Dental plaque is the biofilm which forms on the surface of teeth, comprising a multitude of bacterial species embedded in microbe- and host-derived polymers. Confocal microscopy (CLSM) studies revealed an open architecture in which palisades of biofilm biomass were shown to be interspersed with water channels of lower density. However, the penetration depth of CLSM in these samples is limited to around 20-40 μm, depending on the density of the samples. Therefore, thick biofilms associated with, for example, fissures in the tooth surface, can not be investigated using this technique. The limited depth penetration of CLSM can be overcome by employing two-photon excitation microscopy (TPEM). Due to the use of the longer wavelength excitation light in TPEM, a two to four fold higher penetration into the sample is obtained than in CLSM, see fig. 1. This, together with the reduced photobleaching of the fluorescent probes, makes TPEM well suited for in-depth imaging studies of strongly scattering samples such as biofilm.

The biofilm nature of plaque allows the development of gradients of key biochemical parameters such as ion concentrations. These gradients may result in extreme conditions at certain sites within the plaque that are often associated with the development of the diseased state. Conditions in the oral cavity are overtly aerobic, and the pH of saliva is near neutral.