S. mutans biofilms were grown anaerobically on hydroxylapatite discs in 12-well plates in BM medium supplemented with 10 mM sucrose for 48 hours. Following proper staining using LIVE/DEAD BacLight fluorescent dye, biofilms were subjected to optical dissection using an Olympus laser scanning confocal microscope. Images were taken at 600× using a water immersion objective. Post-acquisition analyses were performed using SLIDEBOOK 5.0 (Olympus) and COMSTAT 2.0. Data presented here are representative xyz, xz and yz images (512×512) of UA159, TW239, TW263 and JB314 from more than three independent experiments.