Microsc. Microanal. 23, 1159–1172, 2017 doi:10.1017/S143192761701265X


© MICROSCOPY SOCIETY OF AMERICA 2017


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


Guillaume Wille,1,* Jennifer Hellal,1 Patrick Ollivier,1 Annie Richard,2 Agnes Burel,3 Louis Jolly,1 Marc Crampon,1 and Caroline Michel1


1BRGM, 3 avenue Claude Guillemin, BP 36009, 45060 Orleans Cedex 2, France 2CME, University of Orleans, 1 Rue de Chartres, BP 6759, 45067 Orleans Cedex 2, France 3MRIC TEM BIOSIT, University of Rennes, 1-2 avenue du Pr Léon Bernard, CS 34317, 35043 Rennes Cedex, France


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.


Key words: Cryo-SEM, STEM-in-SEM,multispecies natural biofilms, lectin-gold, labeling, bio-mineral interactions


INTRODUCTION Soil and groundwater contamination by recalcitrant organic substances such as polycyclic aromatic hydrocarbons or chlor- oethenes (perchloroethene, trichloroethene, dichloroethene, vinyl chloride) is a major health and environmental issue. Despite much progress, new effective methods for “in situ remediation” are still required. Among recent developments, the high reactivity and the large surface/volume ratio of mate- rials with specific properties (active charcoal, nanoparticles) mayofferadvantagescomparedwith existing solutions (Zhang, 2003). Zero-valent iron nanoparticles (nZVI) have demon- strated their efficiency in degrading problematic contaminants at the laboratory scale (e.g., nitrates, chlorinated solvents) (Fu et al., 2014; Stefaniuk et al., 2016). However, in deconta- mination conditions, it has been seen that the efficiency of nZVI is dependent on their reactivity toward the contaminant as well as their accessibility to contaminants (Kocur et al., 2016). These two properties can potentially be affected by the presence of biofilms—that are the main form of living microorganisms


*Corresponding author. g.wille@brgm.fr Received May 29, 2017; accepted October 11, 2017


in the environment as they offer them a protection against various environmental stresses (dehydration, pollution, preda- tion, etc.). These biological structures are composed of micro- organismsembeddedinanextracellular matrix [extracellular polymeric substances (EPS)] and growing at an interface (solid/ liquid, liquid/air, solid/air). Environmental biofilms are generally multispecies biofilms (i.e., biofilms composed of sev- eral species of microorganisms), in contrast to monospecies biofilms which are often used in in vitro studies. Biofilms are one of the components of a groundwater environment, akin to aquifer mineral composition (e.g., clay, sand), and therefore mayhave animpactonthe efficiency of remediation approa- ches such as nanoremediation using nZVI. In this context, the


use of microscopy offers the possibility of visualization of bio- film structures at different scales of magnitude. Coupling sev- eral approaches in microscopy can enable, where possible, imaging of the same sample at different resolutions (Lawrence et al., 2003;Wrede et al., 2008). Biofilm detection, observation, and analysis is possible


with a large choice of microscopy approaches [e.g., fluores- cence microscopy, scanning electron microscopy (SEM), confocal laser scanning microscopy, transmission electron microscopy (TEM)] coupled with a large choice of cell labeling


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