The mobility of the amphiphilic QDs in the biofilm was found to be drastically reduced, as revealed by the distortion of the FCS curves measured at different points through the biofilm in comparison with that obtained for QDs dispersed in water (
Fig. 2). In addition, the diffusion kinetics of QDs inside the biofilm was monitored by time course fluorescence imaging for 75 min. By assessing the diffusion kinetics of QDs inside the biofilm using time course fluorescence imaging for 75 min, we found that the QDs accumulate in every part of the biofilm, with a more pronounced concentration at the periphery than at the center of bacterial aggregates (see Fig. S1 in the supplemental material), contrary to what has been previously shown with hydrophilic QDs (
17).
In order to specify the biofilm parts labeled by the amphiphilic QDs, i.e., extracellular microdomains of
S. oneidensis MR-1 biofilms and/or cell surface of the bacteria, a dual staining was performed with both 2.5 μM Syto 9, a cell-permeant nucleic acid stain which allows easy observation of the cells, and the QDs. Confocal microscopy imaging was performed using a beam line from a continuous argon ion laser as the sole source of excitation at 488 nm (
17). The Syto 9 and CdSe/ZnS–DHLA-Phe QD fluorescence signals were collected at 495 to 530 nm and 580 to 610 nm, respectively. Furthermore, we applied a spectral reassignment procedure based on a Bio-Rad algorithm to reduce the inherent fluorescence overlap of each chromophore.
Figure 3, as well as Fig. S2 in the supplemental material, shows clearly that the cells are easy to recognize due to the green fluorescence of Syto 9 and uniformly distributed in the biofilm, while the amphiphilic QDs are unevenly accumulated and form clusters in the extracellular space between the bacterial cells. Some of the tagged areas (see Fig. S2B in the supplemental material) are large (5 to 15 μm). This QD overaccumulation could be partly attributed to probe-probe interactions. According to previous semivariogram calculations carried out on the whole biofilm (
17), the pseudoperiodic distribution of the amphiphilic QDs should be attributed to the EPS matrix only. The QD accumulation in the exopolymeric matrix of the biofilm reveals irregularly shaped, micrometric (average extent, ca. 4 ± 2.2 μm), closely spaced (2 to 5 μm side to side), and patterned hydrophobic microdomains (
Fig. 4). The frontier of the exopolymeric matrix in
Fig. 4 is based on the outline estimated on the basis of the threshold of gray levels of wide-field images. This consistent information suggests a high density of hydrophobic microdomains per unit volume (up to 10
6 per mm
3) of the
S. oneidensis biofilm matrix. We have also checked the distribution of the hydrophobic domains in biofilms grown under higher (ca. 6 mg/liter) versus lower (ca. 1 mg/liter) dioxygen concentrations, as the concentration of dioxygen has been reported to be one determinant parameter of biofilm cohesiveness (
20). However, similar patterns of QD cluster distribution were observed (data not shown), suggesting the same matrix architecture with respect to hydrophobic microdomains even with the lowest dioxygen concentration tested here.
To summarize, using a surface-functionalized amphiphilic quantum dot we demonstrated the presence of a high density of hydrophobic microdomains with a patterned distribution throughout the exopolymer matrix of S. oneidensis MR-1 biofilms. This hydrophobic texture (i.e., arrangement and size of microdomains) in such a highly hydrated network should allow a protective accumulation of poorly soluble xenobiotics (e.g., steroids, hydrocarbons, etc.) outside the cells.