Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy

Eamonn Kennedy, Edward M. Nelson, Tetsuya Tanaka, John Damiano, and Gregory Timp, 2016

Image courtesy of American Chemical Society

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–/nm2, 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.

Impact Statement

First reported results of live cell processes imaged with LC-STEM. Contrast was enhanced using heavy metal staining and processes such as cellular fission and bacterial phage infection of E.coli were observed in situ.
Keywords: Cells; Live Cells; Bacteriaphage; Electron Dose; Resolution; Staining