title: Critical step-by-step approaches toward correlative fluorescence/soft X-ray cryo-microscopy of adherent mammalian cells.
abstract: Soft X-ray cryo-microscopy/tomography with its extraordinary capability to map vitreous cells with high absorption contrast in their full three-dimensional extent, and at a resolution exceeding super-resolution fluorescence microscopy, is a valuable tool for integrative structural cell biology. Focusing on cell biological applications, its ongoing methodological development gained momentum by combining it with fluorescence cryo-microscopy, thus correlating highly resolved structural and specific information in situ. In this chapter, we provide a basic description of the techniques, as well as an overview of equipment and methods available to carry out correlative soft X-ray cryo-tomography experiments on frozen-hydrated cells grown on a planar support. Our aim here is to suggest ways that biologically representative data can be recorded to the highest possible resolution, while also keeping in mind the limitations of the technique during data acquisition and analysis. We have written from our perspective as electron cryo-microscopists/structural cell biologists who have experience using correlative fluorescence/cryoXM/T at synchrotron beamlines presently available for external users in Europe (HZB TXM at U41-FSGM, BESSY II, Berlin/Germany; Carl Zeiss TXMs at MISTRAL, ALBA, Barcelona/Spain, and B24, DLS, Oxfordshire, UK).
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom; Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Kyle C
authors.initials: KC
authors.lastname: Dent
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hagen
authors.affiliation: Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Kay
authors.initials: K
authors.lastname: Grünewald
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D015925:Cryopreservation; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods*; D014056:Tomography, X-Ray / Q000379:methods
keywords: Correlative microscopy; Fluorescence cryo-microscopy; Integrative structural biology; Structural cell biology; X-ray imaging; soft X-ray cryo-microscopy