The ExoMars Raman spectrometer and the identification of biogeological spectroscopic signatures using a flight-like prototype

The molecular specificity of Raman spectroscopy provides a powerful tool for the analytical interrogation of mineralogical and many biological specimens. The Raman Laser Spectrometer (RLS) is a compact Raman spectrometer under development for deployment on the Martian surface as part of the forthcoming ESA ExoMars mission. This will be the first Raman instrument deployed in space. The scientific interpretation of the data emerging from such an instrument not only addresses the geological and mineral composition of the specimens but also enables an assessment to be made of organic biomaterials that may be preserved in the planetary geological record. The latter evidence centres on the residual and distinctive chemistry relating to the biological adaptation of the geological matrix that has occurred as a result of extremophilic organisms colonizing suitable geological niches for their survival in environmentally stressed habitats on Mars. These biogeological modifications have been studied terrestrially for Mars analogue sites and consist of both a geological component and residual key organic biomarkers, the recognition of which would be a prime factor in life detection surveys of a planetary surface and subsurface. In this paper, the protocols required for the Raman spectral discrimination of key biogeological features that may be detectable on the Martian planetary surface or subsurface are developed using the UK breadboard (UKBB) instrument. This instrument has been constructed to be functionally equivalent to the RLS flight instrument design in order to evaluate the feasible science return of the instrument which will finally be delivered to Mars. Initial Raman measurements using the UKBB are presented and compared with the performance of a commercial laboratory Raman microscope. The initial measurements reported here demonstrate this flight-like prototype achieves straightforward detection of biological signatures contained in geological matrices with Raman band signal to noise ratios high enough to determine sample composition by inspection and without the need for deconvolution or further processing.     

Raman spectroscopy in astrobiology

Raman spectroscopy is proposed as a valuable analytical technique for planetary exploration because it is sensitive to organic and inorganic compounds and able to unambiguously identify key spectral markers in a mixture of biological and geological components; furthermore, sample manipulation is not required and any size of sample can be studied without chemical or mechanical pretreatment. NASA and ESA are considering the adoption of miniaturised Raman spectrometers for inclusion in suites of analytical instrumentation to be placed on robotic landers on Mars in the near future to search for extinct or extant life signals. In this paper we review the advantages and limitations of Raman spectroscopy for the analysis of complex specimens with relevance to the detection of bio- and geomarkers in extremophilic organisms which are considered to be terrestrial analogues of possible extraterrestial life that could have developed on planetary surfaces.     

Critical evaluation of a handheld Raman spectrometer with near infrared (785nm) excitation for field identification of minerals

Handheld Raman spectrometers (Ahura First Defender XL, Inspector Raman DeltaNu) permit the recording of acceptable and good quality spectra of a large majority of minerals outdoors and on outcrops. Raman spectra of minerals in the current study were obtained using instruments equipped with 785 nm diode lasers. Repetitive measurements carried out under an identical instrumental setup confirmed the reliability of the tested Raman spectrometers. Raman bands are found at correct wavenumber positions within ±3 cm(-1) compared to reference values in the literature. Taking into account several limitations such as the spatial resolution and problems with metallic and black and green minerals handheld Raman spectrometers equipped with 785 nm diode lasers can be applied successfully for the detection of minerals from the majority of classes of the mineralogical system. For the detection of biomarkers and biomolecules using Raman spectroscopy, e.g. for exobiological applications, the near infrared excitation can be considered as a preferred excitation. Areas of potential applications of the actual instruments include all kind of common geoscience work outdoors. Modified Raman systems can be proposed for studies of superficial or subsurface targets for Mars or Lunar investigations.     

Study of sub-mJ-excited laser-induced plasma combined with Raman spectroscopy under Mars atmosphere-simulated conditions

Laser-Induced Breakdown Spectroscopy (LIBS) and Raman spectroscopy are complimentary techniques. LIBS yields elemental information while Raman spectroscopy yields molecular information about a sample, and both share similar instrumentation configurations. The combination of LIBS and Raman spectroscopy in a single instrument for planetary surface exploration has been proposed, however challenges exist for developing a combined instrument. We present LIBS and Raman spectroscopy results obtained using a diode pumped, intracavity doubled, Q-switched, Nd:YLF laser operating at 523 nm, which overcomes some of the difficulties associated with a combined instrument. LIBS spectra were obtained with 170 μJ per pulse at 4 Hz repetition rate in a low pressure Mars-simulated atmosphere and Raman spectra produced with 200 mW at 100 kHz. The Nd:YLF laser is switchable between LIBS and Raman spectroscopy modes only by a change in Q-switch repetition rate. Emissions from Ca, Ca II, Fe, Fe II, Mg, Na, and O atom were identified in the μ-LIBS spectrum of oolithic hematite. Evidence was found for a change in plasma dynamics between 7 and 5 Torr that could be explained as a decrease in plasma temperature and electron density below 5 Torr. This is relevant to future Mars exploration using LIBS as the mean surface pressure on Mars varies from 3.75 to 6 Torr. LIBS plasma dynamics should be carefully evaluated at the pressures that will be encountered at the specific Mars landing site.     

Life in the sabkha: Raman spectroscopy of halotrophic extremophiles of relevance to planetary exploration

The Raman spectroscopic biosignatures of halotrophic cyanobacterial extremophiles from sabkha evaporitic saltpans are reported for the first time and ideas about the possible survival strategies in operation have been forthcoming. The biochemicals produced by the cyanobacteria which colonise the interfaces between large plates of clear selenitic gypsum, halite, and dolomitized calcium carbonates in the centre of the salt pans are identifiably different from those which are produced by benthic cyanobacterial mats colonising the surface of the salt pan edges in the intertidal zone. The prediction that similar geological formations would have been present on early Mars and which could now be underlying the highly peroxidised regolith on the surface of the planet has been confirmed by recent satellite observations from Mars orbit and by localised traverses by robotic surface rovers. The successful adoption of miniaturised Raman spectroscopic instrumentation as part of a scientific package for detection of extant life or biomolecular traces of extinct life on proposed future Mars missions will depend critically on interpretation of data from terrestrial Mars analogues such as sabkhas, of which the current study is an example.     

Raman spectroscopic analysis of minerals and organic molecules of relevance to astrobiology

Characteristic geological features and hydrated minerals recently found on the surface of Mars by the NASA planetary rovers Spirit and Opportunity suggest that a possible biosphere could have once existed there. Analytical instrumentation protocols for the unequivocal detection of biomarkers in suitable geological matrices are critical for future unmanned explorations, including the forthcoming ESA-ExoMars mission scheduled for 2018. Raman spectroscopy is currently a part of the Pasteur instrumentation suite of the ExoMars mission scheduled for 2018 for the remote detection of extant or extinct life signatures in the Martian surface and subsurface. Terrestrial analogues of Martian sites have been identified, and the biogeological modifications incurred as a result of extremophilic survival activity have been studied. Polyaromatic hydrocarbons (PAHs) are recognised as a class of degradation product that occur from biological processes terrestrially. In this work, various concentrations of polyaromatic hydrocarbons in matrices of gypsum, calcite and quartz have been investigated by Raman microspectrometry to determine the lowest detectable organic levels. The studies are conceived in simulation of their potential PAHs identification in geobiological conditions in Martian scenarios. Two laser source wavelengths, namely, 785 and 633 nm, were adopted to excite Raman spectra from the PAHs, which represent degraded carbons and therefore potentially provide a key bimolecular marker of ancient life.     

Biogeological Raman spectroscopic studies of Antarctic lacustrine sediments

Analysis of lacustrine sediments is an accepted method for deciphering the palaeoenvironment of a lake's catchment area, as each strata of the sediment gives information about the rock type it was eroded from and also the state of the lake, i.e. oxic or anoxic. Antarctica has long been accepted as a putative analogue for Mars, so the analysis of Antarctic material may give results that can be compared to sediments on Mars. Raman spectroscopy has been selected as the method of analysis as it does not destroy the sample, can be used in situ and requires very little sample preparation. It is a suitable method for analysing both inorganic and organic matter and a miniature spectrometer is currently being developed for use in the field. The results from the spectrometers can serve as a guide for analysing sediments on Mars. It has been shown that Raman spectroscopy can detect and differentiate between oxic and anoxic sediments. Both 1064 and 785 nm wavelengths are suitable for laser excitation of organic and inorganic matter.     

Chemical Modification of Biomarkers through Accelerated Degradation: Implications for Ancient Plant Identification in Archaeo-Organic Residues

Biochemical and biomolecular archaeology is increasingly used to elucidate the consumption, use, origin, and trade of plants in the past. However, it can be challenging to use biomarkers to identify the taxonomic origin of archaeological plants due to limited knowledge of molecular survival and degradation for many key plant compounds in archaeological contexts. To gain a fundamental understanding of the chemical alterations associated with chemical degradation processes in ancient samples, we conducted accelerated degradation experiments with essential oil derived from cedar (Cedrus atlantica) exposed to materials commonly found in the archaeological record. Using GC-MS and multivariate analysis, we detected a total of 102 compounds across 19 treatments that were classified into three groups. The first group comprised compounds that were abundant in fresh cedar oil but would be unlikely to remain in ancient residues due to rapid degradation. The second group consisted of compounds that remained relatively stable or increased over time, which could be potential biomarkers for identifying cedar in archaeological residues. Compounds in the third group were absent in fresh cedar oil but were formed during specific experiments that could be indicative for certain storage conditions. These results show that caution is warranted for applying biomolecular profiles of fresh plants to ancient samples and that carefully designed accelerated degradation experiments can, at least in part, overcome this limitation.     

Bovine serum albumin as a molecular sensor for the discrimination of complex metabolite samples

The potential for using serum albumin (SA) as a broadly applicable molecular sensor was explored in an effort to develop a method for rapid analysis of complex metabolite samples. SA is a protein present at high concentration in blood, which transports a diverse set of compounds including fatty acids, hormones, and drugs. The effectiveness of the bovine ortholog (BSA) as a molecular sensor was tested by analyzing the pool of small molecules bound to the protein after a brief incubation with complex fluids of biological origin. As an initial test, three varietals of red wine were readily distinguished. Further analysis using four varietals of white wine also showed clear separation. In a second analysis using urine, animals in hemorrhagic shock were separated from a group of comparably treated controls. A time course analysis showed that recovery from injury could also be followed using the assay. This finding is significant as there currently is no method or biomarker for predicting the onset of shock. Comparison of samples was based on liquid chromatography mass spectrometry (LCMS) analysis of compounds selectively bound by BSA. Analysis of the samples after protein selection revealed a significant reduction in complexity and clear separation of groups by Principle Component Analysis (PCA). These results show the potential for using cargo-carrying proteins as molecular sensors for screening complex samples without the need for prior knowledge of sample composition or concentration and may streamline elucidation of biomarkers.     

Raman spectroscopic detection of biomolecular markers from Antarctic materials: evaluation for putative Martian habitats

The vital UV-protective and photosynthetic pigments of cyanobacteria and lichens (microbial symbioses) that dominate primary production in Antarctic desert ecosystems auto-fluoresce at short-wavelengths. A long wavelength (1064 nm) near infra-red laser has been used for non-intrusive Raman spectroscopic analysis of their ecologically significant compounds. There is now much interest in the construction of portable Raman systems for the analysis of cyanobacterial and lichen communities in the field; to this extent, Raman spectra obtained with laboratory-based systems operating at wavelengths of 852 and 1064 nm have been evaluated for potential fieldwork applications of miniaturised units. Selected test specimens of the cyanobacterial Nostoc commune, epilithic lichens Acarospora chlorophana, Xanthoria elegans and Caloplaca saxicola and the endolithic Chroococcidiopsis from Antarctic sites have been examined in the present study. Although some organisms gave useable Raman spectra with short-wavelength lasers, 1064 nm was the only excitation that was consistently excellent for all organisms. We conclude that a 1064 nm Raman spectrometer, miniaturised using an InGaAs detector, is the optimal instrument for in situ studies of pigmented communities at the limits of life on Earth. This has practical potential for the quest for biomolecules residual from any former surface or subsurface life on Mars.     

Raman Micro-Spectroscopy of Dental Pulp Stem Cells: An Approach to Monitor the Effects of Cone Beam Computed Tomography Low-Dose Ionizing Radiation

The objective of this study was to determine the molecular and biochemical changes in dental pulp stem cells (DPSCs) due to consecutive low-dose ionizing radiation exposures using label-free Raman micro-spectroscopy (RMS). Ionizing radiation produces biological damage leading to health effects of varying severity. The effects and subsequent health implications caused by exposure to low-dose radiation, such as diagnostic exposure, remain ambiguous. We identified Raman biomarkers characteristic to low-dose cone beam computed tomography (CBCT) irradiation of the DPSCs. The biomarkers were monitored inside the cells using the relative intensity distribution of the 785 and 1734?cm^?1 bands. The control cells presented a higher relative intensity of the nucleic acid specific Raman bands, whereas the irradiated cells revealed an increased intensity of the lipid-induced bands. The results obtained in this study demonstrate the capability of RMS for the detection of cell response to diagnostic radiation dose levels. This may indicate the potential of the technique for future applications such as monitoring the radiation responses in pediatric patients suffering repeated radiological exposures.     

Raman spectra of pure biomolecules obtained using a handheld instrument under cold high-altitude conditions

A handheld Raman spectrometer (Ahura First Defender) was tested for the unambiguous identification of biomolecules (pure amino acids, carboxylic acids, saccharides and trehalose) in the solid state under outdoor conditions (including moderate climate conditions as well as cold temperatures and high altitudes). The biomolecules investigated represent important objects of interest for future exobiological missions. Repetitive measurements carried out under identical instrumental setups confirmed the excellent reliability of the Raman spectrometer. Raman bands are found at correct wavenumbers ±3 cm⁻¹ compared with reference values. This testing represents the first step in a series of studies. In a preliminary, challenging investigation to determine the detection limit for glycine dispersed in a powdered gypsum matrix, 10% was the lowest content confirmed unambiguously. Clearly there is a need to investigate further the detection limits of Raman spectroscopic analyses of biomolecules in more complex samples, to demonstrate the usefulness or disqualify the use of this technique for more realistic outdoor situations, such as eventual future missions to Mars.     

Metabolomics approach to growth-age discrimination in mountain-cultivated ginseng (Panax ginseng C. A. Meyer) using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry

Mountain-cultivated ginseng is typically harvested after 10 years, while ginseng aged over 15 years is considered wild ginseng. This study aims to differentiate mountain-cultivated ginseng by age, as the fraudulent practice of selling low-aged cultivated ginseng disguised as high-aged one is damaging the market. In this study, LC-MS analyzed 98 ginseng samples, and multivariate statistical analysis identified patterns between samples to select influential components. Machine learning models were developed to identify ginseng samples of different ages. The untargeted metabolomic analysis clearly divided samples aged 4-20 years into three age groups. Twenty-two potential age-dependent biomarkers were discovered to differentiate the three sample groups. Three machine learning models were used to predict new samples, and the optimal model was selected. Some biomarkers could determine age phases according to the differentiation of mountain-cultivated ginseng samples. These biomarkers were thoroughly analyzed for variation trends. The machine learning models established using the screened biomarkers successfully predicted the age group of new samples.     

Application of portable Raman instruments for fast and non-destructive detection of minerals on outcrops

Raman spectral signatures have been obtained in situ for a series of minerals using portable Raman instruments. Cerussite, anglesite, wulfenite, titanite, calcite, tremolite, andradite and quartz were detected using portable Raman spectrometer First Defender XL (Ahura). Baryte, almandine and realgar Raman spectra obtained by this instrument in the field were compared to the data measured by the other mobile Raman instrument Inspector Raman (DeltaNu). Bench Raman dispersive microspectrometer (InVia Reflex, Renishaw) was used for comparative purposes. All spectra were obtained using a 785nm diode excitation. Although displaying lower spectral resolution comparing with the laboratory confocal instrument both portable instruments permit unambiguous detection of minerals in the field. These possibilities designate portable Raman machines as excellent tools for field geological applications. Miniaturised Raman instrument combined with LIBS will be included in the payload of the EXO Mars mission and would open interesting research possibilities in other in situ field planetary studies.     

Raman spectroscopic study of mixed carbonate materials

The main parameters for precipitation of mixed carbonate materials have been studied by Raman microscopy. These carbonates are compounds of barium, strontium and calcium. It has been shown that the Raman spectrum of a sample is exclusively controlled by its composition, the precipitation parameters do not affect the crystal structure. Even at relatively low levels, the calcium content of a sample can dominate the vibrational frequencies as measured by Raman spectroscopy. Calcium contents greater than 17% show this effect to a considerable degree, and give the broadest or two Raman peaks and thus the least uniform unit cells. The analysis of the lattice modes demonstrates that each Raman shift observed for a mixed carbonate sample corresponds to a specific crystal structure. Some peaks lie within two or three shifts that are observed for different crystal structures.     

Non-destructive analysis of pigments and other organic compounds in lichens using Fourier-transform Raman spectroscopy: a study of Antarctic epilithic lichens

Lichens in Antarctic habitats are subjected to environmental extremes, including UVB radiation, desiccation and low temperatures, as well as to rapid fluctuations in these. Lichens synthesise a variety of chemical compounds in response to their environmental conditions which contribute towards their colour, and which act as protectants against physiological stresses. The fluorescence generated by the lichens at 532 nm can be used in epifluorescence microscopy to identify their presence on substrata but this can severely affect the Raman spectra using visible excitation. The advantage of the near infrared excitation used in FT-Raman spectroscopy in minimising fluorescence emission facilitates the molecular characterisation of lichen encrustations without having to remove the thallus from its substrate or remove or otherwise damage any part of the thallus. Spectroscopic biomarkers are proposed which allow the lichens to be characterised by the identification of characteristic lichen substances; the use of these biomarkers for the preliminary taxonomic identification of Antarctic lichens is examined and some potential pitfalls are described.     

Headspace solid phase microextraction and gas chromatography-quadrupole mass spectrometry methodology for analysis of volatile compounds of marine salt as potential origin biomarkers

The establishment of geographic origin chemical biomarkers for the marine salt might represent an important improvement to its valorisation. Volatile compounds of marine salt, although never studied, are potential candidates. Thus, the purpose of this work was the development of a headspace solid phase microextraction (SPME) combined with gas chromatography-quadrupole mass spectrometry (HS-SPME/GC-qMS) methodology to study the volatile composition of marine salt. A 65 μm carbowax/divinylbenzene SPME coating fibre was used. Three SPME parameters were optimised: extraction temperature, sample quantity, and presentation mode. An extraction temperature of 60 °C and 16 g of marine salt in a 120 mL glass vial were selected. The study of the effect of sample presentation mode showed that the analysis of an aqueous solution saturated with marine salt allowed higher extraction efficiency than the direct analysis of salt crystals. The dissolution of the salt in water and the consequent effect of salting-out promote the release of the volatile compounds to the headspace, enhancing the sensitivity of SPME for the marine salt volatiles. The optimised methodology was applied to real matrices of marine salt from different geographical origins (Portugal, France, and Cape Verde). The marine salt samples contain ca. 40 volatile compounds, distributed by the chemical groups of hydrocarbons, alcohols, phenols, aldehydes, ketones, esters, terpenoids, and norisoprenoids. These compounds seem to arise from three main sources: algae, surrounding bacterial community, and environment pollution. Since these volatile compounds can provide information about the geographic origin and saltpans environment, this study shows that they can be used as chemical biomarkers of marine salt.     

FT-Raman spectroscopic study of calcium-rich and magnesium-rich carbonate minerals

Calcium and magnesium carbonates are important minerals found in sedimentary environments. Although sandstones are the most common rock colonized by endolith organisms, the production of calcium and magnesium carbonates is important in survival strategies of organisms and as a source for the removal of oxalate ions. Extremophile organisms in some situations may convert or destroy carbonates of calcium and magnesium, which gives important information about the conditions under which these organisms can survive. The identification on the surface of Mars of 'White Rock' formations, in Juventae Chasma or Sabaea Terra, as possibly carbonate rocks makes the study of these minerals a prerequisite of remote Martian exploration. Here, we show the protocol for the identification by Raman spectroscopy of different calcium and magnesium carbonates and we present a database of relevance in the search for life, extinct or extant, on Mars; this will be useful for the assessment of data obtained from remote, miniaturized Raman spectrometers now proposed for Mars exploration.     

Evaluation of portable Raman instrumentation for identification of β-carotene and mellitic acid in two-component mixtures with halite

Recently, portable Raman instrumentation has been in demand for geosciences and for future planetary exploration for the identification of both organic and inorganic compounds in situ on Earth and on other planetary bodies, especially on Mars. Here we present the results of the analysis of halite/β-carotene and halite/mellitic acid mixtures, performed by miniaturized Raman instrumentation equipped with 785 and 1064 nm excitation. Various proportions of organics in the halite matrix were examined. The lowest concentration of β-carotene detected using the 785 nm laser was 1 mg kg(-1), with slightly better signals observed with shorter exposure times compared with the bench instrument using the same excitation wavelength. Mellitic acid was identified at the concentration level 10 g kg(-1). The 1064 nm excitation provided a lower sensitivity towards low concentration when compared with the 785 nm excitation.     

In situ analysis of the Martian soil by gas chromatography: decoding of complex chromatograms of organic molecules of exobiological interest

Gas chromatography-mass spectrometry (GC-MS) will be used in future space exploration missions, in order to seek organic molecules at the surface of Mars, and especially potential chemical indicators of life. Carboxylic acids are among the most expected organic species at the surface of Mars, and they could be numerous in the analysed samples. For this reason, a chemometric method was applied to support the interpretation of chromatograms of carboxylic acid mixtures. The method is based on AutoCovariance Function (ACVF) in order to extract information on the sample--number and chemical structure of the components--and on separation performance. The procedure was applied to standard samples containing targeted compounds which are among the most expected to be present in the Martian soil: n-alkanoic and benzene dicarboxylic acids. ACVF was computed on the obtained chromatograms and plotted versus retention time: peaks of the ACVF plot can be related to specific molecular structures and are diagnostic for chemical identification of compounds.