B. subtilis biofilms were inoculated as described above and incubated for the time period indicated in the legend for each figure. Biofilms were then scraped from the plate surface and separated into single cells using mild sonication, as previously described in (9499). Samples were fixated in 4% paraformaldehyde (Electron Microscopy Sciences) and kept at 4°C until the measurement. Samples were measured using an LSR II cytometer (Becton Dickinson, San Jose, CA, USA) operating a solid-state laser at 488 nm. GFP intensities were collected by 505 LP and 525/50 BP filters. For each sample, 106 events were recorded and analyzed for GFP intensities. The autofluorescence level was determined in each experiment by measuring a biofilm sample from a nonfluorescent strain of the same genetic background. Then, the distribution of GFP intensities was analyzed using a custom MATLAB code.