High-Resolution Imaging of Bacterial Sacculi

Unless otherwise stated data were collected on this instrument. Live cell imaging was carried out in “Tapping Mode” under a buffer comprised of 300 mM KCl, 10 mM Tris, pH 7.8 using Bruker FastScanD probes driven at ~80 kHz with a typical free amplitude of ~1-2 nm and a set-point amplitude typically ~60-90% of the free amplitude^{35 (link)}. This corresponds to average tip-sample forces ~ 100 pN. Imaging of the external surface of sacculi at high resolution with “Tapping Mode” was not achieved, possibly because of the diffuse nature of the surface when relaxed. Sacculi imaging was instead carried out in “PeakForce Tapping” mode³⁶ with a typical amplitude of 100-200 nm, frequency of 2-8 kHz and setpoint force of 1-2 nN with buffer conditions which varied across the samples between HPLC water and 150-300 mM KCl +10mM Tris (pH 8). All images are topographic images unless otherwise stated. All 3D visuals were created using Nanoscope Analysis software.