Application of spherical substrate to observe bacterial motility machineries by Quick-Freeze-Replica Electron Microscopy

Abstract

Abstract 3-D Structural information is essential to elucidate the molecular mechanisms of various biological machineries. Quick-Freeze Deep-Etch-Replica Electron Microscopy is a unique technique to give very high-contrast surface profiles of extra- and intra-cellular apparatuses that bear numerous cellular functions. Though the global architecture of those machineries is primarily required to understand their functional features, it is difficult or even impossible to depict side- or highly-oblique views of the same targets by usual goniometry, inasmuch as the objects (e.g. motile microorganisms) are placed on conventional flat substrates. We introduced silica-beads as an alternative substrate to solve such crucial issue. Elongated Flavobacterium and globular Mycoplasmas cells glided regularly along the bead’s surface, similarly to those on a flat substrate. Quick-freeze replicas of those cells attached to the beads showed various views; side-, oblique- and frontal-views, enabling us to study not only global but potentially more detailed morphology of complicated architecture. Adhesion of the targets to the convex surface could give surplus merits to visualizing intriguing molecular assemblies within the cells, which is relevant to a variety of motility machinery of microorganisms.