Ix81 inverted optical microscope

Correlated optical fluorescence and AFM images were acquired as described (Eskandarian et al., 2017 (link)). Briefly, optical fluorescence images were acquired with an electron-multiplying charge-coupled device (EMCCD) iXon Ultra 897 camera (Andor) mounted on an IX81 inverted optical microscope (Olympus) equipped with an UPLFLN100XO2PH x100 oil immersion objective (Olympus). Transmitted light illumination was provided by a 12V/100W AHS-LAMP halogen lamp. An U-MGFPHQ fluorescence filter cube for GFP with HQ-Ion-coated filters was used to detect GFP fluorescence. The AFM was mounted on top of the inverted microscope, and images were acquired with a Dimension Icon scan head (Bruker) using ScanAsyst fluid cantilevers (Bruker) with a nominal spring constant of 0.7 N m⁻¹ in Peak Force QNM mode at a force setpoint ~1 nN and typical scan rates of 0.5 Hz. Indentation on the cell surface was estimated to be ~10 nm in the Z-axis. Optical fluorescence microscopy was used to identify Wag31-GFP puncta expressed in a wild-type background (Santi et al., 2013 ) in order to distinguish them from cells of the ∆LDT mutant strains.

An aliquot from an exponential phase culture of M. smegmatis was pipetted onto a PDMS-coated glass coverslip^{25 (link)}, mounted in a custom-made coverslip holder with a built-in heating unit^{46 (link)}, and incubated at 37 °C without agitation for ~15 min to allow attachment. Unattached bacteria were removed by rinsing the coverslip surface with 7H9 before imaging. The sample was maintained at 37 °C during imaging using the coverslip heating holder controlled by a TC200 temperature controller (Thorlabs). AFM images were recorded using a customized Icon AFM (Bruker) that was mounted above an inverted optical microscope. Images were recorded at ≈0.5 Hz line rate using ScanAsyst Fluid cantilevers (Bruker) with a nominal spring constant of 0.7 N m⁻¹ in PeakForce quantitative nanomechanical mode (QNM) at an oscillation rate of 1 kHz and a force setpoint <2 nN. Fluorescence images were acquired with an EMCCD iXon Ultra 897 camera (Andor) mounted on an IX81 inverted optical microscope (Olympus) equipped with a 100X oil immersion objective. Illumination was provided by a mercury lamp (U-HGLGPS, Olympus). The AFM was mounted above the inverted microscope and the AFM laser was switched off while acquiring fluorescence images.