Macrophages and bacteria were imaged using an Andor integrated (Andor, Belfast, UK) Metamorph controlled (Molecular Devices, Sunnyvale, CA) Nikon TiE motorized microscope (Nikon Corporation, Tokyo, Japan) with a 20x, 0.75 NA phase objective. For Mtb RFP and mCherry fluorescence, excitation source was a 561-laser line and emission was detected through a Semrock Brightline 607 nm filter (Semrock, Rochester, NY). Images were captured using an 888 EMCCD camera (Andor). Temperature (37°C), humidity and CO2 (5%) were controlled using an environmental chamber (OKO Labs, Naples, Italy). Approximately 40 fields of view were captured every 10 min, one phase contrast image and one fluorescent image per field at every time point. Fluorescence readings after death were confirmed by widefield microscopy, but fluorescence readings before and particularly at the point of cell death were strongly influenced by cell movement in the z-plane. For imaging data after cell death, the fluorescence signal starting 4 hours after the cell death event was used for analysis.18 (link)