1164 Guillaume Wille et al.


This observation could indicate that the biofilm/nZVI inter- actions would be limited due to a small contact area. Only a few bacteria were visible on the sand grain


surfaces in the cryo-SEM images (Fig. 4). When observed as floc (Figs. 5a–5d), the biofilm mainly consisted of a dense


network of connected forms but without having the char- acteristics of distinguished single cells (Figs. 5b–5c). Figure 5d


presents an accumulation of nZVI attached to the biofilm. nZVI was identified by SE and BSE imaging and EDS analysis (EDS spectra not shown) on the Tescan SEM. nZVI was identified on the Hitachi SEM by their size and shape – in comparison with images of nZVI collected on the Tescan SEM. The main hypothesis is that these forms are bacteria embedded in an EPS matrix, and are not really visible for this reason. Cutting slices of the biofilm for TEM/STEM observation can reveal further


morphological information useful to the understanding of bio- film organization/composition and interactions with nZVI.


Figure 4. Cryo-scanning electron microscopy (SEM) observation of a biofilm growth on a sand grain in the presence of nZVI (SE imaging, T=−50°C/P nitrogen=80 Pa) – red dotted arrow: bac- teria/yellow dashed arrow: nZVI.


STEM-in-SEM and (S)TEM (in TEM) General remarks By using the transmission mode in a SEM (STEM or STEM- in-SEM) performed in a SEM at 30 kV, both contrast and resolution are improved due to the lower accelerating voltage (compared with conventional TEM/STEM at, say 80 kV), which increases the cross-sections and reduces the interac- tion volume of the incident electron beam (Golla-Schindler, 2004; Tracy & Alberi, 2004). High contrast of images from STEM-in-SEM results from the use of low voltages. On the other hand, high resolution is available thanks to limited interaction volume and low chromatic aberration (Bogner et al., 2007). In addition to the flexibility of SEM for nano- metric resolution imaging, ease of use compared with TEM allows to extend the usefulness and capabilities of the SEM. However, the imaging resolution is ultimately limited by the spatial broadening of the electron probe at the exit surface of the sample (Golla-Schindler, 2004). STEM-in-SEM is well suited to the observation of low-Z samples (Brown &


Figure 5. Cryo-scanning electron microscopy observations of biofilms. (a–c): Flocs without nZVI; (d) flocs with nZVI (yellow dashed arrow).


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