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.

Unveiling the potentials of biocompatible silver nanoparticles on human lung carcinoma A549 cells and Helicobacter pylori

“…The un-inoculated MHB and untreated bacterial cells were used as negative and positive controls respectively and optical density was measured at 600 nm 70 . Tv-AgNPs induced bacterial cell disruption was observed using high-resolution transmission electron microscopy (HRTEM) 71 . For HRTEM analysis, the MIC of PDK-CE was treated to H. pylori for 24 h at 37 °C.…”

Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures

“…However, a 3D reconstruction may be implemented by combining serial sections (serial tomography) although this is complicated and time consuming. The tomography of thicker samples (up to 1.5 µm) may be obtained using scanning transmission electron microscopy (STEM) with a spatial resolution of 5–10 nm [ 121 , 122 ]. Detailed ultrastructural 3D information is achievable also by using high resolution field emission SEM on resin embedded samples; focus ion beam scanning electron microscopy (FIBSEM) uses a focused ion beam to mill the sample surface and obtain serial images with a z-axis resolution of 3–30 nm whereas serial block face scanning electron microscopy (SBFSEM) uses a built-in diamond knife to cut serial sections that are then imaged with a z-axis resolution of 20–50 nm.…”

Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria

“…This S-layer was essential for the formation of nanopods, which were S-layer protrusions containing outer membrane vesicles (OMV) [ 10, 11 ]. Imaging of D. acidovorans Cs1-4 biofilm by three-dimensional scanning electron microscopy (3D SEM) of thick (1250–1500 nm) sections revealed native structure of intact nanopods as cell-attached, polymorphic structures [ 12 ]. The 3-D SEM data established that nanopods were physically contiguous with S-layers of neighboring cells, and revealed openings into cells at the nanopod junctions.…”