M. tuberculosis replicates in a cellular necrotic environment. (A) Snapshots taken from live-cell imaging of GM-CSF macrophages infected with GFP-Mtb over 4 d; the graph below shows how the growth of GFP-Mtb occurs only after the cell becomes PI positive. (B) SBF SEM and TEM were performed on the same sample as in A. This allowed for the GFP-Mtb signal to be overlaid with the EM images. Subpanels i–iii show magnified regions of the ultrastructure taken from the TEM image. The bottom subpanel shows segmentation of the PM in red using the SBF SEM data (with reference to the adjacent TEM image) with the LM data and TEM image overlay. Black arrows indicate PM damage.