title: Phosphorus detection in vitrified bacteria by cryo-STEM annular dark-field analysis.
abstract: Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low-dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus-containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context.
authors.affiliation: Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Sharon Grayer
authors.initials: SG
authors.lastname: Wolf
authors.affiliation: Department of Physics, Arizona State University, Tempe, Arizona, U.S.A.
authors.forename: Peter
authors.initials: P
authors.lastname: Rez
authors.affiliation: Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Elbaum
mesh_terms: D001419:Bacteria / Q000737:chemistry* / Q000648:ultrastructure*; D002244:Carbon / Q000032:analysis; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000648:ultrastructure*; D004583:Electrons; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D009584:Nitrogen / Q000032:analysis; D010100:Oxygen / Q000032:analysis; D010758:Phosphorus / Q000032:analysis*; D011122:Polyphosphates; D058989:Vitrification
chemical_list: D011122:Polyphosphates; D010758:Phosphorus; D002244:Carbon; D009584:Nitrogen; D010100:Oxygen
keywords: Agrobacterium; EDS; EFTEM; analytical microscopy; cryo-microscopy; scanning transmission electron microscopy