Electron microscopy

In electron microscopy a high-energy, low-wavelength, accelerated electrons beam interacts with matter. Some of those interactions results into scattered or transmitted electrons which can be collected to form an image of the analysed material. As the electrons beam wavelength is many orders of magnitude shorter than that of visible light it is possible to produce images with a resolution power several order greater than what can be obtained in standard optical microscopy, allowing the characterization of details down to the atomic scale.

Furthermore, depending on the emission investigated, electron microscopy can be used to provide insights into the elemental composition, the internal structure and crystallinity of the analyzed sample(Watt, 1997).

In this work three electrons beam based microscopic analyses, electron microprobe, scanning and transmitted electron microscopy, have been applied to the BARB3 chert material. Such techniques have been commonly used on the ancient geological record mainly with the purpose to characterize thin section mineralogy and their microfossils and carbonaceous content (Wacey, 2009; Wacey et al., 2017). However, compared to vibrational spectroscopy, electron microscopic methods are more demanding as they work with solids only and, in most of the cases, require a more invasive sample preparation. Moreover, as mentioned above, they make use of an electron beam which is a high energy, destructive, radiation, especially problematic with concern to amorphous CM, whom structure can be easily altered (Gautam et al., 2005). For this reasons electron beam based analyses should be performed only after the less-destructive in-situ vibrational spectroscopic techniques and applied being aware that primary signatures, especially carbonaceous structures, could be altered permanently.

2.2.3.1 SEM

In scanning electron microscopy a finely focused electron beam is scanned across the surface of an investigated bulk material, the resulting backscattered and secondary electrons are thus collected to produce an image of the analyzed specimen. Specific further equipments

39 can be used to detect X-radiations, cathodoluminescence and other phenomena in order to gain a chemical and elemental distribution information from the investigated sample (Watt, 1997). It results that in-situ topographic and elemental images of the sample surface can be provided having a resolution down to a few microns or even a few nanometres depending on the instrumental capabilities. In ancient life studies SEM associated to energy dispersive X-ray spectrometry (or EDS, which allow a qualitative and semi-quantitative elemental detection within the analyzed volume) has been commonly used as a minero-petrographic tool to investigate the mineralogical context in which possible microbial traces are buried. SEM has been also on HF acid etched silica-rich samples to investigate topographic details of microfossils relieves (Javaux et al., 2004; Wacey, 2009; Agić et al., 2015), however this preparation can easily introduce contamination or alter primary textures and structures. Other thanlimited to the sample surface, with CM and microfossils normally occurring embedded by minerals within the thin sections, another disadvantage of SEM is that, working in a vacuum chamber, the analyzed sample needs to be conductive and thus usually require, especially in the case of highly silicified materials, carbon or gold coating before measurements can be performed. This type of preparation, which in case of carbon coating can contaminate the original CM composition, can be avoided using an environmental SEM (ESEM). In this instrument the sample chamber is kept at a low vacuum allowing the sample surface charging to be suppressed with no need of coating (Reed, 2005).

In this work SEM has been used largely for mineral-petrographic characterization of carbon-rich facies. Further ESEM has been used to map thin sections selected for TEM analyses.

Analytical settings

A selected suite of polished thin section were carbon coated prior to analyses under electrons beam excitation. To preserve them in case of further spectroscopic investigation each section has been only half coated. SEM and EMPA analyses on such prepared material were carried out at Spectrum facilities. Half-coated thin sections were analyzed with a Tescan Vega 3 scanning electron microscope (SEM) coupled with an electron back-scattering detector and an energy dispersive spectrometer (EDS) for elemental mapping and semi-quantitative elemental analyses. The operating conditions were 10 to 14 keV accelerating voltage for imaging and 15 to 17 keV for elemental mapping.

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2.2.3.2 TEM and HRTEM coupled to focused ion beam milling

In transmitted electron microscopy the image is produced after transmitted electrons once the beam has passed through an ultra-thin sample. Objects can be magnified up to 10⁶ times and resolved down to about 0.1 nm, moreover, as for SEM, other induced phenomena can be exploited to perform elemental and crystallographic microanalyses (Watt, 1997;

Egerton, 2005). X-rays can be detected to provide qualitative compositional information in the TEM scanning mode (STEM), furthermore in high resolution transmission electron microscopy (HRTEM), by means of phase contrast imaging, it is possible to produce images of materials at the Ångstrom scale to characterise their crystallinity at the atomic level using magnifications up to 50 million times (Wacey et al., 2017). However, to obtain in-situ data with such a high definition and resolution, an invasive and destructive preparation is required to produce samples having thicknesses in the range between 10 nm to 1 µm which can be traversed by an electrons beam. To realize ultra-thin samples, or at least to thin down some sample areas, preparation include some steps of mechanical thinning followed by less-destructive more precise chemical, electrochemical or ion-beam thinning (Egerton, 2005). Of those latter methods focused ion beam (FIB) milling can be used directly on standard petrographic thin sections to extract ultra-thin foils for TEM and HRTEM analyses(Heaney et al., 2015). In FIB milling a focused beam of gallium ions is used to bombard the sample and remove material at the atomic scale through a sputtering process (Egerton, 2005; Heaney et al., 2015). Such technique is normally combined to SEM facilities allowing nano-scale cutting precision which can be planned and controlled in-situ at specific locations of the thin section through the electrons beam SEM imaging.

Although FIB sample preparation is an invasive and time consuming technique, FIB-TEM and HRFIB-TEM analyses represent important means of characterization of ancient geological materials as they provide in-situ chemical and structural insights at the atomic scale. FIB-TEM (but also FIB-SEM; Westall et al., 2006), have been frequently used to investigate chemically and structurally ancient microfossils previously detected in standard petrographic thin sections(Kempe et al., 2005; Moczydłowska and Willman, 2009; Wacey et al., 2012; Foucher and Westall, 2012). Furthermore the application of HRTEM analyses bears a great value in CM investigation supplying unique in-situ structural information which are complementary to vibrational spectroscopic results. Particularly HRTEM analyses allow the investigation of the CM nanotexture (or nanostructure) which represent the mutual organization in space of its constitutive nanometric polyaromatic C-rich sheets forming the

41 carbonaceous lattice. In HRTEM imaging the profiles of individual polyaromatic planes are visualized as dark fringes and various parameters can be measured including their length, curvature, tortuosity and the inter-layer distance in the basic structural units (BSU) (Olcott Marshall et al., 2014; Apicella et al., 2015; Deldicque et al., 2016). As the carbonaceous nanotexture and lattice characteristics are dependent on the maturational process of the original carbonaceous precursor and its nature, HRTEM represent an important tool in ancient CM studies. A further advantage of CM high resolution studies is the possibility to discriminate minerals in the form of nanoparticles associated to the carbonaceous lattice as those could give insights concerning the original rock protolith and depositional conditions(Rasmussen et al., 2017) or into possible metabolic pathways exploited by microbial communities now represented in the form of carbonaceous remains (Westall et al., 2011).

In this work FIB-TEM and HRTEM microscopy has been used to characterize carbonaceous rich structures for which an hypothetical biological origin has been hypothesised after previous observations through core, optical and vibrational spectroscopic techniques. The process of FIB-TEM sample preparation is illustrated in Fig. 2.3.

FIB preparation of TEM lamellae

Two standard petrographic thin sections, obtained from the BARB3 shallow-platformal ferruginous laminated chert and granular chert (see chapter 4), have been selected for FIB foils extraction. Thin sections have been accurately mapped through optical and ESEM imaging to allow the location of the exact area of interest at the FIB. Optical mapping was performed using the Zeiss Axioplan Microscope equipped with a Nikon HS DS-Vi1 colour camera at BiGeA, while ESEM imaging was carried out at the Centro Interdipardimentale Grandi Strumenti (CIGS) of the University of Modena and Reggio Emilia, Modena, using a Quanta-200 ESEM microscope operating at low-vacuum (≤ 20 Torr) with no need of conductive sample coating.The extraction of six ultra-thin (less than 100 nm to allow also HR analyses)foils bearing carbonaceous structures of interest was performed at the FIB facilities of the Istituto Nazionale di Ricerca Metrologica (INRiM) of Turin, in collaboration with Dr.

Enrico Emanuele. Thin sections were first coated with about 10 nm of gold, than ion cutting was achieved using a Quanta 3D DualBeam microscope equipped with a SEM Field Emission Gun (SEM-FEG), operating between 200V and 30kV with a resolution up to 1.5nm, and a Gallium FIB with a resolution up to 7 nm at 30kV.After trenches milling and foils ion

42 thinning the lamellae where lifted-up from the thin sections and placed on a copper made TEM grid through nanomanipulation. For a review of the FIB foils extraction from thin sections for TEM analyses refer to Wirth (2009).

Analytical settings for TEM and HRTEM analyses

TEM and HRTEM analyses on the six ultra-thin foils were carried out at the Institute for Microelectronics and Microsystems (IMM) of the CNR of Italy in Bologna, in collaboration with Dr. Andrea Parisini. TEM data were obtained using a FEI Tecnai F20 ST transmission electron microscope operating at 200 kV equipped with a Gatan MSC794 CCD camera an EDAX EDS PV9761 SUTW for X-rays EDS spectrometry and a Fischione HAADF detector for STEM. For HRTEM analyses a lower voltage (180 kV) was used to avoid the induction of CM organization by the electron beam energy (Gautam et al., 2005). Measurements where acquired in STEM mode and HR and lamellae were observed before and after short cycles (up to 30 sec) of cleaning with a Fischione 1020 Plasma Cleaner to remove contamination.

43 Figure 2.3 – a)SEM imaging at the FIB facility on a gold coated thin section after the first step of ion milling has been completed. Tranches have been cut around a strip which represent the top of the FIB foil which is going to be later thinned, extracted and positioned on the copper grid. (b) for TEM observation. c) A magnification on the copper grid where it’s possible to observe tiny lamellae which has been extracted from thin sections and positioned on the comb-like structure of the grid for TEM analyses. TEM-FIB foils have a bigger dimension in th eorder of a few micrometer and a thickness of ≤ 100 nm (d).

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Chapter 3

Characterization of the BARB3 carbonaceous-rich

shallow-platformal lithofacies

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