Fluorescence Microscopy for Exponentially Growing Cells

Fluorescence microscopy was performed as described [11 (link)]. Briefly, exponentially growing cells were stained with 1mg mL^-1 DAPI for 10min at 32°C, transferred to a pad containing 1.5% agarose (Cambrex) with TPM buffer (10 mM Tris-HCl pH 7.6, 1 mM KPO₄ pH 7.6, 8 mM MgSO4) supplemented with 0.2% CTT broth on a microscope slide, and covered with a coverslip. A Leica DMi8 inverted microscope was used for imaging, and phase contrast and fluorescence snapshots were acquired using a Hamamatsu ORCA-flash V2 Digital CMOS camera. For image processing, Metamorph v 7.5 (Molecular Devices) was used. Using a custom made Matlab R2020a (MathWorks) script, cells and fluorescent signals were detected automatically using Oufti48 [64 (link)].