title: High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers.
abstract: Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell.
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Pascale
authors.initials: P
authors.lastname: Schellenberger
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK; Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kaufmann
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: C Alistair
authors.initials: CA
authors.lastname: Siebert
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hagen
authors.affiliation: Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, University of Bordeaux SEGALEN, 146 rue Leo Seignat, 33076 Bordeaux, France.
authors.forename: Harald
authors.initials: H
authors.lastname: Wodrich
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. Electronic address: kay@strubi.ox.ac.uk.
authors.forename: Kay
authors.initials: K
authors.lastname: Grünewald
mesh_terms: D002460:Cell Line; D020285:Cryoelectron Microscopy / Q000379:methods*; D004583:Electrons; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008859:Microscopy, Polarization / Q000379:methods
chemical_list: D005456:Fluorescent Dyes
keywords: Cellular tomography; Correlative microscopy; Electron cryo microscopy; Fluorescent cryo microscopy; Fluorescent microspheres