title: Imaging fluorescently labeled complexes by means of multidimensional correlative light and transmission electron microscopy: practical considerations.
abstract: These days the common ground between structural biology and molecular biology continues to grow thanks to the biomolecular insights offered by correlative microscopy, even though the vision of combining insights from different imaging tools has been around for nearly four decades. The use of correlative imaging methods to dissect the cell's internal structure is progressing faster than ever as shown by the boom in the number of methodological approaches available for correlative microscopy studies, each designed to address a specific scientific question. In this chapter, we will present a relatively straightforward approach to combining information from fluorescence microscopy and electron microscopy at the supramolecular level. The method combines live-cell and/or confocal laser microscopy with classical sample preparation for transmission electron microscopy (TEM), thereby allowing the integration of dynamic details of subcellular processes with insights about the organelles and molecular machinery involved. We illustrate the applicability of this multidimensional correlative microscopy approach on cultured Caco-2 colorectal cancer cells exposed to fluorescently labeled cisplatin, and discuss how these methods can deepen our understanding of key cellular processes, such as drug uptake and cell fate.
authors.affiliation: Australian Centre for Microscopy & Microanalysis, The University of Sydney, NSW 2006, Australia.
authors.forename: K
authors.initials: K
authors.lastname: Kobayashi
authors.forename: D
authors.initials: D
authors.lastname: Cheng
authors.forename: M
authors.initials: M
authors.lastname: Huynh
authors.forename: K R
authors.initials: KR
authors.lastname: Ratinac
authors.forename: P
authors.initials: P
authors.lastname: Thordarson
authors.forename: F
authors.initials: F
authors.lastname: Braet
mesh_terms: D018938:Caco-2 Cells; D018929:Cell Culture Techniques; D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism; D055032:Electron Microscope Tomography / Q000379:methods*; D005404:Fixatives / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008857:Microscopy, Interference / Q000379:methods; D015388:Organelles / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D059010:Single-Cell Analysis / Q000379:methods; D013194:Staining and Labeling; D059008:Time-Lapse Imaging; D016707:Tissue Fixation
chemical_list: D005404:Fixatives; D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate; D002945:Cisplatin; D005976:Glutaral