An upright Leica Microsystems (Wetzlar, Germany) TCS
SP5 laser-scanning confocal microscope was used (for data
shown in Figure 1D-I) with a 63× water immersion lens
placed directly into the bulk fluid above biofilms grown on
glass coupons or coverglass. Images were acquired
through the Leica Application Suite Advanced Fluorescence
software platform. Three-dimensional reconstructions
of focal plane slices were generated and analyzed
using IMARIS from Bitplane (Zürich, Switzerland;
Figure 1D-H) or Fiji [106]. An Andor spinning disk confocal
system with a Nikon Eclipse Ti microscope (data
shown in Figure 2C,D,E,M) and Nikon A1R Spectral
Confocal Microscope (Additional file 1: Figure S2) were
also used to image live biofilms within chamber slides.