title: Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy.
abstract: It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kennedy
authors.forename: Edward M
authors.initials: EM
authors.lastname: Nelson
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Protochips, Inc. , Morrisville, North Carolina 27560, United States.
authors.forename: John
authors.initials: J
authors.lastname: Damiano
authors.forename: Gregory
authors.initials: G
authors.lastname: Timp
mesh_terms: D017101:Bacteriophage P1 / Q000472:pathogenicity / Q000648:ultrastructure*; D002462:Cell Membrane / Q000648:ultrastructure; D004926:Escherichia coli / Q000648:ultrastructure* / Q000821:virology; D005407:Flagella / Q000648:ultrastructure; D017348:Microscopy, Electron, Scanning Transmission / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: E. coli; bacteriophage; infection; liquid cell; low-dose biological imaging; scanning transmission electron microscopy