Live Imaging of Microcolony Formation

Confocal imaging was performed using TCS SP5 DMI 6000 CS (Leica Microsystems) with a PL FLUOTAR 16x/0.5 oil objective. For paracellular live imaging experiments, we used microcolonies grown laterally on glass coverslips as in Muscatine et al. (1997) (link) and Venn et al. (2011) (link). Briefly, the microcolony was set in an incubation chamber and analyzed by inverted confocal microscopy from beneath, at the edge of the microcolony where there are gaps in between the growing crystals. Time laps imaging was recorded (one image every 5 s) using appropriate channels. After 3 min 30 s of recording, texasRed-D3K was added to the medium. Videos correspond to screencasts of the LasAM program graphical interface (Leica) displaying the timelaps acquisition (xzyt) in the video mode.