SEM/STEM Observation of Biofilm/Mineral Interface 1167


Figure 7. Observation by scanning transmission electron microscopy (STEM)-in-scanning electron microscopy (Tescan Mira) of a biofilm (80nm microtomic section of a resin-embedded biofilm previously attached to the polyvinyl chloride tube). a: STEM bright field; (b) STEM dark field; (c) backscattered electron detector; (d) SE detector. Yellow dotted arrows indicate gold labeling on the same area.


An example is presented in Figure 9. The SEM resolution was clearly acceptable for biological sample observation at this scale: cells can be easily distinguished (including details of membranes or cells or other nanometric details). Combined with other SEM detectors, it appears to be a great advantage for the study of microorganism–mineral inter- faces. In addition, the use of STEM-in-SEM enables easy access with a continuous magnification and a high image resolution from very low magnification (i.e., millimetric scale, e.g., search of regions of interest) to highmagnification (i.e., nanometric scale) images (Fig. 10). One can also notice the ease of use of the SEM when acquiring a large field of view compared with TEM. In STEM-in-SEM, it is not necessary to change magnification mode, as in the TEM, camera mode or type (say, high-resolution on-axis camera to side mount (“35mm port”) large-field-of-view camera) and there is high contrast for samples with low contrast between phases (Guise et al., 2011). As an example, the same area of an 80nm section of the biofilm observed by STEM-DF is presented in Figure 10 at low (Fig. 10a), medium (Fig. 10b), and high (Fig. 10c) magnification. STEM-in-SEM images (Tescan Mira; Fig. 10d) show bacteria surrounded by gold labeling (yellow dotted arrows) and nZVI (red dashed arrows). Figure 11 shows STEM-DF (in TEM, JEOL ARM) images of gold labeling (Fig. 11a) and nZVI (Fig. 11b). Lectin–gold (here as ConA-Au and PNA-Au) labeling and nZVI can be easily differentiated by their size.


nZVI interaction with biofilm Observations at high magnifications on transversal sections of the biofilm (perpendicular to the internal tube surface) were used to precisely observe the interaction between bacteria and nZVI (Fig. 10d). Lectin–gold labeling and nZVI are clearly distinguished because of their composition (Au versus Fe; EDS) and their difference in size (STEM-DF). Gold labeling appeared everywhere in the biofilm (around cells and in intercellular spaces—Figs. 7a–7c, yellow dotted arrows). However, no labeling was observed in contact with the membrane of the bacteria, but surrounded the cells at a distance of ~0.2–0.5 µm. This suggested that the targeted EPS were mainly capsular type EPS. nZVI were only present on or close to the surface of the biofilm. As with gold labeling, no nZVI were in direct contact with bacteria, which sug- gested that EPS formed a protective layer around them. No nZVI interactions with cell wall/membrane or intracellular nZVI were thus observed. This is in contradiction with previous studies on biofilms with silver zero-valent nano- particles (nZVAg) that showed the nanoparticles closely associated with the bacterial cell surface with apparent invagination of the cell wall/membrane, as well as an intra- cellular location (Fabrega et al., 2009). This could be poten- tially explained by the specific physical and chemical properties of each nanoparticle that would thus influence nanoparticle/biofilm interactions (Peulen & Wilkinson, 2011). The fact that, in our study, nZVI did not directly


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