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Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

DOI: 10.1016/j.ultramic.2013.10.006 DOI Help
PMID: 24238600 PMID Help

Authors: Liz Duke (Diamond Light Source) , Minoo Razi (Cancer Research UK) , Anne Weston (Cancer Research UK) , Peter Guttmann (Helmholtz-Zentrum BerlinfürMaterialienundEnergieGmbH) , Stephan Werner (Helmholtz-Zentrum BerlinfürMaterialienundEnergieGmbH) , Katja Henzler (Helmholtz-Zentrum BerlinfürMaterialienundEnergieGmbH) , Gerd Schneider (Helmholtz-Zentrum BerlinfürMaterialienundEnergieGmbH) , Sharon A. Tooze (Cancer Research UK) , Lucy M. Collinson (Cancer Research UK)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Ultramicroscopy , VOL 143 , PAGES 77-87

State: Published (Approved)
Published: October 2013

Open Access Open Access

Abstract: Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities.

Journal Keywords: Antibodies; Compartments; Endoplasmic Reticulum; Fluorescence; Freezing; Golgi Complexes; Images; Mitochondria; Morphology; Receptors; Sample Preparation; Soft X Radiation; Tomography; Transferrin; Transmission Electron Microscopy

Subject Areas: Biology and Bio-materials

Facility: BESSYII