title: Correlative cryo-fluorescence and cryo-soft X-ray tomography of adherent cells at European synchrotrons.
abstract: Cryo-soft X-ray tomography (cryo-SXT) is a synchrotron-hosted imaging technique used to analyze the ultrastructure of intact, cryo-prepared cells. Correlation of cryo-fluorescence microscopy and cryo-SXT can be used to localize fluorescent proteins to organelles preserved close to native state. Cryo-correlative light and X-ray microscopy (cryo-CLXM) is particularly useful for the study of organelles that are susceptible to chemical fixation artifacts during sample preparation for electron microscopy. In our recent work, we used cryo-CLXM to characterize GFP-LC3-positive early autophagosomes in nutrient-starved HEK293A cells (Duke et al., 2013). Cup-shaped omegasomes were found to form at "hot-spots" on the endoplasmic reticulum. Furthermore, cryo-SXT image stacks revealed the presence of large complex networks of tubulated mitochondria in the starved cells, which would be challenging to model at this scale and resolution using light or electron microscopy. In this chapter, we detail the cryo-CLXM workflow that we developed and optimized for studying adherent mammalian cells. We show examples of data collected at the three European synchrotrons that currently host cryo-SXT microscopes, and describe how raw cryo-SXT datasets are processed into tomoX stacks, modeled, and correlated with cryo-fluorescence data to identify structures of interest.
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, United Kingdom.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Carzaniga
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, United Kingdom.
authors.forename: Marie-Charlotte
authors.initials: MC
authors.lastname: Domart
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Duke
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, United Kingdom.
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Collinson
mesh_terms: D002448:Cell Adhesion; D002467:Cell Nucleus / Q000648:ultrastructure; D015925:Cryopreservation; D011992:Endosomes / Q000000981:diagnostic imaging; D005060:Europe; D057809:HEK293 Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008928:Mitochondria / Q000000981:diagnostic imaging; D010588:Phagosomes / Q000000981:diagnostic imaging; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*; D017356:Synchrotrons; D014056:Tomography, X-Ray / Q000295:instrumentation / Q000379:methods; D014463:Ultrasonography; D014584:User-Computer Interface
keywords: Autophagosome; CLEM; CLXM; Correlative; Cryo-SXT; Cryo-fluorescence; Cryo-soft X-ray tomography; Endosome; Mitochondria; Organelle