Video 1. Macrophage internalization of Mtb aggregates.
Mtb infections of human macrophages were imaged by time-lapse microscopy at a resolution of 10 min between image acquisitions. Macrophage borders and locations were tracked using a custom-written, Matlab based image analysis code (Source code 1). The left panel of the montage is a movie of macrophage infection. The border of the analyzed macrophage is shown as a green outline. RFP-expressing Mtb are shown in red. Mtb internalized by the cell is marked by blue ellipses. Time is hours:minutes. Scale bar is 20 µm. The graph on right shows the fluorescence signal converted to bacterial numbers in the analyzed macrophage. Timing of internalizations of Mtb clumps containing varying numbers of bacilli as detected from the movie are shown as vertical red lines on the graph. Macrophage death is marked by a vertical black line of x’s appearing at the point of cell death.