Visualizing Mycoplasma with Nanometer Resolution without Compromising Viability

Abstract

This work represents a step toward the elucidation of the biological mechanisms underpinning live cell physiology with nanometer resolution. Using low-energy (30 keV), low-fluence, probe-corrected, integrated differential phase-contrast scanning transmission electron microscopy, in conjunction with a liquid flow cell, genetically engineered Mycoplasma strains, M. mobile and M. pneumoniae, which are among the smallest self-replicating cells, were scrutinized with nanometer resolution without compromising viability. Following exposure to a 30 keV electron beam, viability was scored at a lethal fluence to 50% (LF50) of the population at LF50 > 4,700 e–/nm2 by expression of an inducible fluorescent reporter, which is in stark contrast with the LF50 < 57 e–/nm2 observed at a beam energy of 300 keV. The higher LF50 at the lower beam energy of 30 keV afforded a wide window for high-resolution imaging of cell physiology. In this window, the mechanisms for “gliding” motility in Mycoplasma, which are supposed to be essential to infection and mediate attachment to a host, were visualized with nanometer resolution.