Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment

Abstract

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Shift in Natural Groundwater Bacterial Community Structure Due to Zero-Valent Iron Nanoparticles (nZVI)

“…Three distinct steps were carried out and are illustrated in Figure 1. Step 1 consisted in enriching a heterotrophic nitrate-reducing community from a natural groundwater collected from an industrial site by adding a carbon substrate [10 mM sodium acetate (C 2 H 3 NaO 2 )], yeast extracts at 0.2 g L -1 and sodium nitrate (NaNO 3 ) to a volume of groundwater (v/v) and monitoring nitrate reduction (Wille et al, 2017). Total nitrate reduction was obtained after about 48 h, then experiment proceeded to step 2.…”

“…The first is direct nZVI contact with biological components (O’Carroll et al, 2013). In the majority of studies that used electron microscopy to evaluate damage to cell integrity, precipitation of nZVI or iron oxides on the cell wall or inside the bacterial cells was commonly observed, suggesting that direct contact of nZVI with bacterial cells is required for nZVI to exert toxicity (Auffan et al, 2008; Diao and Yao, 2009; An et al, 2010; Barnes et al, 2010; Li et al, 2010; Xiu et al, 2010; Sevcu et al, 2011; Wille et al, 2017; Jiang et al, 2018; Xue et al, 2018). The second is the release of reactive oxidative species (ROS), such as hydroxyl radicals, superoxide radicals and hydrogen peroxide (Barnes et al, 2010), generated by nZVI in the aqueous phase (Liu et al, 2014).…”

“…Finally, the release of ferrous iron from nZVI followed by the Fenton reaction can also affect organisms (Xie and Cwiertny, 2012). Furthermore, although the mechanism of nZVI toxicity at the cellular level has not been fully determined, recent studies support the combined effect of biophysical nanoparticle/cell interactions and oxidative stress (Li et al, 2010; Wille et al, 2017; Crampon et al, 2018).…”

Investigation of the origin of structural colors in calliphoridae flies for bioinspiration

“…This device is used to describe the morphological and structural appearance of the sample under study, as well as its chemical composition, in a vacuum chamber. Its principle is based on the emission of a beam of electrons focused on the sample and generating signals collected after a methodical scan and converted into detailed images represented on the point-by-point display screen [29,30]. Also, SEM has a high resolution that offers us a precise view of the surface microstructures of the Calliphoridae fly.…”