Quantitative laser-matter interaction: a 3D study of UV-fs-laser ablation on single crystalline Ru(0001).

Laser ablation is nowadays an extensively applied technology to probe the chemical composition of solid materials. It allows for precise targeting of micrometer objects on and in samples, and enables chemical depth profiling with nanometer resolution. An in-depth understanding of the 3D geometry of the ablation craters is crucial for precise calibration of the depth scale in chemical depth profiles. Herein we present a comprehensive study on laser ablation processes using a Gaussian-shaped UV-femtosecond irradiation source and present how the combination of three different imaging methods (scanning electron microscopy, interferometric microscopy, and X-ray computed tomography) can provide accurate information on the crater's shapes. Crater analysis by applying X-ray computed tomography is of considerable interest because it allows the imaging of an array of craters in one step with sub-µm accuracy and is not limited to the aspect ratio of the crater. X-ray computed tomography thereby complements the analysis of laser ablation craters. The study investigates the effect of laser pulse energy and laser burst count on a single crystal Ru(0001) sample. Single crystals ensure that there is no dependence on the grain orientations during the laser ablation process. An array of 156 craters of different dimensions ranging from <20 nm to ∼40 µm in depth were created. For each individually applied laser pulse, we measured the number of ions generated in the ablation plume with our laser ablation ionization mass spectrometer. We show to which extent the combination of these four techniques reveals valuable information on the ablation threshold, the ablation rate, and the limiting ablation depth. The latter is expected to be a consequence of decreasing irradiance upon increasing crater surface area. The ion signal generated was found to be proportional to the volume ablated up to the certain depth, which enables in-situ depth calibration during the measurement.

Reduction of surface charging effects in laser ablation ionisation mass spectrometry through gold coating.

In femtosecond Laser Ablation Ionisation Mass Spectrometry (fs-LIMS) short laser pulses are used to ablate, atomise, and ionise solid sample material shot-by-shot. When ablating non-conductive samples electric charging of the surface can occur. Depending on the geometry of the instrument, the surface charge can influence the spread of the ablation plume and reduce spectral quality. Methods to reduce surface charging were investigated using a non-conductive geological sample and a miniature fs-LIMS system with a co-linear ablation geometry. Pausing five seconds between consecutive laser bursts fired on non-coated material improved the spectral quality by giving surface charges more time to dissipate. However, best mass spectrometric results were achieved after the sample was sputter coated with a thin gold layer, as a conductive sample surface hinders charge build-up. Consequently, gold coating allowed operation of the laser system at much higher laser pulse energies improving sensitivity and reliability. It also removed the need to pause between laser bursts, speeding up the measurement acquisition.

Improved plasma stoichiometry recorded by laser ablation ionization mass spectrometry using a double-pulse femtosecond laser ablation ion source.

RATIONALE: Femtosecond (fs) laser ablation ion sources have allowed for improved measurement capabilities and figures of merit of laser ablation based spectroscopic and mass spectrometric measurement techniques. However, in comparison to longer pulse laser systems, the ablation plume from fs lasers is observed to be colder, which favors the formation of polyatomic species. Such species can limit the analytical capabilities of a system due to isobaric interferences. In this contribution, a double-pulse femtosecond (DP-fs) laser ablation ion source is coupled to our miniature Laser Ablation Ionization Mass Spectrometry (LIMS) system and its impact on the recorded stoichiometry of the generated plasma is analyzed in detail. METHODS: A DP-fs laser ablation ion source (temporal delays of +300 to - 300 ps between pulses) is connected to our miniature LIMS system. The first pulse is used for material removal from the sample surface and the second for post-ionization of the ablation plume. To characterize the performance, parametric double- and single-pulse studies (temporal delays, variation of the pulse energy, voltage applied on detector system) were conducted on three different NIST SRM alloy samples (SRM 661, 664 and 665). RESULTS: At optimal instrument settings for both the double-pulse laser ablation ion source and the detector voltage, relative sensitivity coefficients were observed to be closer (factor of ~2) to 1 compared with single-pulse measurements. Furthermore, the optimized settings worked for all three samples, meaning no further optimization was necessary when changing to another alloy sample material during this study. CONCLUSIONS: The application of a double-pulse femtosecond laser ablation ion source resulted in the recording of improved stoichiometry of the generated plasma using our LIMS measurement technique. This is of great importance for the quantitative chemical analysis of more complex solid materials, e.g., geological samples or metal alloys, especially when aiming for standard-free quantification procedures for the determination of the chemical composition.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

UV post-ionization laser ablation ionization mass spectrometry for improved nm-depth profiling resolution on Cr/Ni reference standard.

RATIONALE: Laser ablation combined with mass spectrometry forms a promising tool for chemical depth profiling of solids. At irradiations near the ablation threshold, high depth resolutions are achieved. However, at these conditions, a large fraction of ablated species is neutral and therefore invisible to the instrument. To compensate for this effect, an additional ionization step can be introduced. METHODS: Double-pulse laser ablation is frequently used in material sciences to produce shallow craters. We apply double-pulse UV femtosecond (fs) Laser Ablation Ionization Mass Spectrometry to investigate the depth profiling performance. The first pulse energy is set to gentle ablation conditions, whereas the second pulse is applied at a delay and a pulse energy promoting the highest possible ion yield. RESULTS: The experiments were performed on a Cr/Ni multi-layered standard. For a mean ablation rate of ~3 nm/pulse (~72 nJ/pulse), a delay of ~73 ps provided optimal results. By further increasing the energy of the second pulse (5-30% higher with respect to the first pulse) an enhancement of up to 15 times the single pulse intensity was achieved. These conditions resulted in mean depth resolutions of ~37 and ~30 nm for the Cr and Ni layers, respectively. CONCLUSIONS: It is demonstrated on the thin-film standard that the double-pulse excitation scheme substantially enhances the chemical depth profiling resolution of LIMS with respect to the single-pulse scheme. The post-ionization allows for extraordinarily low ablation rates and for quantitative and stoichiometric analysis of nm-thick films/coatings.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus.

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.

Combining Anisotropic Etching and PDMS Casting for Three-Dimensional Analysis of Laser Ablation Processes.

State-of-the-art laser ablation (LA) depth-profiling techniques (e.g. LA-ICP-MS, LIBS, and LIMS) allow for chemical composition analysis of solid materials with high spatial resolution at micro- and nanometer levels. Accurate determination of LA-volume is essential to correlate the recorded chemical information to the specific location inside the sample. In this contribution, we demonstrate two novel approaches towards a better quantitative analysis of LA craters with dimensions at micrometer level formed by femtosecond-LA processes on single-crystalline Si(100) and polycrystalline Cu model substrates. For our parametric crater evolution studies, both the number of applied laser shots and the pulse energy were systematically varied, thus yielding 2D matrices of LA craters which vary in depth, diameter, and crater volume. To access the 3D structure of LA craters formed on Si(100), we applied a combination of standard lithographic and deep reactive-ion etching (DRIE) techniques followed by a HR-SEM inspection of the previously formed crater cross sections. As DRIE is not applicable for other material classes such as metals, an alternative and more versatile preparation technique was developed and applied to the LA craters formed on the Cu substrate. After the initial LA treatment, the Cu surface was subjected to a polydimethylsiloxane (PDMS) casting process yielding a mold being a full 3D replica of the LA craters, which was then analyzed by HR-SEM. Both approaches revealed cone-like shaped craters with depths ranging between 1 and 70 µm and showed a larger ablation depth of Cu that exceed the one of Si by a factor of about 3.

Insights into Laser Ablation Processes of Heterogeneous Samples: Toward Analysis of Through-Silicon-Vias.

State-of-the-art three-dimensional very large-scale integration (3D-VLSI) relies, among other factors, on the purity of high-aspect-ratio Cu interconnects such as through-silicon-vias (TSVs). Accurate spatial chemical analysis of electroplated TSV structures has been proven to be challenging due to their large aspect ratios and their multimaterial composition (Cu and Si) with distinct physical properties. Here, we demonstrate that these structures can be accurately analyzed by femtosecond (fs) laser beam ablation techniques in combination with ionization mass spectrometry (LIMS). We specifically report on novel preparation approaches for the postablation analysis of craters formed upon TSV depth profiling. The novel TSV sample preparation is based on deep and material-selective reactive-ion etching of the Si matrix surrounding the Cu interconnects thus facilitating systematic focused-ion-beam (FIB) investigations of the high-aspect-ratio TSV structures upon ablation. The particular structure of the TSV analyte combined with the ⌀beam > ⌀Cu-TSV condition allowed for an in-depth investigation of fundamental laser ablation processes, particularly focusing on the redeposition of ablated material at the inner side-walls of the LIMS craters. This phenomenon is of imminent importance for the ultimate quantification in any laser ablation-based depth profiling. In addition, we have developed a new method which allows the unambiguous determination of the crossing-point of the Si/Cu||bare Si interface upon Cu-TSV depth profiling which is based on pronounced, depth-dependent changes in the mass-spectrometric detection of those Si xy+ species formed upon the LIMS depth erosion.

Depth Profiling and Cross-Sectional Laser Ablation Ionization Mass Spectrometry Studies of Through-Silicon-Vias.

Through-silicon-via (TSV) technology enables 3D integration of multiple 2D components in advanced microchip architectures. Key in the TSV fabrication is an additive-assisted Cu electroplating process in which the additives employed may get embedded in the TSV body. This incorporation negatively influences the reliability and durability of the Cu interconnects. Here, we present a novel approach toward the chemical analysis of TSVs which is based on femtosecond laser ablation ionization mass spectrometry (fs-LIMS). The conditions for LIMS depth profiling were identified by a systematic variation of the laser pulse energy and the number of laser shots applied. In this contribution, new aspects are addressed related to the analysis of highly heterogeneous specimens having dimensions in the range of the probing beam itself. Particularly challenging were the different chemical and physical properties of which the target specimens were composed. Depth profiling of the TSVs along their main axis (approach 1) revealed a gradient in the carbon (C) content. These differences in the C concentration inside the TSVs could be confirmed and quantified by LIMS analyses of cross-sectionally sliced TSVs (approach 2). Our quantitative analysis revealed a C content that is ∼1.5 times higher at the TSV top surface compared to its bottom. Complementary Scanning Auger Microscopy (SAM) data confirmed a preferential embedment of suppressor additives at the side walls of the TSV. These results demonstrate that the TSV filling concept significantly deviates from common Damascene electroplating processes and will therefore contribute to a more comprehensive, mechanistic understanding of the underlying mechanisms.

Toward Three-Dimensional Chemical Imaging of Ternary Cu-Sn-Pb Alloys Using Femtosecond Laser Ablation/Ionization Mass Spectrometry.

Femtosecond laser ablation/ionization mass spectrometry (LIMS) has been applied to probe the spatial element composition of three ternary Cu-Sn-Pb model bronze alloys (lead bronzes: CuSn10Pb10, CuSn7Pb15, and CuSn5Pb20), which were recently identified as high-performance cathode materials in the context of electro-organic synthesis (dehalogenation, deoxygenation) of pharmaceutically relevant building blocks. The quantitative and spatially resolved element analysis of such cathode materials will help in understanding the observed profound differences in their electrochemical reactivity and stability. For that purpose, we developed a measurement procedure using the LIMS technique which allows analyzing the element composition of these ternary alloys in all three spatial dimensions. Their chemical composition was determined spotwise, by ablating material from various surface locations on a 4 × 4 raster array (50 µm pitch distance, ablation crater diameter of ∼20 µm). The element analyses show significant chemical inhomogeneities in all three ternary bronze alloys with profound local deviations from their nominal bulk compositions and indicate further differences in the nature and origin of these compositional inhomogeneities. In addition, the element analyses showed specific compositional correlations among the major elements (Cu, Sn, and Pb) in these alloys. On selected sample positions minor (Ni, Zn, Ag, and Sb) and trace elements (C, P, Fe, and As) were quantified. These results are in agreement with inductively coupled plasma collision/reaction interface mass spectrometry (ICP-CRI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) reference measurements, thus proving the LIMS depth profiling technique as a powerful alternative methodology to conventional quantification techniques with the advantage, however, of a highly localized measurement capability.

Towards matrix-free femtosecond-laser desorption mass spectrometry for in situ space research.

RATIONALE: There is an increasing interest in the quest for low molecular weight biomarkers that can be studied on extra-terrestrial objects by direct laser desorption mass spectrometry (LD-MS). Although molecular structure investigations have recently been carried out by direct LD-MS approaches, there is still a lack of suitable instruments for implementation on a spacecraft due to weight, size and power consumption demands. In this contribution we demonstrate the feasibility of LD-MS structural analysis of molecular species by a miniature laser desorption-ionization mass spectrometer (instrument name LMS) originally designed for in situ elemental and isotope analysis of solids in space research. METHODS: Direct LD-MS studies with molecular resolution were carried out by means of a Laser Ablation/Ionization Mass Spectrometry (LIMS) technique. Two polymer samples served as model systems: neutral polyethylene glycol (PEG) and cationic polymerizates of imidazole and epichlorohydrin (IMEP). Optimal conditions for molecular fragmentation could be identified for both polymers by tuning the laser energy and the instrument-sample distance. RESULTS: PEG and IMEP polymers show sufficient stability over a relatively wide laser energy range. Under mild LD conditions only moderate fragmentation of the polymers takes place so that valuable structural characterization based on fragment ions can be achieved. As the applied laser pulse energy rises, the abundance of fragment ions increases, reaches a plateau and subsequently drops down due to more severe fragmentation and atomization of the polymers. At this final stage, usually referred to as laser ablation, only elemental/isotope analysis can be achieved. CONCLUSIONS: Our investigations demonstrate the versatility of the LMS instrument that can be tuned to favourable laser desorption conditions that successfully meet molecule-specific requirements and deliver abundant fragment ion signals with detailed structural information. Overall, the results show promise for use in similar studies on planetary surfaces beyond Earth where no or minimal sample preparation is essential.

Laser Ablation/Ionisation Mass Spectrometry: Sensitive and Quantitative Chemical Depth Profiling of Solid Materials.

Direct quantitative and sensitive chemical analysis of solid materials with high spatial resolution, both in lateral and vertical direction is of high importance in various fields of analytical research, ranging from in situ space research to the semiconductor industry. Accurate knowledge of the chemical composition of solid materials allows a better understanding of physical and chemical processes that formed/altered the material and allows e.g. to further improve these processes. So far, state-of-the-art techniques such as SIMS, LA-ICP-MS or GD-MS have been applied for chemical analyses in these fields of research. In this report we review the current measurement capability and the applicability of our Laser Ablation/Ionisation Mass Spectrometer (instrument name LMS) for the chemical analysis of solids with high spatial resolution. The most recent chemical analyses conducted on various solid materials, including e.g. alloys, fossils and meteorites are discussed.

High-resolution chemical depth profiling of solid material using a miniature laser ablation/ionization mass spectrometer.

High-resolution chemical depth profiling measurements of copper films are presented. The 10 µm thick copper test samples were electrodeposited on a Si-supported Cu seed under galvanostatic conditions in the presence of particular plating additives (SPS, Imep, PEI, and PAG) used in the semiconductor industry for the on-chip metallization of interconnects. To probe the trend of these plating additives toward inclusion into the deposit upon growth, quantitative elemental mass spectrometric measurements at trace level concentration were conducted by using a sensitive miniature laser ablation ionization mass spectrometer (LIMS), originally designed and developed for in situ space exploration. An ultrashort pulsed laser system (τ ∼ 190 fs, λ = 775 nm) was used for ablation and ionization of sample material. We show that with our LIMS system, quantitative chemical mass spectrometric analysis with an ablation rate at the subnanometer level per single laser shot can be conducted. The measurement capabilities of our instrument, including the high vertical depth resolution coupled with high detection sensitivity of ∼10 ppb, high dynamic range ≥10(8), measurement accuracy and precision, is of considerable interest in various fields of application, where investigations with high lateral and vertical resolution of the chemical composition of solid materials are required, these include, e.g., wafers from semiconductor industry or studies on space weathered samples in space research.

Automated, 3-D and Sub-Micron Accurate Ablation-Volume Determination by Inverse Molding and X-Ray Computed Tomography.

Ablation of materials in combination with element-specific analysis of the matter released is a widely used method to accurately determine a material's chemical composition. Among other methods, repetitive ablation using femto-second pulsed laser systems provides excellent spatial resolution through its incremental removal of nanometer thick layers. The method can be combined with high-resolution mass spectrometry, for example, laser ablation ionization mass spectrometry, to simultaneously analyze chemically the material released. With increasing depth of the volume ablated, however, secondary effects start to play an important role and the ablation geometry deviates substantially from the desired cylindrical shape. Consequently, primarily conical but sometimes even more complex, rather than cylindrical, craters are created. Their dimensions need to be analyzed to enable a direct correlation with the element-specific analytical signals. Here, a post-ablation analysis method is presented that combines generic polydimethylsiloxane-based molding of craters with the volumetric reconstruction of the crater's inverse using X-ray computed tomography. Automated analysis yields the full, sub-micron accurate anatomy of the craters, thereby a scalable and generic method to better understand the fundamentals underlying ablation processes applicable to a wide range of materials. Furthermore, it may serve toward a more accurate determination of heterogeneous material's composition for a variety of applications without requiring time- and labor-intensive analyses of individual craters.

Multiwavelength Ablation/Ionization and Mass Spectrometric Analysis of 1.88 Ga Gunflint Chert.

The investigation of chemical composition on planetary bodies without significant sample processing is of importance for nearly every mission aimed at robotic exploration. Moreover, it is a necessary tool to achieve the longstanding goal of finding evidence of life beyond Earth, for example, possibly preserved microbial remains within martian sediments. Our Laser Ablation Ionization Mass Spectrometer (LIMS) is a compact time-of-flight mass spectrometer intended to investigate the elemental, isotope, and molecular composition of a wide range of solid samples, including e.g., low bulk density organic remains in microfossils. Here, we present an overview of the instrument and collected chemical spectrometric data at the micrometer level from a Precambrian chert sample (1.88 Ga Gunflint Formation, Ontario, Canada), which is considered to be a martian analogue. Data were collected from two distinct zones-a silicified host area and a carbon-bearing microfossil assemblage zone. We performed these measurements using an ultrafast pulsed laser system (pulse width of ∼180 fs) with multiple wavelengths (infrared [IR]-775 nm, ultraviolet [UV]-387 nm, UV-258 nm) and using a pulsed high voltage on the mass spectrometer to reveal small organic signals. We investigated (1) the chemical composition of the sample and (2) the different laser wavelengths' performance to provide chemical depth profiles in silicified media. Our key findings are as follows: (1) microfossils from the Gunflint chert reveal a distinct chemical composition compared with the host mineralogy (we report the identification of 24 elements in the microfossils); (2) detection of the pristine composition of microfossils and co-occurring fine chemistry (rare earth elements) requires utilization of the depth profiling measurement protocol; and (3) our results show that, for analysis of heterogeneous material from siliciclastic deposits, siliceous sinters, and cherts, the most suitable wavelength for laser ablation/Ionization is UV-258 nm.

On Topological Analysis of fs-LIMS Data. Implications for in Situ Planetary Mass Spectrometry.

In this contribution, we present results of non-linear dimensionality reduction and classification of the fs laser ablation ionization mass spectrometry (LIMS) imaging dataset acquired from the Precambrian Gunflint chert (1.88 Ga) using a miniature time-of-flight mass spectrometer developed for in situ space applications. We discuss the data generation, processing, and analysis pipeline for the classification of the recorded fs-LIMS mass spectra. Further, we define topological biosignatures identified for Precambrian Gunflint microfossils by projecting the recorded fs-LIMS intensity space into low dimensions. Two distinct subtypes of microfossil-related spectra, a layer of organic contamination and inorganic quartz matrix were identified using the fs-LIMS data. The topological analysis applied to the fs-LIMS data allows to gain additional knowledge from large datasets, formulate hypotheses and quickly generate insights from spectral data. Our contribution illustrates the utility of applying spatially resolved mass spectrometry in combination with topology-based analytics in detecting signatures of early (primitive) life. Our results indicate that fs-LIMS, in combination with topological methods, provides a powerful analytical framework and could be applied to the study of other complex mineralogical samples.

Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

Isotope abundance ratio measurements using femtosecond laser ablation ionization mass spectrometry.

Accurate isotope ratio measurements are of high importance in various scientific fields, ranging from radio isotope geochronology of solids to studies of element isotopes fractionated by living organisms. Instrument limitations, such as unresolved isobaric inferences in the mass spectra, or cosampling of the material of interest together with the matrix material may reduce the quality of isotope measurements. Here, we describe a method for accurate isotope ratio measurements using our laser ablation ionization time-of-flight mass spectrometer (LIMS) that is designed for in situ planetary research. The method is based on chemical depth profiling that allows for identifying micrometer scale inclusions embedded in surrounding rocks with different composition inside the bulk of the sample. The data used for precise isotope measurements are improved using a spectrum cleaning procedure that ensures removal of low quality spectra. Furthermore, correlation of isotopes of an element is used to identify and reject the data points that, for example, do not belong to the species of interest. The measurements were conducted using IR femtosecond laser irradiation focused on the sample surface to a spot size of ~12 µm. Material removal was conducted for a predefined number of laser shots, and time-of-flight mass spectra were recorded for each of the ablated layers. Measurements were conducted on NIST SRM 986 Ni isotope standard, trevorite mineral, and micrometer-sized inclusions embedded in aragonite. Our measurements demonstrate that element isotope ratios can be measured with accuracies and precision at the permille level, exemplified by the analysis of B, Mg, and Ni element isotopes. The method applied will be used for in situ investigation of samples on planetary surfaces, for accurate quantification of element fractionation induced by, for example, past or present life or by geochemical processes.

The Detection of Elemental Signatures of Microbes in Martian Mudstone Analogs Using High Spatial Resolution Laser Ablation Ionization Mass Spectrometry.

The detection and identification of biosignatures on planetary bodies such as Mars in situ is extremely challenging. Current knowledge from space exploration missions suggests that a suite of complementary instruments is required in situ for a successful identification of past or present life. For future exploration missions, new and innovative instrumentation capable of high spatial resolution chemical (elemental and isotope) analysis of solids with improved measurement capabilities is of considerable interest because a multitude of potential signatures of extinct or extant life have dimensions on the micrometer scale. The aim of this study is to extend the current measurement capabilities of a miniature laser ablation ionization mass spectrometer (LIMS) designed for space exploration missions to detect signatures of microbial life. In total, 14 martian mudstone analogue samples were investigated regarding their elemental composition. Half the samples were artificially inoculated with a low number density of microbes, and half were used as abiotic controls. The samples were treated in a number of ways. Some were cultured anaerobically and some aerobically; some abiotic samples were incubated with water, and some remained dry. Some of the samples were exposed to a large dose of γ radiation, and some were left un-irradiated. While no significant elemental differences were observed between the applied sample treatments, the instrument showed the capability to detect biogenic element signatures of the inoculated microbes by monitoring biologically relevant elements, such as hydrogen, carbon, sulfur, iron, and so on. When an enrichment in carbon was measured in the samples but no simultaneous increase in other biologically relevant elements was detected, it suggests, for example, a carbon-containing inclusion; when the enrichment was in carbon and in bio-relevant elements, it suggests the presences of microbes. This study presents first results on the detection of biogenic element patterns of microbial life using a miniature LIMS system designed for space exploration missions.

Biosignature Analysis of Mars Soil Analogs from the Atacama Desert: Challenges and Implications for Future Missions to Mars.

The detection of biosignatures on Mars is of outstanding interest in the current field of astrobiology and drives various fields of research, ranging from new sample collection strategies to the development of more sensitive detection techniques. Detailed analysis of the organic content in Mars analog materials collected from extreme environments on Earth improves the current understanding of biosignature preservation and detection under conditions similar to those of Mars. In this article, we examined the biological fingerprint of several locations in the Atacama Desert (Chile), which include different wet and dry, and intermediate to high elevation salt flats (also named salars). Liquid chromatography and multidimensional gas chromatography mass spectrometry measurement techniques were used for the detection and analysis of amino acids extracted from the salt crusts and sediments by using sophisticated extraction procedures. Illumina 16S amplicon sequencing was used for the identification of microbial communities associated with the different sample locations. Although amino acid load and organic carbon and nitrogen quantities were generally low, it was found that most of the samples harbored complex and versatile microbial communities, which were dominated by (extremely) halophilic microorganisms (most notably by species of the Archaeal family Halobacteriaceae). The dominance of salts (i.e., halites and sulfates) in the investigated samples leaves its mark on the composition of the microbial communities but does not appear to hinder the potential of life to flourish since it can clearly adapt to the higher concentrations. Although the Atacama Desert is one of the driest and harshest environments on Earth, it is shown that there are still sub-locations where life is able to maintain a foothold, and, as such, salt flats could be considered as interesting targets for future life exploration missions on Mars.