title: Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy.
abstract: Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resolution. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was observed between microbes, suggesting multicellular communication in biofilms. A universal immuno-labelling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was determined (e.g., straight curli, flagella, and excreted cytoplasmic molecular chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community.
authors.affiliation: Department of Bacteriology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.|Jikei Center for Biofilm Science and Technology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Sugimoto
authors.affiliation: Department of Bacteriology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.|Jikei Center for Biofilm Science and Technology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Okuda
authors.affiliation: Department of Bacteriology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.
authors.forename: Reina
authors.initials: R
authors.lastname: Miyakawa
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto, 860-0811, Japan.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Arita-Morioka
authors.affiliation: Department of Bacteriology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Chiba
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto, 860-0811, Japan.
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Yamanaka
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto, 860-0811, Japan.
authors.forename: Teru
authors.initials: T
authors.lastname: Ogura
authors.affiliation: Department of Bacteriology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.|Jikei Center for Biofilm Science and Technology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Mizunoe
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Chikara
authors.initials: C
authors.lastname: Sato
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D005089:Exocytosis; D005109:Extracellular Matrix / Q000382:microbiology; D006090:Gram-Negative Bacteria / Q000166:cytology* / Q000502:physiology; D006094:Gram-Positive Bacteria / Q000166:cytology* / Q000502:physiology; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation; D012996:Solutions
chemical_list: D012996:Solutions