Plan apo lambda phase objective

For microscopy analyses, cells were grown in M9-Glucose media supplemented with casamino acids. For Snapshot analysis, 1 µL of an exponentially growing cell culture was spotted on the agarose pad (1% w/v) and phase contrast and GFP signals were acquired using a DM6000-B (Leica Microsystems, Wetzlar, Germany) microscope. For the microfluidics, the culture was prepared with M9-Glucose media supplemented with 0.4% casamino acids and Amp at 37 °C overnight. Cells were back-diluted to a starting OD_{600 nm} of 0.05 in fresh media without Amp selection. After 2 h of growth to allow the potential emergence of plasmid-free cells, the culture was loaded into a B04A microfluidic chamber (ONIX, CellASIC, EMD Milipore) preheated at 37 °C. A continuous flow of fresh media without Amp was injected at 2 psi into the microfluidic chamber throughout the experiment (2 h) at 37 °C. Wide-field microscopy imaging was carried out on an Eclipse Ti-E microscope (Nikon, Tokyo, Japan), equipped with × 100/1.45 oil Plan Apo Lambda phase objective, FLash4 V2 CMOS camera (Hamamatsu Photonics, Hamamatsu, Japan), and using NIS software (Nikon) for image acquisition. Phase contrast and mCherry images were acquired every 15 min with 50 ms and 100 ms exposure, respectively Resulting images were analyzed with Fiji/ImageJ software [27 (link)].

Conventional wide-field fluorescence microscopy imaging was carried out on an Eclipse Ti-E microscope (Nikon), equipped with ×100/1.45 oil Plan Apo Lambda phase objective, FLash4 V2 CMOS camera (Hamamatsu), and using NIS software for image acquisition. Acquisition setting was 10 ms for Syto9, 100 ms for GFP, 10 ms for propidium iodide, and 100 ms for mCherry, using 50% power of a fluo LED Spectra X light source at 488- and 560-nm excitation wavelengths, respectively. Cell counting and length analysis were performed using MicrobeJ (Ducret et al., 2016 (link)).