Pulse cell wall labeled mycobacteria in a microfluidic device.
Time lapse imaging of pulse-labeled M. smegmatis cells: After pulse labeling M. smegmatis cells with amine reactive dye (green, see protocol), the cells were placed in rich media to resume growth. The recovery media includes a lipophilic dye that stains cell membranes and septa (FM4-64FX; red). Cells are imaged in a microfluidic device every 10 minutes. Brightfield images are shown pseudocolored blue and are overlayed with the red and green fluorescent images. A few cells are dead and have taken up a lot of the FM4-64 (red) dye. After the initial division, asymmetric growth is observed with the old pole growing at a faster rate than the newly septated pole. We can quantify this effect in many cells by measuring the elongation of the cell from each pole relative to the green bands of the pulse labeled “old” cell wall. See Aldridge et al., Science, 2012 for more information. These movies and a similar protocol are also available at the Fortune lab.