SEM/STEM Observation of Biofilm/Mineral Interface 1165
Westwood, 2003; Barkay et al., 2009; Guise et al., 2011). However, the sample is thin, so the analyzed volume is highly reduced. Moreover, EDS geometry in the SEM is often not optimized for EDS analysis while using the STEM detector, on thin TEM-type samples. The collection solid angle is low, from a few millisteradian (msrad) to a few tens of msrad (except for special flat detectors positioned under the SEM pole piece). For example, for the EDS system on Tescan SEM, the solid angle is 5 msrad (with a detection area of 10mm2). As a result, mapping on thin samples observed by STEM-in-SEM is difficult and requires long collection peri- ods, which can cause problems at high magnification, due to stage drift. Another problem is the signal collection from the support and/or detecting diodes of the STEMdetector of the SEM (e.g., a noticeable signal from Ti is observed in EDS spectra collected while using the STEMdetector in the Mira, due to the sample holder of that detector). TEM EDS systems have been optimized over time and in particular, recent “large solid angle” EDS detectors have markedly increased the EDS efficiency (e.g., the JEOL Centurio detector on the system used in this study has a solid angle of 0.98 srad). STEM-in-SEM has been applied successfully on TEM
thin samples of biological samples (García-Negrete et al., 2015; Ferroni et al., 2016), but to the authors knowledge, no application of STEM-in-SEM on biofilms has, hitherto, been reported in literature.
Influence of sample preparation The electron microscopy observations [SEM, STEM- in-SEM, (S)TEM (in TEM)] of bio-mineral interfaces is challenged by sample preparation. For biological samples alone (without a solid growth substratum), several prepara- tion, and observation methods have already been developed and evaluated (Lane, 1970; Alhede et al., 2012; Karcz et al., 2012). High-vacuum SEM observation of biological samples, especially biofilms, is limited by the requirement of sample dehydration during preparation. SEM requires a complex and multi-step preparation which involves fixation, dehydration, drying, and coating with metal or carbon. A prefixation step involving incubation with glutaraldehyde in cacodylate buffer may also be necessary for samples that cannot undergo the fixation step immediately after sampling. This is the case for biofilms sampled in the field. All these steps may inflict damage to samples and morphological information can be altered, as, for instance, hydrated biofilms subjected to glutaraldehyde fixation and postfixa- tion (osmium tetroxide), followed by ethanol or acetone
dehydration, critical-point drying, and metal coating. The fixation with crosslinking agents (i.e., glutaraldehyde fixation) is used to stabilize the network structure of the
biofilm. This step, together with the postfixation, is designed to prevent breakdown of biological structures. However, in some cases, swelling or shrinkage at the same time has been reported by some authors (Karcz et al., 2012). Alternative methods have been applied, such as environmental scanning electron microscopy (ESEM; Stokes, 2008) and cryo-SEM. ESEM is an effective technique for imaging hydrated
bacterial biofilms by preserving the EPS component without introducing common SEM chemical preparation artifacts. It is based on the application of differential-pumping systems and pressure-limiting apertures, which enables the introduction of gases (e.g., water vapor) into the specimen chamber at quasi-ambient pressure (5–20 Torr). ESEM has been applied successfully for the study of biofilms without the artifacts introduced by chemical preparation for conventional SEM (Little et al., 1991; Priester et al., 2007) and provides accurate images of humid biological samples without any preparation, and may also allow EDS micro- analysis. However, Egerton-Warburton & Griffin (1994) have noticed some variations in the anion content during ESEM analysis, this phenomenon has been attributed by these authors to interactions between anions and electrons in the interaction volume, and possible dehydration effects. Alternatively, cryo-SEM is the combination of high-vacuum or variable pressure SEM coupled to a cryo-stage for the observation of frozen samples by maintaining water in a quasi-stable solid state. This technique preserves the integ- rity of the biofilm structure, and fewer artifacts are noticed compared with dehydration–fixation techniques (Richard & Turner, 1984). The main artifact noticed in this technique is the potential presence of a thin layer of ice formed during sample transfer that can be removed by a controlled pre- observation step (temporary elevation of temperature to sublimate the superficial ice in the preparation chamber or the SEM chamber). Another artifact introduced to samples during freezing is the formation of ice crystals, when the freezing rate is too slow. For this reason, specific techniques such as plunge freezing and high-pressure freezing were developed (Moor et al., 1980). Cryo-SEM techniques allow fractionation and/or metal coating of the sample inside the preparation chamber. Cryo-fractionation is a good way to access the interior of the biofilm (visualization of the EPS network). Commercial Cryo-STEM-in-SEM detectors are not
available and, to date, only “home-made” solutions exist, and consequently STEM-in-SEM observation requires chemical sample preparation (Dobberstein et al., 2006; Robins, 2015).
Influence of accelerating voltage STEM observations were performed between 10 and 30kV on a dozen microtome sections of each sample. However, it appears that such biological samples are very sensitive to carbon contamination. Contamination is particularly visible in STEM imaging at low voltage (10kV or less), compared with higher voltage (20–30 kV) that were thus chosen for STEM imaging in this study (data not shown). Then it is necessary to set up the SEM (focus, stigmatism…) next to the area of interest (by using beam shift, for example) before any image collection. Moreover, STEM resolution was strongly affected by accelerating voltage. Monte-Carlo simulations (CASINO 2.48 software; Drouin et al., 2007) were performed on thin samples (carbon sample, 80 nm, 2000 electron trajectories, beam radius 10 nm) at 5, 15, and 25kV and compared with STEM BF/DF images obtained on
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