Diffusion and Binding Measurements within Oral Biofilms Using Fluorescence Photobleaching Recovery Methods

Numerous studies have postulated that bacteria which reside in a biofilm differ from planktonic bacteria. These differences are thought to affect biofilm permeability and, indirectly, the susceptibility of biofilm bacteria to antibacterial agents. In this study fluorescence recovery after photobleaching (FRAP) was used to monitor the diffusion and binding characteristics of a set of size fractionated fluorescein isothiocyanate (FITC)-conjugated dextrans over small areas (ca. 10 $\mu $ m) in bacterial biofilms. From these measurements it was straightforward to calculate apparent diffusion rates. Initial studies on the concentration dependence of dextran interaction with planktonic bacteria showed that no irreversible interaction was occurring, however, anomalous faster than free solution diffusion rates were obtained. This phenomenon was modelled using novel analytical and numerical methods which incorporate reversible binding with associated fluorescence changes. Apparent diffusion rates measured in biofilms were highly dependent on biofilm preparation. Sucrose starved biofilms produced an apparent slow-down of two- to fivefold depending on dextran molecular mass and location within the biofilm, indicating that diffusion within the biofilm is hindered. Sucrose supplemented biofilms produced apparent diffusion rates close to those in free solution, suggesting less hindered diffusion. Ex vivo plaque showed diffusion and binding similar to the sucrose supplemented biofilms. The FRAP approach provides a fast and convenient method for determining diffusion rates over small areas within bacterial biofilms. This study reinforces the importance of considering the influence of reversible binding and associated fluorescence changes, as these may have a marked effect on the measured apparent diffusion rate.