Single-Molecule Imaging of Labeled Cells

Single-molecule imaging was realized by under-labelling the cells with fluorophore. Strains were cultured in MM to OD₆₀₀ = 0.4, at which point 2.5 nM Janelia Fluor 646 HaloTag ligand (Promega) was added and the cells cultured for another 20 min. Cells were washed five times with PY2 medium, supplemented with 0.5 ng ml⁻¹ SynaptoGreen (Biotium) to label the OM, and 2 μl spotted onto PY2 agar pads. Where indicated, 25 μg ml⁻¹ chloramphenicol was added to the cells 30 min before imaging.
All imaging data were acquired using HiLo (glancing TIRF) illumination on a Nanoimager (Oxford Nanoimaging) equipped with a 640 nm 1W DPSS laser. Optical magnification was provided by a ×100 oil-immersion objective (Olympus, numerical aperture (NA) 1.4) and images were acquired using an ORCA-Flash4.0 V3 CMOS camera (Hamamatsu). All fluorescence images were collected at 15% laser power.
Raw data were analysed using the Fiji plugin ThunderSTORM^{51 (link)} to determine single-molecule localizations. Cell outlines were determined using custom Python codes and single-molecule trajectories within cells computed using the Trackpy Python package (https://zenodo.org/record/3492186#.Y3ZWpH2ZNPY). Finally, apparent diffusion coefficients were determined using custom Matlab codes.