1162 Guillaume Wille et al. The use of a high-vacuum SEM at low voltage enabled


good-quality SE images. However, low voltage conditions are not well adapted for performing backscattered electron (BSE) images, and most of all, energy dispersive X-ray spectrometry (EDS) analysis on noncoated samples. Thus, a Peltier stage (up to −50°C) was used in a VP-FE-SEM (Tescan Mira3XMU, Brno, Czech Republic). The samples were first frozen in a nitrogen slush at −210°C, then transferred to the cooled stage under low-vacuum conditions. Water was kept under solid state by using low-vacuum conditions (typical conditions used were T= −50°C and P=80Pa nitrogen). Observations were performed at 15–25kV using a low-vacuum SE detector (Jacka et al., 2003). This SE detector is adapted for working under variable pressure conditions, enabling organic matter visualization and the higher voltages allowed use of a BSE detector for the discrimination of mineral particles with high atomic numbers, that is, nZVI and lectin–gold labeling. EDS was used for chemical analysis using an EDAX Team EDS systemwith a silicon drift detector EDAX Apollo XPP (EDAX, Mahwah, NJ, USA) at a working distance of 15mm (detector area 10mm2, collection solid angle 5msrad).


STEM-in-SEM and (S)TEM (in TEM)


Samples (biofilms attached to the inner face of PVC tubes) were prefixed with glutaraldehyde after lectin–gold labeling while still attached to their support, thus limiting manipulations for biofilm sampling (detachment from the PVC tube) and subsequent perturbation of its structure. It appeared that the biofilms then came off their support during the fixation process. This appeared to be an advantage


for observations, as it was easier to make cross-sections on the fixed biofilm after its detachment from the growth support. STEM-in-SEM analysis was conducted with a STEM


detector used in the VP-FE-SEM using bright field (BF) and dark field (DF) imaging (Fig. 2) in combination with SE and BSE detectors. EDS analysis was performed for identifying particles using an EDAX Team EDS system. TEMobservations in combination with EDS analysiswere


performed on a Philips CM20 (Eindhoven, The Netherlands) LaB6 operated at 120 kV and a JEOL ARM200F (Tokyo,


Japan) Cold-FEG equipped with an EDS system JEOL Centurio (detection area 100mm2, collection solid angle 0.98 srad) (JEOL, Tokyo, Japan) operated at 80 kV.


RESULTS AND DISCUSSION


Fluorescent Microscopy The interaction of biofilms (as flocs) with nZVI was initially analyzed using fluorescent microscopy after DAPI and lectins (as PNA-FITC and ConA-FITC) labeling, to target cells and EPS (as exopolysaccharides), respectively (Michel et al., 2016). The two tested lectins-FITC positively labeled the studied flocs (Fig. 3). Lectin labeling was detected close to/ around cells, strongly suggesting the presence of capsular exopolysaccharides (Fig. 3). nZVI aggregates (as black aggregates detected only in flocs in contact with nZVI) were embedded in the biofilm structure as shown on Figure 3, strongly suggesting that biofilms were able to interact with nZVIs and acted as a trap. However, with this technique, it was not possible to detect nZVIs as single particles, but as 0.5–5 µm aggregates. With varying focus, nZVI aggregates


could not be observed. They were absent in deep zones of the flocs where cell density was very high, suggesting that they were only located at the periphery of flocs. Fluorescent microscopy can thus be considered here as an interesting, rapid and low-cost screening approach, but it was not precise enough to appreciate EPS and nZVI distribution in the stu- died biofilm (Table 2). Biofilm’s EPS labeling by lectins can also be achieved using electron microscopy. In this case, lec- tins have to be associated to a metal instead of a fluorescent dye. PNA and ConA (this time as PNA and ConA coupled to


gold nanoparticles), as they gave a positive labeling using fluorescent microscopy, were thus tested for the labeling of the extracellular matrix of the studied biofilm in STEM experiments (Kämper et al., 2004). This approach takes more time and is more expensive than fluorescent microscopy but it offers the advantage of higher resolutions (Tables 1 and 2).


Cryo-SEM


Figure 2. Scanning transmission electron microscopy (STEM)-in- scanning electron microscopy (SEM) detector on the Tescan Mira stage. BSE, backscattered electron; TEM, transmission electron microscopy; BF, bright field; DF, dark field.


Cryo-SEM was applied to observe biofilm development and location of EPS (via lectins–gold labeling) and nZVI, and also to observe bacterial attachment onto sand grains. SEM observations were performed on bulk samples (with or without fractionation) of the sample in the preparation chamber on the Hitachi SEM. SE imaging enables the observation of the organic molecules and thus the biofilm on its own, biofilm colonization of sand grains, and their interactions with nZVI. In contrast, the location and iden- tification of lectin–gold labels and nZVI is more efficient using a BSE detector and EDS microanalysis. Indeed, a gold coating layer (available on the cryo-SEM chamber of this SEM) could be a problem for applying EDS analysis for the identification of gold nanoparticles (from lectin–gold label- ing) as well as the location of lectin–gold labeling and nZVI. This therefore required the use of a variable pressure SEM.


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