Imaging the Smallest Living Things with Nanometer-Resolution Without Compromising Viability

This work represents a first step towards the illumination of the biological mechanisms underpinning live cell physiology with nanometer resolution. Using low-energy (30 keV), low-dose, probe-corrected, <u>i</u>ntegrated <u>d</u>ifferential <u>p</u>hase-<u>c</u>ontrast <u>s</u>canning <u>t</u>ransmission <u>e</u>lectron <u>m</u>icroscopy (iDPC-STEM) in conjunction with a liquid flow cell, two genetically engineered <i>Mycoplasma</i> species, <i>M. mobile</i> and <i>M. pneumoniae,</i> which are among the smallest, simplest, self-replicating bacteria, were scrutinized with nanometer-resolution without compromising cell viability. The viability was scored at a <u>l</u>ethal <u>d</u>ose to <u>50</u>% of the population at LD₅₀ &gt; 3600 <i>e</i><i>^-</i>/nm² at a beam energy of 30 keV by expression of an inducible fluorescent reporter following exposure to the electron beam in genetically engineered strains of the bacteria, which is in stark contrast with the LD₅₀ &lt; 56 <i>e</i><i>^-</i>/nm² observed at 300 keV. The higher LD₅₀ at a beam energy of 30 keV opened a wide window for high-resolution imaging of cell physiology. Within this window, the mechanisms for gliding motility in <i>Mycoplasma, </i>which are essential to infection and mediate attachment to a host, were elucidated with &lt; 3 nm resolution.