Imaging of Vanadium in Microfossils: A New Potential Biosignature.

The inability to unambiguously distinguish the biogenicity of microfossil-like structures in the ancient rock record is a fundamental predicament facing Archean paleobiologists and astrobiologists. Therefore, novel methods for discriminating biological from nonbiological chemistries of microfossil-like structures are of the utmost importance in the search for evidence of early life on Earth. This, too, is important for the search for life on Mars by in situ analyses via rovers or sample return missions for future analysis here on Earth. Here, we report the application of synchrotron X-ray fluorescence imaging of vanadium, within thermally altered organic-walled microfossils of bona fide biological origin. From our data, we demonstrate that vanadium is present within microfossils of undisputable biological origin. It is well known in the organic geochemistry literature that elements such as vanadium are enriched and contained within crude oils, asphalts, and black shales that have been formed by diagenesis of biological organic material. It has been demonstrated that the origin of vanadium is due to the diagenetic alteration of precursor chlorophyll and heme porphyrin pigment compounds from living organisms. We propose that, taken together, microfossil-like morphology, carbonaceous composition, and the presence of vanadium could be used in tandem as a biosignature to ascertain the biogenicity of putative microfossil-like structures. Key Words: Microfossils-Synchrotron micro-X-ray fluorescence-Vanadium-Tetrapyrrole-Biosignature. Astrobiology 17, 1069-1076.

Challenges Analyzing Gypsum on Mars by Raman Spectroscopy.

Raman spectroscopy can provide chemical information about organic and inorganic substances quickly and nondestructively with little to no sample preparation, thus making it an ideal instrument for Mars rover missions. The ESA ExoMars planetary mission scheduled for launch in 2018 will contain a miniaturized Raman spectrometer (RLS) as part of the Pasteur payload operating with a continuous wave (CW) laser emitting at 532 nm. In addition, NASA is independently developing two miniaturized Raman spectrometers for the upcoming Mars 2020 rover mission, one of which is a remote (stand-off) Raman spectrometer that uses a pulse-gated 532 nm excitation system (SuperCam). The other is an in situ Raman spectrometer that employs a CW excitation laser emitting at 248.6 nm (SHERLOC). Recently, it has been shown with analyses by Curiosity that Gale Crater contains significantly elevated concentrations of transition metals such as Cr and Mn. Significantly, these transition metals are known to undergo fluorescence emission in the visible portion of the electromagnetic spectrum. Consequently, samples containing these metals could be problematic for the successful acquisition of fluorescence-free Raman spectra when using a CW 532 nm excitation source. Here, we investigate one analog environment, with a similar mineralogy and sedimentology to that observed in martian environments, as well as elevated Cr contents, to ascertain the best excitation wavelength to successfully collect fluorescence-free spectra from Mars-like samples. Our results clearly show that CW near-infrared laser excitation emitting at 785 nm is better suited to the collection of fluorescence-free Raman spectra than would be a CW laser emitting at 532 nm.

Selection of Portable Spectrometers for Planetary Exploration: A Comparison of 532 nm and 785 nm Raman Spectroscopy of Reduced Carbon in Archean Cherts.

Knowledge and understanding of the martian environment has advanced greatly over the past two decades, beginning with NASA's return to the surface of Mars with the Pathfinder mission and its rover Sojourner in 1997 and continuing today with data being returned by the Curiosity rover. Reduced carbon, however, is yet to be detected on the martian surface, despite its abundance in meteorites originating from the planet. If carbon is detected on Mars, it could be a remnant of extinct life, although an abiotic source is much more likely. If the latter is the case, environmental carbonaceous material would still provide a source of carbon that could be utilized by microbial life for biochemical synthesis and could therefore act as a marker for potential habitats, indicating regions that should be investigated further. For this reason, the detection and characterization of reduced or organic carbon is a top priority for both the ESA/Roscosmos ExoMars rover, currently due for launch in 2018, and for NASA's Mars 2020 mission. Here, we present a Raman spectroscopic study of Archean chert Mars analog samples from the Pilbara Craton, Western Australia. Raman spectra were acquired with a flight-representative 532 nm instrument and a 785 nm instrument with similar operating parameters. Reduced carbon was successfully detected with both instruments; however, its Raman bands were detected more readily with 785 nm excitation, and the corresponding spectra exhibited superior signal-to-noise ratios and reduced background levels.

Raman spectroscopy as a screening tool for ancient life detection on Mars.

The search for sp(2)-bonded carbonaceous material is one of the major life detection strategies of the astrobiological exploration programmes of National Aeronautics and Space Administration and European Space Agency (ESA). The ESA ExoMars rover scheduled for launch in 2018 will include a Raman spectrometer with the goal of detecting sp(2)-bonded carbonaceous material as potential evidence of ancient life. However, sp(2)-bonded carbonaceous material will yield the same Raman spectra of well-developed G and D bands whether they are synthesized biologically or non-biologically. Therefore, the origin and source of sp(2)-bonded carbonaceous material cannot be elucidated by Raman spectroscopy alone. Here, we report the combined approach of Raman spectroscopy and gas chromatography-mass spectrometry biomarker analysis to Precambrian sedimentary rocks, which taken together, provides a promising new methodology for readily detecting and rapidly screening samples for immature organic material amenable to successful biomarker analysis.

Raman hyperspectral imaging of microfossils: potential pitfalls.

Initially, Raman spectroscopy was a specialized technique used by vibrational spectroscopists; however, due to rapid advancements in instrumentation and imaging techniques over the last few decades, Raman spectrometers are widely available at many institutions, allowing Raman spectroscopy to become a widespread analytical tool in mineralogy and other geological sciences. Hyperspectral imaging, in particular, has become popular due to the fact that Raman spectroscopy can quickly delineate crystallographic and compositional differences in 2-D and 3-D at the micron scale. Although this rapid growth of applications to the Earth sciences has provided great insight across the geological sciences, the ease of application as the instruments become increasingly automated combined with nonspecialists using this techique has resulted in the propagation of errors and misunderstandings throughout the field. For example, the literature now includes misassigned vibration modes, inappropriate spectral processing techniques, confocal depth of laser penetration incorrectly estimated into opaque crystalline solids, and a misconstrued understanding of the anisotropic nature of sp² carbons.

Field-based Raman spectroscopic analyses of an Ordovician stromatolite.

Raman spectrometers are being miniaturized for future life-detection missions on Mars. Field-portable Raman spectrometers, which have similar spectral parameters to the instruments being developed for Mars rovers, have been used to examine extant biosignatures, but they have not yet been used to examine ancient biosignatures. Here, a portable Raman spectrometer was used to analyze an Ordovician stromatolite at the outcrop, revealing both its mineralogy and the presence of sp² carbonaceous material. As stromatolites are often used as proof of the presence of life in Archean rocks and are searched for on Mars, the ability to analyze them in the field with no sample preparation has important ramifications for future Mars missions. However, these results also reveal that a 785 nm excitation source, rather than the 532 nm excitation source planned for future missions, might be a better choice in the search for fossil biosignatures.

Raman spectroscopic documentation of Oligocene bladder stone.

Discovery of a fossil (30-35 million-year-old) urolith from Early Oligocene deposits in northeastern Colorado provides the earliest evidence for the antiquity of bladder stones. These are spherical objects with a layered phosphatic structure and a hollow center. Each layer is composed of parallel crystals oriented perpendicular to the surface. Macroscopic and microscopic examination and X-ray diffraction analysis, along with comparison with 1,000 contemporary uroliths, were used as evidence in the confirmation of this diagnosis. Raman microspectroscopy verified the presence of organic material between layers, confirming its biologic origin.

Multiple generations of carbon in the apex chert and implications for preservation of microfossils.

While the Apex chert is one of the most well-studied Archean deposits on Earth, its formation history is still not fully understood. Here, we present Raman spectroscopic data collected on the carbonaceous material (CM) present within the matrix of the Apex chert. These data, collected within a paragenetic framework, reveal two different phases of CM deposited within separate phases of quartz matrix. These multiple generations of CM illustrate the difficulty of searching for signs of life in these rocks and, by extension, in other Archean sequences.

Hematite and carbonaceous materials in geological samples: a cautionary tale.

Over the last few decades Raman spectroscopy has been increasingly applied as an analytical tool in geoscience research. Raman spectroscopy is a powerful tool for geologists as it is non-destructive, requires little to no sample preparation, and can be undertaken in situ on various irreplaceable geological samples. Also, this technique is useful in the identification of minerals and geo-organic material. However, despite this ease of application, there are some facets of Raman spectroscopy data that can lead to erroneous interpretations. For instance, there is much confusion in the geological literature distinguishing the difference between the hematite vibrational mode at ca. 1320 cm(-1) and the disordered sp(2) carbonaceous material D band at 1340 cm(-1). Furthermore, geologists will often collect 2 spectra, one in the mineral finger print region (200-800 cm(-1)) and then a spectrum in the carbon first-order region (1000-1800 cm(-1)), rather than performing a full-region scan. This allows the misidentification of the hematite mode at 1320 cm(-1) as the D band from disordered carbonaceous material. Here we show that it is best practice for geologists to collect spectra between 200 and 1800 cm(-1) to better distinguish between hematite and disordered carbonaceous material, materials that often co-occur in geological samples.

The potential of Raman spectroscopy for the analysis of diagenetically transformed carotenoids.

Recently, carotenoids have received much attention as target compounds for astrobiological prospecting principally because they are a group of molecules that display unique diagnostic Raman spectra that can be assigned to organic material of unequivocal biological origin. However, no work has been performed on assessing the potential of Raman spectroscopic detection of carotenoids from fossilized microbes. Here, we report the first Raman spectra acquired from 'perhydro' derivatives of beta-carotene and lycopene formed by hydrogenation of the polyene chain during diagenesis, resulting in much less specific fossil hydrocarbons such as beta-carotane and lycopane, respectively. We propose here that diagenetically altered carotenoids formed by hydrogenation reactions during the fossilization processes also provide unique diagnostic spectra that can be interpreted as a biological signature.

Silicon nitride as a versatile growth substrate for microspectroscopic imaging and mapping of individual cells.

Herein is described a general sampling protocol that includes culture, differentiation and fixing of cells in their preferred morphology on the one sample substrate (Si(3)N(4)) to enable subsequent diverse modern microspectroscopic analyses. The protocol enables unprecedented correlated and complementary information on the intracellular biochemistry of metabolic processes, diseases and their treatment, which offers the opportunity to revolutionize our understanding of cell and tissue biology at a molecular level. The culture of adherent cells onto inexpensive Si(3)N(4) membranes allows microspectroscopic analyses across the electromagnetic spectrum, from hard X-ray fluorescence (both XRF and XANES), through to visible and fluorescence light microscopies, and infrared microspectroscopy without substrate interference. Adherent mammalian cell lines (3T3-L1 adipocytes and H9c2 cardiac myocytes) illustrate the in vitro application of these protocols. The cells adhered strongly to Si(3)N(4) membranes and visually displayed normal proliferative and phenotypic growth; more importantly, rapid alcohol fixation of cells did not affect their structural integrity for subsequent analyses.

Understanding the application of Raman spectroscopy to the detection of traces of life.

Investigating carbonaceous microstructures and material in Earth's oldest sedimentary rocks is an essential part of tracing the origins of life on our planet; furthermore, it is important for developing techniques to search for traces of life on other planets, for example, Mars. NASA and ESA are considering the adoption of miniaturized Raman spectrometers for inclusion in suites of analytical instrumentation to be placed on robotic landers on Mars in the near future to search for fossil or extant biomolecules. Recently, Raman spectroscopy has been used to infer a biological origin of putative carbonaceous microfossils in Early Archean rocks. However, it has been demonstrated that the spectral signature obtained from kerogen (of known biological origin) is similar to spectra obtained from many poorly ordered carbonaceous materials that arise through abiotic processes. Yet there is still confusion in the literature as to whether the Raman spectroscopy of carbonaceous materials can indeed delineate a signature of ancient life. Despite the similar nature in spectra, rigorous structural interrogation between the thermal alteration products of biological and nonbiological organic materials has not been undertaken. Therefore, we propose a new way forward by investigating the second derivative, deconvolution, and chemometrics of the carbon first-order spectra to build a database of structural parameters that may yield distinguishable characteristics between biogenic and abiogenic carbonaceous material. To place Raman spectroscopy as a technique to delineate a biological origin for samples in context, we will discuss what is currently accepted as a spectral signature for life; review Raman spectroscopy of carbonaceous material; and provide a historical overview of Raman spectroscopy applied to Archean carbonaceous materials, interpretations of the origin of the ancient carbonaceous material, and a future way forward for Raman spectroscopy.

Organic-walled microfossils in 3.2-billion-year-old shallow-marine siliciclastic deposits.

Although the notion of an early origin and diversification of life on Earth during the Archaean eon has received increasing support in geochemical, sedimentological and palaeontological evidence, ambiguities and controversies persist regarding the biogenicity and syngeneity of the record older than Late Archaean. Non-biological processes are known to produce morphologies similar to some microfossils, and hydrothermal fluids have the potential to produce abiotic organic compounds with depleted carbon isotope values, making it difficult to establish unambiguous traces of life. Here we report the discovery of a population of large (up to about 300 mum in diameter) carbonaceous spheroidal microstructures in Mesoarchaean shales and siltstones of the Moodies Group, South Africa, the Earth's oldest siliciclastic alluvial to tidal-estuarine deposits. These microstructures are interpreted as organic-walled microfossils on the basis of petrographic and geochemical evidence for their endogenicity and syngeneity, their carbonaceous composition, cellular morphology and ultrastructure, occurrence in populations, taphonomic features of soft wall deformation, and the geological context plausible for life, as well as a lack of abiotic explanation falsifying a biological origin. These are the oldest and largest Archaean organic-walled spheroidal microfossils reported so far. Our observations suggest that relatively large microorganisms cohabited with earlier reported benthic microbial mats in the photic zone of marginal marine siliciclastic environments 3.2 billion years ago.

Carotenoid analysis of halophilic archaea by resonance Raman spectroscopy.

Recently, halite and sulfate evaporate rocks have been discovered on Mars by the NASA rovers, Spirit and Opportunity. It is reasonable to propose that halophilic microorganisms could have potentially flourished in these settings. If so, biomolecules found in microorganisms adapted to high salinity and basic pH environments on Earth may be reliable biomarkers for detecting life on Mars. Therefore, we investigated the potential of Resonance Raman (RR) spectroscopy to detect biomarkers derived from microorganisms adapted to hypersaline environments. RR spectra were acquired using 488.0 and 514.5 nm excitation from a variety of halophilic archaea, including Halobacterium salinarum NRC-1, Halococcus morrhuae, and Natrinema pallidum. It was clearly demonstrated that RR spectra enhance the chromophore carotenoid molecules in the cell membrane with respect to the various protein and lipid cellular components. RR spectra acquired from all halophilic archaea investigated contained major features at approximately 1000, 1152, and 1505 cm(-1). The bands at 1505 cm(-1) and 1152 cm(-1) are due to in-phase C=C (nu(1) ) and C-C stretching ( nu(2) ) vibrations of the polyene chain in carotenoids. Additionally, in-plane rocking modes of CH(3) groups attached to the polyene chain coupled with C-C bonds occur in the 1000 cm(-1) region. We also investigated the RR spectral differences between bacterioruberin and bacteriorhodopsin as another potential biomarker for hypersaline environments. By comparison, the RR spectrum acquired from bacteriorhodopsin is much more complex and contains modes that can be divided into four groups: the C=C stretches (1600-1500 cm(-1)), the CCH in-plane rocks (1400-1250 cm(-1)), the C-C stretches (1250-1100 cm(-1)), and the hydrogen out-of-plane wags (1000-700 cm(-1)). RR spectroscopy was shown to be a useful tool for the analysis and remote in situ detection of carotenoids from halophilic archaea without the need for large sample sizes and complicated extractions, which are required by analytical techniques such as high performance liquid chromatography and mass spectrometry.

Stromatolite reef from the Early Archaean era of Australia.

The 3,430-million-year-old Strelley Pool Chert (SPC) (Pilbara Craton, Australia) is a sedimentary rock formation containing laminated structures of probable biological origin (stromatolites). Determining the biogenicity of such ancient fossils is the subject of ongoing debate. However, many obstacles to interpretation of the fossils are overcome in the SPC because of the broad extent, excellent preservation and morphological variety of its stromatolitic outcrops--which provide comprehensive palaeontological information on a scale exceeding other rocks of such age. Here we present a multi-kilometre-scale palaeontological and palaeoenvironmental study of the SPC, in which we identify seven stromatolite morphotypes--many previously undiscovered--in different parts of a peritidal carbonate platform. We undertake the first morphotype-specific analysis of the structures within their palaeoenvironment and refute contemporary abiogenic hypotheses for their formation. Finally, we argue that the diversity, complexity and environmental associations of the stromatolites describe patterns that--in similar settings throughout Earth's history--reflect the presence of organisms.