Fluorescence microscopy as a tool for in situ life detection

The identification of extant and, in some cases, extinct bacterial life is most convincingly and efficiently performed with modern high-resolution microscopy. Epifluorescence microscopy of microbial autofluorescence or in conjunction with fluorescent dyes is among the most useful of these techniques. We explored fluorescent labeling and imaging of bacteria in rock and soil in the context of in situ life detection for planetary exploration. The goals were two-fold: to target non-Earth-centric biosignatures with the greatest possible sensitivity and to develop labeling procedures amenable to robotic implementation with technologies that are currently space qualified. A wide panel of commercially available dyes that target specific biosignature molecules was screened, and those with desirable properties (i.e., minimal binding to minerals, strong autofluorescence contrast, no need for wash steps) were identified. We also explored the potential of semiconductor quantum dots (QDs) as bacterial and space probes. A specific instrument for space implementation is suggested and discussed.     

The Urey instrument: an advanced in situ organic and oxidant detector for Mars exploration

The Urey organic and oxidant detector consists of a suite of instruments designed to search for several classes of organic molecules in the martian regolith and ascertain whether these compounds were produced by biotic or abiotic processes using chirality measurements. These experiments will also determine the chemical stability of organic molecules within the host regolith based on the presence and chemical reactivity of surface and atmospheric oxidants. Urey has been selected for the Pasteur payload on the European Space Agency's (ESA's) upcoming 2013 ExoMars rover mission. The diverse and effective capabilities of Urey make it an integral part of the payload and will help to achieve a large portion of the mission's primary scientific objective: "to search for signs of past and present life on Mars." This instrument is named in honor of Harold Urey for his seminal contributions to the fields of cosmochemistry and the origin of life.     

Survival and germinability of Bacillus subtilis spores exposed to simulated Mars solar radiation: implications for life detection and planetary protection

Bacterial spores have been considered as microbial life that could survive interplanetary transport by natural impact processes or human spaceflight activity. Deposition of terrestrial microbes or their biosignature molecules onto the surface of Mars could negatively impact life detection experiments and planetary protection measures. Simulated Mars solar radiation, particularly the ultraviolet component, has been shown to reduce spore viability, but its effect on spore germination and resulting production of biosignature molecules has not been explored. We examined the survival and germinability of Bacillus subtilis spores exposed to simulated martian conditions that include solar radiation. Spores of B. subtilis that contain luciferase resulting from expression of an sspB-luxAB gene fusion were deposited on aluminum coupons to simulate deposition on spacecraft surfaces and exposed to simulated Mars atmosphere and solar radiation. The equivalent of 42 min of simulated Mars solar radiation exposure reduced spore viability by nearly 3 logs, while germination-induced bioluminescence, a measure of germination metabolism, was reduced by less than 1 log. The data indicate that spores can retain the potential to initiate germination-associated metabolic processes and produce biological signature molecules after being rendered nonviable by exposure to Mars solar radiation.     

Terahertz circular dichroism spectroscopy: a potential approach to the in situ detection of life's metabolic and genetic machinery

We propose a terahertz (far-infrared) circular dichroism-based life-detection technology that may provide a universal and unequivocal spectroscopic signature of living systems regardless of their genesis. We argue that, irrespective of the specifics of their chemistry, all life forms will employ well-structured, chiral, stereochemically pure macromolecules (>500 atoms) as the catalysts with which they perform their metabolic and replicative functions. We also argue that nearly all such macromolecules will absorb strongly at terahertz frequencies and exhibit significant circular dichroism, and that this circular dichroism unambiguously distinguishes biological from abiological materials. Lastly, we describe several approaches to the fabrication of a terahertz circular dichroism spectrometer and provide preliminary experimental indications of their feasibility. Because terahertz circular dichroism signals arise from the molecular machinery necessary to carry out life's metabolic and genetic processes, this life-detection method differs fundamentally from more well-established approaches based on the detection of isotopic fractionation, "signature" carbon compounds, disequilibria, or other by-products of metabolism. Moreover, terahertz circular dichroism spectroscopy detects this machinery in a manner that makes few, if any, assumptions as to its chemical nature or the processes that it performs.     

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5?m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2?m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5?g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260?g kg(-1)) and perchlorate (41.13?μg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14?μg g(-1)) or formate (76.06?μg g(-1)) as electron donors, and sulfate (15875?μg g(-1)), nitrate (13490?μg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.     

Intensity/frequency indicator for detection in space: the high values of the incident solar or laser optical radiation in comparison with the appropriate maximum permissible exposure.

The solar or laser optical radiation impact to humans in space depends on the intensity, on the exposure type (direct or indirect) & duration and on the matching of radiation wavelength to tissue characteristics. The main protection factor in space is the application of exposure limits. This paper describes the main biological optical interaction parameters, the optical exposure hazards and the development of a small active lightweight indicator, with output beeper rate depended to the ratio of optical irradiance/exposure limit. The indicator may be used as warning element on the side of helmets, goggles, spectacles, etc, with low power consumption. Electronically the indicator is an intensity/frequency converter, based on the value of the ratio of exposure/exposure limits, with audio & light beepers like the indication output of the ionizing (radioactive) radiation monitors.     

Discovery of abundant cellulose microfibers encased in 250 Ma Permian halite: a macromolecular target in the search for life on other planets

In this study, we utilized transmission electron microscopy to examine the contents of fluid inclusions in halite (NaCl) and solid halite crystals collected 650 m below the surface from the Late Permian Salado Formation in southeastern New Mexico (USA). The halite has been isolated from contaminating groundwater since deposition approximately 250 Ma ago. We show that abundant cellulose microfibers are present in the halite and appear remarkably intact. The cellulose is in the form of 5 nm microfibers as well as composite ropes and mats, and was identified by resistance to 0.5 N NaOH treatment and susceptibility to cellulase enzyme treatment. These cellulose microfibers represent the oldest native biological macromolecules to have been directly isolated, examined biochemically, and visualized (without growth or replication) to date. This discovery points to cellulose as an ideal macromolecular target in the search for life on other planets in our Solar System.     

Formation of replicating saponite from a gel in the presence of oxalate: implications for the formation of clay minerals in carbonaceous chondrites and the origin of life

The potential role of clay minerals in the abiotic origin of life has been the subject of ongoing debate for the past several decades. At issue are the clay minerals found in a class of meteorites known as carbonaceous chondrites. These clay minerals are the product of aqueous alteration of anhydrous mineral phases, such as olivine and orthopyroxene, that are often present in the chondrules. Moreover, there is a strong correlation in the occurrence of clay minerals and the presence of polar organic molecules. It has been shown in laboratory experiments at low temperature and ambient pressure that polar organic molecules, such as the oxalate found in meteorites, can catalyze the crystallization of clay minerals. In this study, we show that oxalate is a robust catalyst in the crystallization of saponite, an Al- and Mg-rich, trioctahedral 2:1 layer silicate, from a silicate gel at 60°C and ambient pressure. High-resolution transmission electron microscopy analysis of the saponite treated with octadecylammonium (n(C)=18) cations revealed the presence of 2:1 layer structures that have variable interlayer charge. The crystallization of these differently charged 2:1 layer silicates most likely occurred independently. The fact that 2:1 layer silicates with variable charge formed in the same gel has implications for our understanding of the origin of life, as these 2:1 clay minerals most likely replicate by a mechanism of template-catalyzed polymerization and transmit the charge distribution from layer to layer. If polar organic molecules like oxalate can catalyze the formation of clay-mineral crystals, which in turn promote clay microenvironments and provide abundant adsorption sites for other organic molecules present in solution, the interaction among these adsorbed molecules could lead to the polymerization of more complex organic molecules like RNA from nucleotides on early Earth.     

Navigating in a Virtual 3D Maze: Body and Gravity,Two Possible Reference Frames for Perceiving and Memorizing

Although recent studies have brought new insight into the mechanisms of spatial memory and cognitive strategies during navigation, most of these studies have concerned two-dimensional navigation and little is known regarding the problem of three-dimensional (3D) spatial memory. We found previously that memorizing complex 3D-structured corridors was easier with natural self-motion that included only yaw turns, and vertical translations facing the walls at vertical sections. This suggests that when only sideways (yaw) mental rotations had to be performed in order to shift from the experienced egocentric to the allocentric reference frame where recognition was tested, memorization of such corridors was improved. In the present investigation we studied the effect of tilting separately subject's body axis and self-motion's rotation axis relative to gravity. With a computerized 3D reconstruction task of the maze, we examined whether having any single rotation axis was enough to facilitate this reference shift or, if not, what aspect of the terrestrial condition-where visual displacement rotation, gravity and body axes were aligned-led to better performance. Field dependent (FD) and independent (FI) subjects, as determined by the rod and frame test, showed distinct effects of the navigation conditions. The FD group performance was markedly impaired when gravity and body axis were in conflict, independently of the rotation axis, whereas FI performance only slightly worsened when the body was tilted and the rotation axis remained aligned with gravity. Moreover, tilting the body in the control condition only worsened performance for the FD group.     

Search for the OH (X(2)Pi) Meinel band emission in meteors as a tracer of mineral water in comets: detection of N(2)(+) (A-X)

We report the discovery of the N(2)(+) A-X Meinel band in the 780-840 nm meteor emission from two Leonid meteoroids that were ejected less than 1000 years ago by comet 55P/Tempel-Tuttle. Our analysis indicates that the N(2)(+) molecule is at least an order of magnitude less abundant than expected, possibly as a result of charge transfer reactions with meteoric metal atoms. This new band was found while searching for rovibrational transitions in the X(2)Pi electronic ground state of OH (the OH Meinel band), a potential tracer of water bound to minerals in cometary matter. The electronic A-X transition of OH has been identified in other Leonid meteors. We did not detect this OH Meinel band, which implies that the excited A state is not populated by thermal excitation but by a mechanism that directly produces OH in low vibrational levels of the excited A(2)Sigma state. Ultraviolet dissociation of atmospheric or meteoric water vapor is such a mechanism, as is the possible combustion of meteoric organics.