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Critical Step-by-Step Approaches Toward Correlative Fluorescence/Soft X-Ray Cryo-Microscopy of Adherent Mammalian Cells

DOI: 10.1016/B978-0-12-801075-4.00009-4 DOI Help
PMID: 25287842 PMID Help

Authors: Kyle C. Dent (Diamond Light Source; University of Oxford) , Christoph Hagen (University of Oxford) , Kay Grunewald (Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Methods In Cell Biology , VOL 124 , PAGES 179–216

State: Published (Approved)
Published: October 2014

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).

Journal Keywords: Correlative microscopy; Fluorescence cryo-microscopy; Integrative structural biology; Structural cell biology; X-ray imaging; soft X-ray cryo-microscopy

Subject Areas: Biology and Bio-materials


Technical Areas: Data acquisition