μ slide well glass bottom

Cell Growth. Strains BHF3 and 1803 were streaked onto LB plates. Single colonies were grown overnight in LB medium at 30 °C, 220 rpm.
Live Cell Sample Preparation. Strain 1803 was inoculated 1:100 into fresh LB medium the next morning and grown at 30 °C and 150 rpm until OD₆₀₀ = 0.5. An amount of 200 μL of cell culture was centrifuged for 20–30 s at 2000 xg. A cellular pellet was resuspended in 3 μL of LB medium and spotted on a 1.5% (w/v) agarose pad. A glass coverslip was placed on the agarose pad and cells were immediately imaged.
Fixed Cell Sample Preparation. Strain BHF3 was grown, fixed, and immobilized as performed in Stockmar et al. (2018). An amount of 2% paraformaldehyde was used for fixation. The maximum cellular density per fixation reaction was OD₆₀₀ = 0.25. Cells were immobilized in multi-well chambers (μ-Slide Well Glass Bottom, Ibidi 80827, Gräfeling, Germany).

Fixed cells were immobilized either on poly-L-lysine coated coverslips or in multi-well chamber (μ-Slide Well Glass Bottom, Ibidi 80827). Coverslips or chamber were incubated with 1:10 diluted poly-L-lysine solution (Sigma P8920) for 30 min or overnight at 4 °C. Poly-L-lysine solution was removed and coverslips (or chamber) were rinsed three times with Milli-Q water. 50 µL of the fixed cells and 1 µL of *fiducial particles (40 nm diameter gold particles) were deposited on the slide (or into the chamber), and centrifuged for 10 min at 3,700 g. Non-immobilized cells were removed by washing three times with PBSG and cells were stored in 200 μl of PBSG until imaging. *Approximately 5 fiduciary particles were present per field of view.