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.

The role of electron irradiation history in liquid cell transmission electron microscopy

“…Investigating biological systems has additionally been a major interest of LC-TEM ( 31 , 32 ), although the role of irradiation history on soft and organic samples is of even greater concern than for other systems. Thus, determining whether structural and morphological changes are actual physiological events or artifacts of the electron beam damaging the sample is a necessary consideration for any LC-TEM experiment on a biological sample.…”

<p>Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology</p>

“…57 Previous works have shown that the effects of changes in pressure, temperature, free radical generation, and exposure to the electron beam itself are non-significant under low electron doses for large samples, such as those containing E. coli, as implemented here (SI notes). 57,59,61,[65][66][67][68][69][70][71][72][73][74][75][76] Thus, previous works indicate that E. coli 1) maintain their metabolic processes, 2) maintain their cell envelopes, and 3) continue to undergo binary fission within liquid-phase TEM. 57,59,61,[65][66][67][68][69][70][71][72][73][74][75][76] Liquid-phase TEM (Figures 3 and S2) provides further evidence of the penetration of nanoprotrusions in E.coli, as also observed forensically with conventional TEM (Figures 2 and S1).…”

Investigating Dynamic Processes of Nanomaterials Using in Situ Liquid Phase TEM Technologies: 2014-2019

“…Liquid cell TEM has been used alone or associated with optical microscopy to image in vivo systems, such as magnetotactic bacteria, 54 the infection of a living biological cell with virus, 55 movements of Au nanoparticles embedded in MDCK cellular matrix in solution, 56 delivery of nanoparticles into cancer cells, 57 etc. For examples, in 2014, Woel et al have combined liquid cell TEM with fluorescence microscopy to image magnetotactic bacteria in liquid.…”