title: Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.
abstract: Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described.
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Roman I
authors.initials: RI
authors.lastname: Koning
authors.affiliation: Molecular Biotechnology, Institute of Biology Leiden, Leiden University, PO Box 9505, Leiden, The Netherlands.
authors.forename: Katherine
authors.initials: K
authors.lastname: Celler
authors.affiliation: Molecular Biotechnology, Institute of Biology Leiden, Leiden University, PO Box 9505, Leiden, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Willemse
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Bos
authors.affiliation: Molecular Biotechnology, Institute of Biology Leiden, Leiden University, PO Box 9505, Leiden, The Netherlands.
authors.forename: Gilles P
authors.initials: GP
authors.lastname: van Wezel
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Abraham J
authors.initials: AJ
authors.lastname: Koster
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods; D015925:Cryopreservation; D004269:DNA, Bacterial / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods; D025301:Hyphae / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000379:methods; D013302:Streptomyces / Q000648:ultrastructure*
chemical_list: D004269:DNA, Bacterial
keywords: Cryo-correlative light and electron microscopy; Cryo-electron microscopy; Cryo-electron tomography; Cryo-light microscopy; Streptomyces