Microscopic Imaging of E. coli Cells

Exponentially growing cells were transferred to slides containing a thin pad of 1.5% agarose (Cambrex) with TPM buffer (10 mM Tris-HCl pH 7.6, 1 mM KH₂PO₄ pH 7.6, 8 mM MgSO₄, 0.2% CTT, covered with a coverslip and imaged with a temperature-controlled Leica DMi8 inverted microscope. Phase contrast and fluorescence images were acquired using a Hamamatsu ORCA-flash V2 Digital CMOS camera. Cells in phase contrast images were automatically detected using Oufti software^{72 (link)}. Fluorescence signals were identified and analyzed using a custom-made Matlab v2016b (MathWorks) script. E. coli cells were induced with 0.05 mM isopropyl-β-D-thiogalactopyranosid (IPTG) for 2 h and treated with 30 μg ml⁻¹ chloramphenicol for 30 min before DAPI staining. For DAPI staining, cells were incubated with 1 mg ml⁻¹ DAPI for 10 min at 32 °C prior to start of microscopy. Image processing was performed using Metamorph_ v 7.5 (Molecular Devices).