Fluorescence Microscopy Imaging and Analysis

Fluorescence microscopy was performed as described^{31 (link)}. Briefly, exponentially growing cultures were transferred to slides with a thin 1.0% agarose pad (SeaKem LE agarose, Cambrex) with TPM buffer (10 mM Tris-HCl pH 7.6, 1 mM KH₂PO₄ pH 7.6, 8 mM MgSO₄), covered with a coverslip and imaged using a temperature-controlled Leica DMi6000B inverted microscope with a 100× HCX PL FLUOTAR objective at 32 °C. Phase-contrast and fluorescence images were recorded with a Hamamatsu ORCA-flash 4.0 sCMOS camera using the LASX software (Leica Microsystems). Time-lapse microscopy was performed as described⁶⁴ . Briefly, cells were transferred to a coverslip mounted on a metallic microscopy slide and covered with a pre-warmed 1% agarose pad supplemented with 0.2% casitone in TPM buffer. Slides were covered with parafilm to retain the humidity of the agarose, and live-cell imaging was performed at 32 °C. Image processing was performed with Metamorph v 7.5 (Molecular Devices). For image analysis, cellular outlines were obtained from phase-contrast images using Oufti^{66 (link)} and manually corrected if necessary. Fluorescence microscopy image analysis was performed with a custom-made Matlab script (Matlab R2018a, MathWorks) as described^{34 (link)}.