to obtain these high-throughput correlative nanoscopic arrays will be visually explored and outlined in detail. The authors will demonstrate the feasibility of the method on cultured Caco-2 colorectal cancer cells that are labeled for filamentous actin. The presented images, morphometric data, and generated models illustrate the strengths of our correlative approach for future advanced structural-biology-oriented questions. Correlative nanoscopy applications can be readily found in which there is a need to reveal biomolecular information at unprecedented resolution on subcellular behavior in various biological and pathobiological processes.
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology)-The Bosch Institute, The University of Sydney, Sydney, NSW, Australia; Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia; Australian Centre for Microscopy & Microanalysis, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Filip
authors.initials: F
authors.lastname: Braet
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology)-The Bosch Institute, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Delfine
authors.initials: D
authors.lastname: Cheng
authors.affiliation: Australian Centre for Microscopy & Microanalysis, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Minh
authors.initials: M
authors.lastname: Huynh
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology)-The Bosch Institute, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Henriquez
authors.affiliation: School of Medical Sciences (Discipline of Anatomy and Histology)-The Bosch Institute, The University of Sydney, Sydney, NSW, Australia.
authors.forename: Gerry
authors.initials: G
authors.lastname: Shami
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany; Department of Translational Pulmonology, Translational Lung Research Center (TLRC), University of Heidelberg, Heidelberg, Germany.
authors.forename: Marko
authors.initials: M
authors.lastname: Lampe
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure; D018938:Caco-2 Cells; D055032:Electron Microscope Tomography / Q000379:methods; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods
keywords: Actin; Colon cancer; Correlating single molecules; Correlative morphomics; Correlative nanoscopy; Correlative pointillism; Cytoskeleton; Fine structure immunocytochemistry; Fluorescent probes; Ground state depletion; Nanoscopy; Simulation; Three-dimensional modeling