title: Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.
abstract: Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.
authors: O.N. Allen Laboratory for Soil Microbiology, Department Soil Science, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.
authors: William J
authors: WJ
authors: Hickey
authors: O.N. Allen Laboratory for Soil Microbiology, Department Soil Science, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.
authors: Ameesha R
authors: AR
authors: Shetty
authors: Electron Microscope Facility, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.
authors: Randall J
authors: RJ
authors: Massey
authors: O.N. Allen Laboratory for Soil Microbiology, Department Soil Science, University of Wisconsin-Madison, Madison, Wisconsin, U.S.A.
authors: Daniel B
authors: DB
authors: Toso
authors: Department Molecular Genetics and Cell Biology and Advanced Electron Microscopy Facility, University of Chicago, Chicago, Illinois, U.S.A.
authors: Jotham
authors: J
authors: Austin
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D020605:Delftia acidovorans / Q000254:growth & development*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D049329:Nanostructures
keywords: Biodegradation; PAH; STEM; biofilm; nanostructure; tomography