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

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 10nm 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 modalitie