ReviewConfocal microscopy imaging of the biofilm matrix

•Confocal laser scanning microscopy is a valuable tool to study the biofilm matrix.•Various extracellular compounds can be labeled fluorescently and visualized in 3D.•Concentrations of diffusing molecules in the matrix can be monitored in real-time.•Diffusion of solutes through the matrix can be quantified.

The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the concentration of solutes and the diffusive properties of the biofilm matrix.