Guillaume Wille
email: g.wille@brgm.fr

Jennifer Hellal

Patrick Ollivier

Annie Richard

Agnès Burel

Louis Jolly

Marc Crampon

Caroline Michel

Agnes Burel

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

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

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INTRODUCTION

Soil and groundwater contamination by recalcitrant organic substances such as polycyclic aromatic hydrocarbons or chloroethenes (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 materials with specific properties (active charcoal, nanoparticles) may offer advantages compared with existing solutions [START_REF] Zhang | Nanoscale iron particles for environmental remediation: An overview[END_REF]. Zero-valent iron nanoparticles (nZVI) have demonstrated their efficiency in degrading problematic contaminants at the laboratory scale (e.g., nitrates, chlorinated solvents) [START_REF] Fu | The use of zero-valent iron for groundwater remediation and wastewater treatment: A review[END_REF][START_REF] Stefaniuk | Review on nano zerovalent iron (nZVI): From synthesis to environmental applications[END_REF]. However, in decontamination 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 [START_REF] Kocur | Long-term field study of microbial community and dechlorinating activity following carboxymethyl cellulose-stabilized nanoscale zero-valent iron injection[END_REF]. These two properties can potentially be affected by the presence of biofilms-that are the main form of living microorganisms in the environment as they offer them a protection against various environmental stresses (dehydration, pollution, predation, etc.). These biological structures are composed of microorganisms embedded in an extracellular 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 several 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 may have an impact on the efficiency of remediation approaches such as nanoremediation using nZVI. In this context, the use of microscopy offers the possibility of visualization of biofilm structures at different scales of magnitude. Coupling several approaches in microscopy can enable, where possible, imaging of the same sample at different resolutions [START_REF] Lawrence | Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms[END_REF][START_REF] Wrede | Correlative light/electron microscopy for the investigation of microbial mats from black sea cold seeps[END_REF].

Biofilm detection, observation, and analysis is possible with a large choice of microscopy approaches [e.g., fluorescence microscopy, scanning electron microscopy (SEM), confocal laser scanning microscopy, transmission electron microscopy (TEM)] coupled with a large choice of cell labeling [e.g., fluorescent in situ hybridization, 4',6-diamidino-2phenylindol (DAPI)] and exopolymeric substance labeling (e.g., lectins for EPS, dichlorodimethylacridinone for eDNA) [START_REF] Surman | Comparison of microscope techniques for the examination of biofilms[END_REF][START_REF] Lawrence | Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms[END_REF][START_REF] Kämper | Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy[END_REF][START_REF] Zhang | Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus[END_REF]. Each approach has its specific advantages, but also its drawbacks, the main two being the potential impacts of sample preparation (fixation, drying) on native morphologies and cell structures and the difficulties observing a biofilm associated with its growth substratum (solid), often leading to the removal of biofilms from substrata before observation [START_REF] Surman | Comparison of microscope techniques for the examination of biofilms[END_REF][START_REF] Priester | Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy[END_REF].

To overcome the these drawbacks, the present study investigates the possibilities of coupling several approaches, taking into account that sample preparation and labeling differ according to the type of microscopy [START_REF] Wrede | Correlative light/electron microscopy for the investigation of microbial mats from black sea cold seeps[END_REF]. The methodology was applied to an example of a multispecies biofilm interacting with nZVI. Biofilms were grown for several weeks either as flocs or on a solid surface [sand, polyvinyl chloride (PVC) tube]. The sampling, labeling, and treatment strategies were developed and adapted for each type of microscopy to access the surface or the inside of the biofilm, biofilm structure, and metal location. Special attention was given to the different problems caused by these specific samples, which are a mixture of "soft" hydrated biological samples and "high hardness" mineral particles. Due to the size of the bacteria and EPS filaments in the biofilm (a few microns at most), their observation is limited with optical microscopy. Thus, we applied high-vacuum SEM, variable pressure field-emission scanning electron microscopy (VP-FE-SEM), cryo-SEM, scanning transmission electron microscopy in the SEM (STEM-in-SEM) and TEM techniques.

MATERIALS AND METHODS

Table 1 summarizes the approaches tested in this study, in terms of biofilm growth, sampling, labeling, and treatment according to the microscopy techniques used.

Culture and sampling of biofilms

Biofilms were grown either in PVC tubes (inner Ø: 4.8 mm) or a continuous up-flow fixed-bed bioreactor (laboratoryscale column) (20 cm high; 2.5 cm in diameter) filled with sand (d50 = 0.51 mm) (Fig. 1). The main advantage of PVC tubes is the opportunity to easily and quickly obtain a large amount of biological material as flocs or biofilms attached to a smooth surface (i.e., the inner face of the PVC tube). The column approach was used in order for the biofilm to attach to the sand grains such as in a natural aquifer. However, the intrinsic characteristics of sand grains (rough and hard) limit the microscopic approaches that can be applied. For biofilm culture, a groundwater sample was used as the inoculum and microbial development was favored by supplementing the water with a nutritive solution (sodium acetate 10 mM and yeast extract 0.2 g/L) and an electron acceptor (NO 3 -, solution of NaNO 3 10 mM) [START_REF] Hellal | Interaction entre biofilms bactériens et nanoparticules de fer réactives dans le cadre de procédés de nanoremediation[END_REF]. Anaerobic conditions (under N 2 bubbling) were maintained as in a deep contaminated aquifer. The incubation temperature was maintained at 20°C.

First the biofilms were left to grow during 6 weeks without nZVI. Then a nZVI suspension (1 g/L, NANOFER 25 S, mean particle size ~50-80 nm; NanoIron, Rajhrad, Czech Republic) was continuously introduced into the PVC tube or column (1 g/L, 10 mL/min, for five poral volumes). Finally, biofilms were sampled as flocs in the PVC tube or as attached biofilms on the inner face of PVC tubes, or collected as attached to sand grains in the column. Biofilms attached to the inner face of the PVC tube were used either still attached to the PVC tubes, or detached from the tubes by removing the biofilm with a sterile scalpel (this allows to access the external face of the biofilm) and placing them on a SEM copper stub.

Biofilm labeling for fluorescence microscopy

Biofilms as flocs were first stained with DAPI for cell labeling and then with lectin for EPS detection [START_REF] Michel | Role of exopolymeric substances (EPS) in the stability of the biofilm of Thiomonas arsenivorans grown on a porous mineral support[END_REF]. To avoid out of focus images and to decrease background fluorescence, a z-stack approach for fluorescent labeling imaging of the flocs was applied. Using this approach, 15 images of the same area in the biofilm were taken at different focus/ depths over an overall distance of 0.3 µm, and then merged.

Lectin-gold labeling and sample preparation for observation for electron microscopy

Labeling with lectins coupled to gold particles (15 and 40 nm) (PNA and ConA; EY Laboratories, San Mateo, CA, USA-ready-to-use lectin solutions at a concentration of 10-20 µg/mL) was applied to the samples before SEM observations. Labeling was performed on the fresh biofilm immediately after sampling. Biofilms were washed once using PBS 1× buffer. They were then covered with the readyto-use solutions of lectin coupled to gold clusters for 90 min in the dark. After removing the lectin-gold solution, biofilms were washed three times with PBS 1× buffer. For STEM-in-SEM and (S)TEM (in TEM), labeling was performed before glutaraldehyde fixation.

For STEM-in-SEM and (S)TEM (in TEM) observations, samples (lectin-gold labeled biofilms) were washed three times with cacodylate buffer (0.1 M, pH 7.2) and submitted to a prefixation step. For this, cells were fixed in 2.5% glutaraldehyde in Cacodylate buffer 0.15 M at pH 7.2 for 2 h, under vacuum. Samples were then postfixed in 1% osmium tetroxide for 1 h and extensively rinsed before inclusion in 2% agar gel (bacteriological quality) at 60°C. Agar was used to stabilize biofilm samples. Stabilizing samples in gel make them easy to cut into small pieces of 1 mm 3 before dehydration and inclusion. Samples were dehydrated through a series of graded ethanol solutions from 70 to 100%. The specimens were infiltrated with a mixture of acetone-Epon (50/50) for 3 h then with pure Epon for 16 h. Finally, the specimens were embedded in DMP30-Epon for 24 h at 60°C. Ultrathin sections (80 nm) were obtained on a Leica UC6 microtome (Wetzlar, Germany), collected on copper hexagonal grids.

For STEM-in-SEM observations, samples (biofilm sections on copper grids) were coated with 2 nm Carbon in a Cressington 208C (Cressington, Watford, UK) or 0.5 nm Pt/Pd in a Cressington 208HR. Indeed, noncoated section on grids did not withstand the electron beam energy and serious degradation was observed when increasing magnification.

Cryo-SEM

Two different cryo-SEM options were used. The first option is the use of a high-vacuum Hitachi S4500 (Tokyo, Japan) cold-FEG SEM equipped with a Quorum Technology Polaron LT 7400 (Lewes, UK) cryo-SEM preparation system (including cryo-fracturation, gold coating). The samples were frozen in nitrogen slush at -210°C, and then transferred to the preparation chamber. No coating was applied on the samples. Cryo-fractionation (if necessary) was processed in the preparation chamber at -180°C. Cryo-fractionation allows observation of the internal organization (including the EPS network) of the biofilm. Then, SEM observations were performed at -108°C at low voltage (1 kV) using a secondary electron (SE) detector. A preobservation step at -70°C was applied in the SEM chamber to remove the ice layer formed on the surface of the sample during its transfer. 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 = 80 Pa nitrogen).

Observations were performed at 15-25 kV using a low-vacuum SE detector [START_REF] Jacka | A differentially pumped secondary electron detector for low-vacuum scanning electron microscopy[END_REF]. 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 system with a silicon drift detector EDAX Apollo XPP (EDAX, Mahwah, NJ, USA) at a working distance of 15 mm (detector area 10 mm 2 , collection solid angle 5 msrad).

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.

TEM observations in combination with EDS analysis were performed on a Philips CM20 (Eindhoven, The Netherlands) LaB 6 operated at 120 kV and a JEOL ARM200 F (Tokyo, Japan) Cold-FEG equipped with an EDS system JEOL Centurio (detection area 100 mm 2 , 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 [START_REF] Michel | Analysis of biofilm-nanoparticles interaction using microscopy (fluorescence, MEB[END_REF]. 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 studied biofilm (Table 2). Biofilm's EPS labeling by lectins can also be achieved using electron microscopy. In this case, lectins 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 [START_REF] Kämper | Methods for in situ detection and characterization of extracellular polymers in biofilms by electron microscopy[END_REF]. This approach takes more time and is more expensive than fluorescent microscopy but it offers the advantage of higher resolutions (Tables 1 and2).

Cryo-SEM

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 identification 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 labeling) as well as the location of lectin-gold labeling and nZVI. This therefore required the use of a variable pressure SEM. Biofilms grown on sand grains, put in contact with nZVI and then observed by cryo-SEM, are presented on Figure 4. SE image clearly shows the presence of a biofilm on the sand grain, and the interaction of nZVI with bacteria. EDS analysis was used to confirm the aggregates were nZVI and not ice. Sand grains are millimeter-sized and thus cannot be observed at this scale (view field 25.3 µm maximum). It appears that nZVI remained aggregated and did not disperse in the biofilm. This observation could indicate that the biofilm/nZVI interactions 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 characteristics 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 shapein 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 biofilm organization/composition and interactions with nZVI.

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

General remarks By using the transmission mode in a SEM (STEM or STEMin-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 interaction volume of the incident electron beam [START_REF] Golla-Schindler | STEM-Unit measurements in a scanning electron microscope[END_REF][START_REF] Tracy | Adopting low-voltage STEM and automated sample prep to perform IC failure analysis[END_REF]. 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 [START_REF] Bogner | A history of scanning electron microscopy developments: Towards Wet-STEM imaging[END_REF]. In addition to the flexibility of SEM for nanometric 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 & Westwood, 2003; [START_REF] Barkay | Three-dimensional characterization of drug-encapsulating particles using STEM detector in a FEG-SEM[END_REF][START_REF] Guise | STEM-in-SEM method for morphology analysis of polymer systems[END_REF]. 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 10 mm 2 ). As a result, mapping on thin samples observed by STEM-in-SEM is difficult and requires long collection periods, 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 STEM detector of the SEM (e.g., a noticeable signal from Ti is observed in EDS spectra collected while using the STEM detector 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 [START_REF] García-Negrete | STEM-in-SEM high resolution imaging of gold nanoparticles and bivalve tissues in bioaccumulation experiments[END_REF][START_REF] Ferroni | Biological application of compressed sensing tomography in the scanning electron microscope[END_REF], 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, STEMin-SEM, (S)TEM (in TEM)] of bio-mineral interfaces is challenged by sample preparation. For biological samples alone (without a solid growth substratum), several preparation, and observation methods have already been developed and evaluated [START_REF] Lane | The environmental control stage[END_REF][START_REF] Alhede | Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition FEMS[END_REF][START_REF] Karcz | Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy[END_REF]. 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 postfixation (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 [START_REF] Karcz | Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy[END_REF]. Alternative methods have been applied, such as environmental scanning electron microscopy (ESEM; [START_REF] Stokes | A Brief Historical Overview, in Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy[END_REF] 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 [START_REF] Little | Biofilms: an ESEM evaluation of artifacts introduced during SEM preparation[END_REF][START_REF] Priester | Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy[END_REF] and provides accurate images of humid biological samples without any preparation, and may also allow EDS microanalysis. However, [START_REF] Egerton-Warburton | Levels of mineral nutrients in fresh-and frozen bulk hydrated biological specimens: A comparison of EDS data collected in the environmental SEM and a conventional Cryo-SEM[END_REF] 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 integrity of the biofilm structure, and fewer artifacts are noticed compared with dehydration-fixation techniques [START_REF] Richard | A comparative study of techniques for the examination of biofilms by scanning electron microscopy[END_REF]. 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 preobservation 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 [START_REF] Moor | The influence of high pressure freezing on mammalian nerve tissue[END_REF]. 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 [START_REF] Dobberstein | Development of Cryo-STEM for scanning electron microscopy[END_REF][START_REF] Robins | Ongoing developments of the Cryo-SEM/ STEM technique[END_REF].

Influence of accelerating voltage

STEM observations were performed between 10 and 30 kV 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 (10 kV 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; [START_REF] Drouin | CASINO V2.42 -A fast and easy-to-use modeling tool for scanning electron microscopy and microanalysis users[END_REF] were performed on thin samples (carbon sample, 80 nm, 2000 electron trajectories, beam radius 10 nm) at 5, 15, and 25 kV and compared with STEM BF/DF images obtained on the same area on one biofilm floc sample under the same imaging conditions (accelerating voltage, beam width) (Fig. 6). These simulations show the influence of the accelerating voltage on the STEM-in-SEM resolution and enabled comparison of the STEM-DF images obtained from the same area on one biofilm sample at these accelerating voltage conditions. Due to the low mean Z of the biological sample (composed mainly of carbon, hydrogen, nitrogen, oxygen), most of the electrons are transmitted with little or no energy loss at high accelerating voltage, but the dispersion of the electron trajectories increased strongly when lowering the high voltage. This effect provides an increase of the image contrast, as does the addition of osmium tetroxide as a contrasting agent for cell membranes during sample preparation. An accelerating voltage of 30 kV was chosen because it provided the best resolution and good contrast in STEM-in-SEM on biological samples.

Imaging with STEM-in-SEM STEM in the SEM offers the ability to simultaneously collect images using the STEM, SE, and BSE detectors. This combination of detectors is a great advantage for the discrimination of contrasts in the STEM-DF/BF images. An example of STEM-in-SEM observation (DF, BF) of the same area of a biofilm (an 80 nm microtomic section of a resin embedded biofilm floc sample) is presented in Figure 7, and compared with BSE and SE images (all images were simultaneously collected). Some bright particles are clearly visible in BF or DF images (with reverse contrast in the other). Due to the origin of contrast in STEM images, these bright particles can only be associated to a difference in composition or to diffraction contrast. BSE images of these particles can be used to discriminate between a strong diffraction contrast and high Z. SE image contrast was weak, no contrast was observed using this detector. This can be explained by an absence of topography and a very low difference in mean atomic Z which contributes a little to the signal detected by the SE detector. On this image, bright particles observed on the BSE image, together with DF images, were analyzed by EDS point analysis (on STEM-in-SEM-data not shown) and mapping (on STEM in TEM) (Fig. 8). The particles were composed of calcium and phosphorus.

A comparison of TEM (Philips CM20 -120 kV) and STEM-in-SEM (Tescan Mira -30 kV) was performed. An example is presented in Figure 9. The SEM resolution was clearly acceptable for biological sample observation at this scale: cells can be easily distinguished (including details of membranes or cells or other nanometric details). Combined with other SEM detectors, it appears to be a great advantage for the study of microorganism-mineral interfaces. In addition, the use of STEM-in-SEM enables easy access with a continuous magnification and a high image resolution from very low magnification (i.e., millimetric scale, e.g., search of regions of interest) to high magnification (i.e., nanometric scale) images (Fig. 10). One can also notice the ease of use of the SEM when acquiring a large field of view compared with TEM. In STEM-in-SEM, it is not necessary to change magnification mode, as in the TEM, camera mode or type (say, high-resolution on-axis camera to side mount ("35 mm port") large-field-of-view camera) and there is high contrast for samples with low contrast between phases [START_REF] Guise | STEM-in-SEM method for morphology analysis of polymer systems[END_REF]. As an example, the same area of an 80 nm section of the biofilm observed by STEM-DF is presented in Figure 10 at low (Fig. 10a), medium (Fig. 10b), and high (Fig. 10c) magnification. STEM-in-SEM images (Tescan Mira; Fig. 10d) show bacteria surrounded by gold labeling (yellow dotted arrows) and nZVI (red dashed arrows). Figure 11 shows STEM-DF (in TEM, JEOL ARM) images of gold labeling (Fig. 11a) and nZVI (Fig. 11b). Lectin-gold (here as ConA-Au and PNA-Au) labeling and nZVI can be easily differentiated by their size.

nZVI interaction with biofilm

Observations at high magnifications on transversal sections of the biofilm (perpendicular to the internal tube surface) were used to precisely observe the interaction between bacteria and nZVI (Fig. 10d). Lectin-gold labeling and nZVI are clearly distinguished because of their composition (Au versus Fe; EDS) and their difference in size (STEM-DF). Gold labeling appeared everywhere in the biofilm (around cells and in intercellular spaces-Figs. 7a-7c, yellow dotted arrows). However, no labeling was observed in contact with the membrane of the bacteria, but surrounded the cells at a distance of ~0.2-0.5 µm. This suggested that the targeted EPS were mainly capsular type EPS. nZVI were only present on or close to the surface of the biofilm. As with gold labeling, no nZVI were in direct contact with bacteria, which suggested that EPS formed a protective layer around them. No nZVI interactions with cell wall/membrane or intracellular nZVI were thus observed. This is in contradiction with previous studies on biofilms with silver zero-valent nanoparticles (nZVAg) that showed the nanoparticles closely associated with the bacterial cell surface with apparent invagination of the cell wall/membrane, as well as an intracellular location [START_REF] Fabrega | Interaction of silver nanoparticles with Pseudomonas putida biofilms[END_REF]. This could be potentially explained by the specific physical and chemical properties of each nanoparticle that would thus influence nanoparticle/biofilm interactions [START_REF] Peulen | Diffusion of nanoparticles in a biofilm[END_REF]. The fact that, in our study, nZVI did not directly interact with cells could potentially lead to a less toxic effect of nZVI on biofilms compared with those described for nZVAg [START_REF] Choi | Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures[END_REF][START_REF] Peulen | Diffusion of nanoparticles in a biofilm[END_REF][START_REF] Mallerve | Pseudomonas putida biofilm dynamics following a single pulse of silver nanoparticles[END_REF]. nZVI were found as aggregates embedded in the biofilm structure and located on the outside of the biofilm in contact with the medium. This is in agreement with results obtained by [START_REF] Choi | Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures[END_REF], who showed that nZVAg aggregated with an average size of about 800 nm could penetrate to ~40 µm in a thick biofilm after 1h exposure. In our study, nZVI aggregates were bigger in size (about 1-5 µm). This could explain the shorter distance of nZVI aggregates penetration [7-11 µm in this study compared with 40 µm in the study of [START_REF] Choi | Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures[END_REF]].

Using STEM-in-SEM imaging only, it was not possible to conclude-or exclude-a co-location of nZVI and gold labeling, that is, a connection and an interaction between nZVI and EPS. For this, elemental mapping appeared to be difficult or impossible due to the low gold content. EDS and/ or electron energy loss spectroscopy (EELS) in the TEM are required for such analyses.

The presence of gold labeling together with nZVI was confirmed by EDS spectra (not shown) and mapping on Fe and Au on a nZVI aggregate in the JEOL ARM200 (Fig. 12). This co-location Fe/Au suggests an interaction between nZVI and EPS. Moreover, these results confirm that EPS are also present in intercellular spaces inside the biofilm. As in previous studies, this work underlined the role of EPS in nanoparticles/biofilm interaction [START_REF] Moreau | Extracellular proteins limit the dispersal of biogenic nanoparticles[END_REF][START_REF] Peulen | Diffusion of nanoparticles in a biofilm[END_REF]. Here, the role of EPS labeled by lectins ConA and PNA was confirmed, but the presence and the role in nanoparticle interactions with other extracellular components, such as exopolysaccharides nontargeted by the two tested lectins or extracellular protein or DNA, can, of course, not be excluded. As an example, [START_REF] Moreau | Extracellular proteins limit the dispersal of biogenic nanoparticles[END_REF] demonstrated the role of extracellular proteins. No, or very few, individual iron particles were detected. It also suggests that nZVI were present in the biofilm as aggregates in contact with EPS, but not directly with bacteria. It also confirms that no direct interaction between nZVI and microorganisms can be considered, because of the presence of a thick layer of EPS surrounding the bacteria. Biofilm/EPS composition thus plays an important role in biofilm/nanoparticle interactions, as well as biofilm structure. In particular, density of both exopolymers and bacteria has been demonstrated to be an important parameter controlling the diffusion of nanoparticles inside the biofilm [START_REF] Peulen | Diffusion of nanoparticles in a biofilm[END_REF]. Growth conditions that can influence biofilm density could thus influence biofilm/nanoparticle interactions [START_REF] Peulen | Diffusion of nanoparticles in a biofilm[END_REF]. In general terms, diffusion in a biofilm has been shown to be reduced due to the presence of microbial cells, extracellular polymers, and abiotic particles or gas bubbles that are trapped in the biofilm [START_REF] Stewart | Diffusion in biofilms[END_REF]. Bacterial cell wall hydrophobicity was also demonstrated to play a role in the diffusion of nanoparticles in biofilms [START_REF] Habimana | Diffusion of nanoparticles in biofilms is altered by bacterial cell wall hydrophobicity[END_REF]. In this study, TEM observations coupled to lectin labeling suggested that our biofilm was quite dense in terms of cells and EPS concentrations. In addition to the nanoparticle (NP)/EPS interaction suggested by our results, the high EPS/bacteria density could also explain that nZVI did not diffuse into the overall biofilm. Last, it can be hypothesized that even if the biofilm is very porous, the size of nZVI aggregates observed in biofilms is so big that the biofilm pores are rapidly plugged by the aggregates, thus preventing them from further entering the biofilm structure. Advantages and drawbacks of the tested microscopy methods for biofilm/nZVI observation studies A comparison of the advantages and drawbacks of the microscopy methods applied to the study of biofilms/nZVI interaction are summarized in Table 2. Our results show that coupling EDS analysis with fluorescent, SEM and TEM microscopy techniques offers the possibility to access the inside and outside of the biofilm and thus enables biofilm/nZVI characterization at (i) the biofilm level, with the location and nature of nZVI (here as aggregates) at the surface of the biofilm structure, (ii) the cell level with the absence of cells/nZVI interaction, and (iii) the molecular level with the lectin labeling strongly suggesting a major role of EPS in nZVI/biofilm interaction. Moreover, the present study demonstrated that the three microscopy methods used are complementary approaches for a complete study of biofilm/nZVI interactions.

Each microscopy technique has its advantages and drawbacks that can be bypassed by the use of other techniques (Table 2). Moreover, preparing biofilm samples for accurate observations, especially when interacting with sand or metal elements such as nZVI, can prove difficult without modifying their structure. Many studies have been carried out using biofilms developed on a smooth surface (glass or flow cell experiments) and protocols that are developed are thus not applicable to most biofilms/substrata directly taken from the environment. Working with flocs using fluorescent microscopy is also not easy. Indeed, samples are thick and water rich (compared with biofilm grown on a substratum). Observation of flocs between slide and cover slide can result to the flocs moving during analysis (because of the presence of water inside the sample), and the thickness makes fluorescence detection in the deeper zones of the biofilm possible, thus leading to fluorescence that is out of focus. The coupling of several approaches in microscopy is thus an obligate approach and the best route to study biofilm/NP interaction with biofilms still attached to a "natural" substratum (sand grains in this study) that are usually porous, not flat, voluminous, and impossible to section.

CONCLUSION

Understanding biofilm organization and interactions with surrounding substratum and pollutants or particles can benefit from the array of existing microscopy tools such as fluorescent microscopy, cryo-SEM, STEM-in-SEM or (S) TEM (in TEM), and EDS. Microscopy is indeed a very powerful approach to characterize biofilms and has previously been used to study biofilm/nZVI interaction.

Cryo-SEM samples exhibited a low number of bacteria visible in the biofilm, but showed rather a network with forms whose shape and size are similar to that of bacteria. We suggested that these forms may be bacteria embedded in an EPS network, as was shown by lectin labeling. This hypothesis was confirmed by STEM-in-SEM observations on the microtome sections of the biofilm with lectin-gold labeling for EPS detection, which showed that the bacteria were encapsulated in a matrix of EPS. STEM-in-SEM analyses also showed nZVI aggregates on or near the surface (up to 10 µm) of the biofilm, but not in direct contact with bacteria. Complementary (S)TEM (in TEM)/EDS analysis also revealed that nZVI are connected to the EPS matrix, as confirmed by EDS mapping of gold and iron which exhibit co-location of lectin-gold (EPS markers) with iron aggregates. Then, the combination of fluorescent microscopy, cryo-SEM and STEM-in-SEM, supplemented by TEM/EDS analysis appeared to be an excellent tool for understanding the biofilm/mineral interface and interaction.

These observations enabled access to the inside and outside of the biofilm at different scales of magnitude, allowing to understand the behavior and the internal structure of biofilms in contact with mineral or metallic particles, and the ability of biofilms to fix nZVI or to bind to sand particles. This approach allows nZVI characterization (in terms of size distribution and aggregation) and location inside the biofilm (intracellular location, association with cells wall/membrane).

Figure 1 .

 1 Figure 1. Device developed for the growth of biofilms as flocs [in polyvinyl chloride (PVC) tube] or attached on a surface (inner face of PVC tube or sand surface by sampling in the column).

Figure 2 .

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

Figure 3 .

 3 Figure 3. Fluorescent microscopy observations of biofilm (floc) with (b,d) or without (a,c) nZVI contact. Flocs were labeled with DAPI (blue) and Lectin [(a),(b) in red: ConA. (c), (d) in pink: PNA; images were recolored for better observation/contrast of both labeling (DAPI and lectin) on the same image]. Flocs were labeled with DAPI (blue) and Lectin (here ConA) (red).

Figure 4 .

 4 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: bacteria/yellow dashed arrow: nZVI.

Figure 5 .

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

Figure 6 .

 6 Figure 6. Monte-Carlo simulation of the beam dispersion on a carbon samples (thickness 80 nm, 2000 electrons, beam radius 10 nm) at (a) 5, (b) 15, and (c) 25 kV, and bright field (BF)/dark field (DF) images of a biofilm in the same conditionsdetail (white rectangle) is a magnified view of the dotted white rectangle.

Figure 7 .

 7 Figure 7. Observation by scanning transmission electron microscopy (STEM)-in-scanning electron microscopy (Tescan Mira) of a biofilm (80 nm microtomic section of a resin-embedded biofilm previously attached to the polyvinyl chloride tube). a: STEM bright field; (b) STEM dark field; (c) backscattered electron detector; (d) SE detector. Yellow dotted arrows indicate gold labeling on the same area.

Figure 8 .

 8 Figure 8. Energy dispersive X-ray spectrometry mapping on a bacteria (TEM 80 kV -JEOL ARM): (a) Scanning transmission electron microscopy-bright field; (b) calcium Kα; (c) oxygen Kα; (d) phosphorus Kα.

Figure 9 .

 9 Figure 9. Comparison of scanning transmission electron microscopy (STEM)-in-scanning electron microscopy (Tescan Mira) at 30 kV and transmission electron microscopy (TEM) (Philips CM20) at 120 kV. a: STEM-bright field 30 kV; (b) STEM-dark field 30 kV; (c) TEM 120 kV (80 nm microtomic section of a resin-embedded biofilm floc sample).

Figure 10 .

 10 Figure 10. Distribution of nZVI (dashed red arrows) and EPS gold labeling (dotted yellow arrows) in a biofilm by scanning transmission electron microscopy-in-scanning electron microscopy (Tescan Mira): a: view field 148 µm (a complete grid hole); (b) 67.8 µm; (c) 12.6 µm; (d) 10.1 µm (a, b, c: images from the same area, d: images from a different area).

Figure 11 .

 11 Figure 11. Differentiation of lectin (PNA and ConA)-Au labeling and nZVI by their size (scanning transmission electron microscopy -JEOL ARM200).

Figure 12 .

 12 Figure 12. nZVI and lectin (PNA and ConA)-Au labeling co-location: Scanning transmission electron microscopy (JEOL ARM200) [(a) bright field, (b) dark field] and energy dispersive X-ray spectrometry mapping [(c) Fe Kα and (d) Au Lα].

Table 1 .

 1 Sample Selection and Preparation and Observation Characteristics According to the Microscopy Used for Biofilm/nZVI Interaction Analysis.

	Microscopy	Fluorescence	Cryo-SEM + EDS	STEM-in-SEM + EDS	TEM + EDS
	Sample	Floc	Floc or biofilm grown on sand grain	Biofilm attached on PVC tube
	Sample preparation	Incubation with nZVI. Washing	Incubation with nZVI. Washing step	Incubation with nZVI. Washing step for non-fixed NP
		step for non-fixed NPs	for non-fixed NP elimination	elimination
		elimination
	Labeling	DAPI + lectin (PNA or ConA) -	Lectin PNA-Au and Lectin ConA-Au	Lectin PNA-Au (40 nm) and Lectin ConA-Au (15 nm)
		FITC	(gold size 15 to 40 nm)
	Sample treatment	None	Sample frozen in nitrogen slush	Prefixation and postfixation. Ultrathin sectioning (80 nm)
			at -210°C. Cryo-fracturation
			(if necessary)
	Resolution	µm	nm	nm (better than cryo-SEM) <nm
	Imaging mode(s)		SE: morphology	SE: morphology	Transmitted e-BF
			BSE: chemical composition	BSE: chemical composition	HAADF
				BF/DF
	Chemistry		EDS	EDS	EDS EELS (data not shown)
	SEM, scanning electron microscopy; EDS, energy dispersive X-ray spectrometry; STEM, scanning transmission electron microscopy; TEM, transmission
	electron microscopy; PVC, polyvinyl chloride; nZVI, zero-valent iron nanoparticles; NP, nanoparticle; DAPI, 4',6-diamidino-2-phenylindol; SE, secondary
	electron; BSE, backscattered electron; BF, bright field; HAADF, high angle annular dark field; DF, dark field; EELS, electron energy loss spectroscopy.

4 0.24 g/L) and labeled with lectin PNA-FITC [Peanut Aglutinin, lectin from Arachis hypogaea (peanut) FITC conjugates; Sigma-Aldrich] or ConA-FITC [lectin from Canavalia ensiformis FITC conjugate; Sigma-Aldrich, St. Louis, MO, USA] (both lectins at 50 µg/mL in PBS 1× buffer) in the dark for 20 min. Samples were washed before observation in a fluorescent microscope (Zeiss Axio Imager Z1, Plan Neofluar ×100 objective (immersion); AxioVision 4.6 software).

Table 2 .

 2 Advantages and Drawbacks of Each of the Microscopy Technique Applied to the Biofilm/NP Interaction Studies[START_REF] Michel | Analysis of biofilm-nanoparticles interaction using microscopy (fluorescence, MEB[END_REF].

	Techniques Fluorescence microscopy	Cryo-SEM	STEM-in-SEM and TEM
	Advantages Rapid, easy, and low-cost technique	Observation of the surface of biofilms.	Overview as well as details of the
	Induces no or little perturbation on biofilm	Possibility to analyze the biofilm on its	inside of the biofilm and the
	structure	growth substratum, even on granular and	microorganisms
	Large choice of labels. Overview of the	porous materials. Biofilm labeling	Biofilm labeling.
	biofilm/NP interaction. Screening/	Nanometric scale imaging, various	Nanometric scale imaging and
	preliminary approach for electronic	observation modes.	elemental analysis, various
	microscopy preparation: validation of	Can be coupled to EDS analysis	observation modes
	biofilm/NP interaction and lectin choice	(micrometric scale)	Can be coupled to EDS/EELS
		Ability to work on humid non-frozen	analysis
		sample (environmental SEM)
	Drawbacks Analysis of flocs and biofilms grown on a	The chemical preparation or freezing step	Expensive technique
	smooth surface such as glass: no	can modify the structure of the biofilm	Long procedure (fixation steps).
	granular material as substratum	and lead to misinterpretation	Potential detachment of the
			biofilm from the substratum
			during the fixation steps

SEM, scanning electron microscopy; STEM, scanning transmission electron microscopy; TEM, transmission electron microscopy; NP, nanoparticle; EDS, X-ray spectrometry; EELS, electron energy loss spectroscopy.

ACKNOWLEDGMENTS

This work is in the projects BIOMEB and BIOSORP supported by BRGM Research Direction in collaboration with project NANOREM (FP7 2007-2013 Grand agreement No. 309517). The authors gratefully acknowledge the financial support provided to the PIVOTS project by the Région Centre-Val de Loire (ARD 2020 program and CPER 2015-2020) and the French Ministry of Higher Education and Research (CPER 2015-2020 and public service subsidy to Brgm). Cryo-SEM (Hitachi) and TEM (CM20) observations were performed at the Electron Microscopy Center at the University of Orleans (France) by A.R. and A.S.. TEM and STEM preparation were carried out by MRic TEM Biosit electron microscopy platform at the University of Rennes (France). TEM /EDS/EELS (Jeol ARM200F) analysis were performed at the Centre Raymond Castaing (Toulouse, France) and at the demonstration center of JEOL Ltd (Tokyo, Japan).


B Jodar

D Loison

Y Yokoyama

E Lescoute

M Nivard

L Berthe

J.-C Sangleboeuf

Localized Atomic Segregation in the Spalled Area of a Zr 50 Cu 40 Al 10 Bulk Metallic Glasses Induced by Laser-shock Experiment

Laser-shock experiments were performed on a ternary Zr 50 Cu 40 Al 10 Bulk Metallic Glass. Spalling process was studied through post-mortem analyses conducted on recovered sample and spall. Scanning Electron Microscopy magnification of fracture surfaces revealed the presence of a peculiar feature known as cup-cone. Cups are found on sample fracture surface while cones are observed on spall. Two distinct regions can be observed on cups and cones: a smooth viscous-like in the center and a flat one with large vein-pattern in periphery. Energie Dispersive Spectroscopy measurements conducted on these features emphasized atomic distribution discrepancies both on sample and spall. We propose a mechanism for the initiation and the growth of these features but also a process for atomic segregation during spallation. Cup and cones would originate from cracks arising from shear bands formation (softened paths). These shear bands result from a quadrupolarshaped atomic disorder engendered around an initiation site by shock wave propagation. This disorder turns into shear band when tensile front reaches spallation plane. During the separation process, temperature gain induced by shock wave and shear bands generation decreases material viscosity leading to higher atomic mobility. Once in a liquid-like form, atomic clusters migrate and segregate due to inertial effects originating from particle velocity variation (interaction of release waves). As a result, a high rate of copper is found in sample cups and high zirconium concentration is found on spall cones.

I. INTRODUCTION

Unique and intriguing properties of amorphous alloys have nourished several scientific minds during the past decades. Since their discovery in 1960 by Klement et al [1] and their development in bulk glass-form [2], extensive works have been provided on their synthesis, glass forming ability and structure design [3] related to various icosahedral configurations distributions into an amorphous medium depending on the composition. Regarding the mechanical behavior, substantial studies were reported on deformation mechanisms based mostly on the free volume and Shear Transformation Zone (STZ) theories. Cao et al [22] used molecular dynamics to correlate the evolution of icosahedral structures under quasistatic loading conditions of a binary Cu-Zr Bulk Metallic Glass (BMG) to the localized shear flow process. Maas et al studied experimentally at low strain rates (10 -3 s -1 ) the influence of temperature on the shear bands propagation [23] and velocity [24] to determine how the local structure, that is very temperature sensitive, governs the shear band formation and propagation. Thurneer et al [25] performed compression at strain rate between 10 -4 s -1 and 10 -3 s -1 experiments on several compositions of a ternary ZrCuAl BMG at various sub-ambient temperatures to measure the activation energy for shear band propagation and determine how it is linked to atomic bonding. In contrast, only few studies were published on the dynamic behavior of BMG. Few equations of states (EOS) and Hugoniot curves were reported by Martin et al on a Zr 57 N b 5 Cu 15.4 N i 12.6 Al 10 using single and bi-stage gas guns for pressures up to 122 GP a [8], by Togo et al on a Zr 55 Al 10 N i 5 Cu 30 also by plate-impact experiments for pressure range lower than 45 GP a [9] and Xi et al on a Zr 51 T i 5 N i 10 Cu 25 Al 9 up to 110 GP a [10].

From these studies, BMG revealed several phases transitions that are composition dependent. The dynamic elasto-plastic transition was previously studied by Turneaure et al on a Zr 56.7 Cu 15.3 N i 12.5 N b 5.0 Al 10.0 Y 0.5 [11,12] were the Hugoniot Elastic Limit (HEL) was measured to be around 7 GP a. The shock wave instability related to pressure just above the HEL highlighted a strain-softening response that is well known in static to be a characteristic response upon loadings exceeding the elastic domain. Smirnov 4). Behind shock front, motion is provided to matter. When arriving at free-surface, shock wave turns into releases waves and pressure drops by the interaction of incident and reflected release waves. Subsequent strain and stress states are no longer compressed but tensed, directly impacting strain rate and inducing loading asymmetry within specimen. Matter separation (spalling) occurs when the dynamic tensile threshold of the material is reached, resulting in the ejection of matter (spall) (6).

for pressures range around the elasto-plastic transition to determine how some icosahedral configurations contribute to strain-softening [13] The dynamic tensile limits with strain rate in range of 10 4 s -1 to 10 6 s -1 were also reported to be in range of 2 -4 GP a by several authors [16,18,19] for various Zr-based BMG depending on the compositions. Spalling process induced by planar shock and resulting fracture surfaces were also studied. Zhuang et al [16] and Yang et al [17] performed Scanning Electron Microscopy on recovered target that showed shear band on the cross section and vein-pattern on the fracture surface. Light emission was observed by Sun et al during spallation [20]. Escobedo et al [19] and Lu et al [21] also reported vein-pattern on fracture surface that also contained cup-cones formations.

For decades, laser-shock has been used to study spallation in metals [26][27][28] and glass [29] but also to study the mixed-mod shock wave loading at high strain rate on polymers [30]. Loadings induced by nanoseconds laser plasma can be reached up to 10 2 GP a with high strain rate (10 6 to 10 9 s -1 ). This technique with soft low density gel (∼ 0.9 g.cm -3 ) allows sample and spall recovery for post-mortem analysis [31].The generated loading shape is triangular (1) as illustrated in Fig. 1. When reaching the rear surface, the shock wave is reflected into a release wave (5). The meeting of the incident and reflected releases engenders an inverted pressure front. Ahead of this front, the specimen is still under compression, while tension is observed behind (close to the free surface). It is essential to notice that the strain rate in compression differs from the one in tension, due to release waves spread. Once the dynamic tensile threshold of the material is reached, spallation occurs, leading to matter separation and ejection (6). Finally, to avoid edge effects in laser shock induced spallation, thin samples are machined to ensure uniaxial strain state.

In this paper, we study spallation at high strain rates and high pressures of a Zr 50 Cu 40 Al 10 BMG using shock produced by laser. Especially, this paper deals with the formation of cupcone features observed on recovered sample and spall. A discussion focuses on atomic segregation phenomena observed in fracture areas. First part introduces the experimental setup and methods. The second part reports post-mortem observations and measurements.

In the last part, results are discussed.

II. EXPERIMENTAL SETUP

A single master ingot of ternary Zr 50 Cu 40 Al 10 BMG was prepared by arc-melting mixtures of high purity Zr, Cu and Al under an argon atmosphere. The Zr crystal rod used here contains an oxygen concentration less than 0.05 atomic percent to avoid crystallization during casting process. The master ingot was cast using the tilt-casting method [32] into a 10 mm diameter rod and 45 mm long. As reported by Yokoyama et al [32], this technique reduces the formation of cold shuts that act as crack-initiation sites. According to initial properties, the density of the Zr 50 Cu 40 Al 10 BMG rod was measured by the Archimedes method using purified water and was found to be 6.809 ± 0.006 g.cm -3 . The cast structure was also controlled by Optical Microscopy (OM) and Scanning Electron Microscopy (SEM) and the phase was characterized by X-ray diffractometry exhibiting no Bragg peaks of any crystalline phase. The ultrasonic echography technique was used to obtain Poisson ratios, Young's and shear modulus which were found to be correspondingly equal to 0.369 ± 0.02, 89.3 ± 1 GP a and 32.6 ± 1 GP a. The final target was cut from this single master ingot and polished into a 215 µm thickness slice with a 1 µm flatness controlled by confocal FIG. 2. Photography of the sample holder with the soft recovery device used for collecting ejectas.

The complete system is set in a secondary vacuum chamber (10 -3 P a is achieved) to avoid laser breakdown with air. In this situation, laser is irradiating the front face of the sample (not visible here) from the right.

profilometry.

Laser shock experiments were performed on the LULI2000 facility (UMR 7605, Ecole Polytechnique, Palaiseau, France). Laser provides a 4.6 ns duration high power square pulse at 1.06 µm-wavelength focused onto the specimen surface (spot diameter of 2.9 mm).

Samples are set in a secondary vacuum chamber (10 -3 P a is achieved) to avoid laser breakdown with air. Provided energy is 800 J maximum resulting in laser intensities up to 2.60 T W .cm -2 . Spalls ejected from target free-surface were collected into a soft recovery device.

The experimental setup is displayed in Fig.

III. RESULTS

In the Fig. 3 are displayed the typical central spalled area of a 215 µm thick sample (a) irradiated by a 2.45 T W .cm -2 intensity equivalent to a ∼ 60 GP a pressure obtained by laser-matter interaction simulations using one dimensional Lagrangian code ESTHER [33]. Its corresponding recovered spall is showed in Fig. 4 (a). First, one may distinguish on both surfaces the presence of multiple objects, referred as cup-cones. Several authors also reported such characteristic pattern in case of plate-impact experiments [19] in a Zr 56.7 Cu 15.3 N i 12.5 N b 5.0 Al 10.0 Y 0.5 and [21] in a Zr 50 Cu 40 Al 10 BMG. Main differences between these studies and present one regard the number, the size, the morphologies but also the location of cup-cones on spall or sample. Reported cup-cones diameters were on average ten times larger and ten time less numerous for a same area than the one showed here. Lu et al [21] reported smoother cup and cones surfaces. Recovered spall and sample surfaces differ one another respectively from the presence of cups on sample and cones on spall. However, in previous studies spalls were not recovered so that direct confrontation is not possible.

At cup-cones scale, cups emphasize a smooth and viscous-like central region (Fig. 3 (b) and (c), circles in yellow lines). Flat peripheral rings with large vein-pattern can be distinguished (Fig. 3 (b) and (c), circles in blue lines). This vein-pattern is usually observed on fracture surfaces in metallic glasses and results from instabilities in the liquid-like layer that forms during shear banding [5]. Small molten droplets can also be observed (Fig. 3 (c)) mostly in the outlying region, suggesting viscosity drop (or melting). On the contrary, cones appear clearly grainier in the central area (Fig. 4 (b), yellow circle) with fewer or no droplets. The characteristic vein pattern is here thinner than observed on cups central area. Outlying rings with large vein-patterns are sometimes observed on the bottom of cones (Fig. 4 (b), blue circle). According to Deibler et al [34], these various vein pattern sizes can be attributed to a viscosity discrepancy, where a large pattern is synonym of low viscosity, and thin pattern size to high viscosity. This apparent viscosity difference may be explained by Pampillo work [35] that provided evidences of structural disordering effect within shear bands. Most specifically, it suggested that chemical potential may be altered relatively to the bulk within the bands. In addition, it was reported by Leamy et al [36] that the flow and fracture surface process of metallic glasses in quasi-static tension show a smooth and a veined regions, similar to the two distinct areas observed both in cups and cones.

At atomic scale, EDS measurements on surfaces of cups (Fig. 5 By taking account of EDS accuracy (±5%), cups appear rich in copper (Zr: ∼25%, Cu: ∼70%, Al: ∼5%) while cones are rich in zirconium (Zr: ∼70%, Cu: ∼25%, Al: ∼5%). Once outside of these features, atomic content tend to the initial rate, as depicted by Fig. 5 (e). Magnifications of probed cup and cone are depicted by red lines in (c) and (d) respectively. The atomic distribution of Zr, Cu and Al depending on the position is displayed for a cup (e) and a cone (f). Cups seem to be rich in copper and poor in zirconium. Its the opposite for cones that are rich in Zr and lack of Cu. Aluminum content remains approximatively constant. Outside of the cup-cone feature, the initial composition rate is found.

IV. DISCUSSION

In metallic glasses, several studies with quasi-static loadings have provided key elements relative to its plasticity and were regrouped as a deformation map proposed by Schuh et al [4]. It is now well established that metallic glasses exhibit two regimes of plastic flows: homogeneous at high temperature and low strain rate, inhomogeneous at room temperature and high strain rate that is associated with shear bands. In this last domain, plasticity and yielding are controlled by the initiation, the propagation and the evolution of shear bands that usually lead to failure.

In the present study, i.e. in a dynamic loading situation, strain rate is very high and temperature remains an opened question. Nonetheless, according to shock wave nature (that is strongly discontinuous), it is presumably reasonable to consider that plastic flow under shock is inhomogeneous. This in mind, initiation and propagation of cup-cones correlated with a process for atomic segregation will be discussed according to the shock wave theory but also results reported by quasi-static studies on metallic glasses.

A. Cup-Cone initiation and propagation

If one considers a peculiar site from which plasticity can be initiated Fig. 6 (1), when the shock wave (schematized by the blue front traveling from left to right) interacts with this site, a surrounding displacement field is induced. This local atomic displacement is larger than the one of the surrounding matrix leading to local shearing. This region of local atomic displacement and shearing has been referred in the literature as Shear Transformation [37] or Shear Transformation Zone (STZ) [38] and is assumed to be responsible for plasticity triggering. Experimental [39] and numerical [40] studies emphasized the quadrupolar character of the created strain field (red areas in Fig. 6) which decays with the distance from the STZ center to the surrounding matrix (Fig. 6 (1)). Greer et al [5] proposed a two-stage scenario for shear band initiation. Once a STZ is activated (first stage) the atomic structure is disordered and atoms gain mobility.

At that point, shear strain is small and temperature raise not substantial. Hence, it forms a softened path for sliding and shear to concentrate (second stage). Since matter is softened along a privileged path, large plastic strain accumulates and localizes to form a shear band.

From this localization, significant local heating results thanks to thermodynamics equation.

In the present case, an initiation site is firstly activated by the arrival of the compression front and it engenders atomic disorder in a quadrupolar way, as is illustrated by steps (1)→( 2) in Fig. 6. Since there is no clear evidence that cup-cones originate from STZ triggering, we rather consider any sort of sites from which shear strain may concentrate.

The shock-and release waves rarefaction turning the compression into tension triggers the second stage where shear bands initiates (3)→(4). Once the spallation plane is reached, shear 2), shock wave induces a displacement field that disorders atomic structure locally and activates an initiation site. Matter is softened along a band. After rarefaction, tension occurs and large plastic strain accumulates within disordered band and engenders the formation of shear bands (3)→4). At spallation plane, matter starts sliding and crack follow softened paths resulting from shear bands complete formation (5). Then a cup and cone feature is created. During sliding, temperature evolution modifies viscosity within bands so that two distinct areas can be observed on fracture surface: smooth and vein-pattern (6).

bands on the right are fully formed and sliding begins (5). Finally, when crack initiates, it propagates along softened paths described by fully developed shear bands on the right side. In addition, in the central spot area the strain state is uniaxial (due to initial loading conditions). From this uniaxial strain state and the quadrupolar form of shear concentration results the formation of cups on samples and cones on spall. According to smooth and veinpattern areas observed both on cups and cones, one may consider the temperature evolution during sliding. While sliding, temperature may rise until possibly reaching melting point.

At early stage of sliding, temperature is lower (high viscosity) than at later stages (low viscosity). Therefore, it conducts to the formation of two distinct areas on cup and cones fracture surface.

B. Atomic segregation process

Atomic segregation pointed out by EDS measurements may be the result of a segregation that takes place during shear banding events. Thermodynamically, shock wave is considered as an adiabatic transformation that induces a jump of pressure and a decrease in volume resulting in a gain of free energy and temperature. Then localized plasticity responsible for cups and cones formation from initiation sites (metallurgical defect, free volume or STZ) also contribute to temperature and free energy elevation. This temperature rise (or free energy) is responsible for the drop of viscosity (glass transition or melting) allowing atomic mobility within a softened local neighborhood. Once the matter is locally in a liquid-like state along with the particle motion provided by shock wave, atoms could migrate. Since our material is made of various compounds possessing different masses (Zr: 91u, Cu: 63u, Al: 27u) inertial effects could emerge.

For an identical particle velocity, Zr atoms possess more kinetics energy than Cu and Al.

Hence, segregation would take place during a phase of velocity decrease, where Cu and Al atoms would decelerate more rapidly than Zr atoms. To understand how such a scenario could happen, one may refer to Fig. 7 illustrating the variation of pressure and particle velocity during reflection and overlapping.

In early stages, matter is being compressed by the shock wave propagation that induces pressure, temperature and velocity jump. Once shock wave reaches the free-surface, reflection occurs and the particle velocity rises (more or less doubled) while pressure drops to zero and temperature decreases (but stay higher than initial one). By overlapping at spallation plane, incident and reflected releases modify substantially particle velocity. Hence, the particle velocity increases at a velocity u t1 and tensile stresses result from overlapping.

Assuming a stress level above a tensile threshold σ T (relative to plasticity activation, i.e. shear band initiation), localization and viscosity drop take place soon after t 1 and promote migration. Taking account of hydrodynamic damping and release waves spreading, particle velocity decreases to u t2 at t 2 . As a result, the particle velocity u t2 at t 2 is below u t1 at t 1 .

In short, after reaching σ T the current viscosity drops (cf section IV A) and particle velocity rises to u t1 resulting in a motion of atomic clusters. A little while after, particle velocity decreases to u t2 and inertial effects forecast to happen due to atomic mass differences: Cu and Al are slowed and Zr atoms continue on their path. This hypothesis is sufficient to understand why high Zr and Cu rate are respectively found on spall and sample fracture surfaces but not enough to explain aluminum constant rate on both surfaces.

According to Molecular Dynamic simulations of Cheng et al [41] on a Zr 47 Cu 46 Al 7 showing Partial Differential Functions (PDF) for these compounds, Cu-Al and Zr-Al are the strongest bonds. Then Cu-Zr, Cu-Cu and finally Zr-Zr bonds are the weakest. Assuming that Cu-Al and Zr-Al are highly energetic compared to other bonds and that cracks propagate along paths minimizing energy, one may suggest that during separation crack would neglect as much as possible these bonds areas. Thus, only Cu-Cu, Zr-Zr and Cu-Zr bonds remain. Since Al atoms are the lighter but also strongly bonded to Cu and Zr, our results seem to suggest that crack split mostly Cu-Zr bonds during or after the segregation phenomenon.

As regards morphology, the composition segregation is possibly responsible for the cups and cones aspects. Zirconium possesses the highest melting temperature of the three: T Zr = 2130 K, T Cu = 1350 K, T Al = 933 K. Furthermore, Yokoyama et al [42] showed that Zr-rich ternary ZrCuAl (for a fixed value of Al) possess highest melting temperatures. Hence for a fixed temperature, areas highly concentrated in zirconium will show a higher viscosity than Cu-rich areas. This is clearly in agreement with our observation but also with the correlation of vein pattern size and viscosity level exposed by Deibler et al [34].

V. CONCLUSION

In summary, spalling process induced by laser has been studied on a Zr 50 Cu 40 Al 10 BMG.

Post-mortem analyses were conducted on recovered sample and spall. Scanning Electron Microscopy images revealed the presence of cups (on sample) and cones (on spall) features with various morphological aspects. Two distinct regions can be observed both on cups and cones: a smooth viscous-like in the center and a flat one with large vein-pattern in periphery.

Energy Dispersive Spectroscopy measurements performed on fracture surface exhibited an atomic segregation that could take place during spallation. Cups appear rich in copper while cones are rich in zirconium. Outside of these features the initial composition rate is found. Atomic migration and segregation took place at spallation plane into the shear band initiation. On one hand, viscosity drop resulting from temperature increase and atomic disorder would favor atomic mobility. On the other hand, shock-and release waves propagation and interaction engender particle velocity variations. These two aspects promote atomic segregation through inertial effects.

The initiation and the propagation of cups and cones result from shear bands initiation and asymmetrical loading conditions at spalling plane induced by shock wave rarefaction. shear bands initiation originates from a quadrupolar-shaped atomic disorder firstly engendered by the compression front that would turn into shear bands when tensile front reaches spallation plane. During the separation process, crack arises from shear bands (softened paths) and creates cups and cones features.

  FIG. 1. Illustration of a shock wave propagation. Once generated, shock waves induced by laser become triangular (1). During propagation, release waves spread and peak pressure decays with travelled distance (1)→(4). Behind shock front, motion is provided to matter. When arriving at

  2. Post-mortem observations and analysis were performed on recovered fracture surfaces both on sample and spall by a Scanning Electron Microscopy (SEM) and atomic content was probed by Energy Dispersive Spectroscopy (EDS) on the ScanMat facility (UMS 2001, Rennes, France). Atomic distribution profiles were established in several positions, i.e. in the form of measurements lines made of 200 points with a spatial resolution of 1 µm 3 for each points, to follow the evolution of atomic distribution according to position in cup and cones.

FIG. 3 .

 3 FIG. 3. SEM pictures of recovered sample. Magnifications in picture (a) (squares in red line) reveal a complex fracture surface with cups. Pictures (b) and (c) exhibit two distinct areas on cups: a smooth central area (yellow circle) and a flat outlying one (blue circle). On magnification (c) melted droplets can be distinguished.

FIG. 4 .

 4 FIG. 4. SEM images of recovered spall. Fracture surface is here made of multiple cones (a). Magnification of image (a) (red square) also emphasizes in picture (b) two distinct regions on cones (similarly to cups). The smooth central area is depicted by the yellow circle and the flat area by the blue circle.

  (a) -red line) and cones (Fig. 5 (b) -red line) revealed atomic composition discrepancies in comparison with original one (Zr: 50%, Cu: 40%, Al: 10%). Altogether, 50 cups and 50 cones were probed. According to position, atomic distribution in cups (Fig. 5 (e)) and cones (Fig. 5 (f)) varies strongly.

FIG. 5 .

 5 FIG. 5. EDS measurements of atomic distribution on cup-cones fracture surface. SEM magnifications of sample cups (a) cones on recovered spall (b) with the probed positions (red lines).

FIG. 6 .

 6 FIG. 6. Schematic representation of sites initiation kinetic during shock wave propagation and reflection leading to cup-cone pattern generation. In compression phase (1)→(2), shock wave

FIG. 7 .

 7 FIG. 7. Illustrations of shock wave propagation and reflection inducing pressure and particle velocity changes. In schema (a) are plotted particle velocity and pressure versus position. In schema (b) is plotted the particle velocity at spall plane during time.

VI. ACKNOWLEDGMENTS

The authors express their gratitude to all the LULI and SCANMAT staffs for technical support. The access to the LULI facility was provided through the Institute Laser Plasma (ILP, FR2707). This work was supported by DGA and ARED grants.