Raman spectroscopy plays a major role in robust detection of biomolecules and mineral signatures in halophile research. An overview of Raman spectroscopic investigations in halophile research of the last decade is given here to show advantages of the approach, progress made as well as limits of the technique. Raman spectroscopy is an excellent tool to monitor and identify microbial pigments and other biomolecules in extant and extinct halophile biomass. Studies of bottom gypsum crusts from salterns, native evaporitic sediments, halite inclusions, and endoliths as well as cultures of halophilic microorganisms permitted to understand the content, distribution, and behavior of important molecular species. The first papers describing Raman spectroscopic detection of microbiological and geochemical key markers using portable instruments are highlighted as well.
Raman spectroscopy; halophilic; salterns; gypsum crusts; compatible solutes; carotenoids
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
Raman spectroscopy is being adopted as a nondestructive instrumentation for the robotic exploration of Mars to search for traces of life in the geological record. Here, miniaturized Raman spectrometers of two different types equipped with 532 and 785 nm lasers for excitation, respectively, were compared for the detection of microbial biomarkers in natural halite from the hyperarid region of the Atacama Desert. Measurements were performed directly on the rock as well as on the homogenized, powdered samples prepared from this material—the effects of this sample preparation and the excitation wavelength employed in the analysis are compared and discussed. From these results, 532 nm excitation was found to be superior for the analysis of powdered specimens due to its high sensitivity toward carotenoids and hence a higher capability for their detection at relatively low concentration in bulk powdered specimens. For the same reason, this wavelength was a better choice for the detection of carotenoids in direct measurements made on the rock samples. The 785 nm excitation wavelength, in contrast, proved to be more sensitive toward the detection of scytonemin. Key Words: Miniaturized portable Raman—Atacama—Mars—Biomarker detection. Astrobiology 12, 1095–1099.
Halophilic archaebacteria (haloarchaea) thrive in environments with salt concentrations approaching saturation, such as natural brines, the Dead Sea, alkaline salt lakes and marine solar salterns; they have also been isolated from rock salt of great geological age (195–250 million years). An overview of their taxonomy, including novel isolates from rock salt, is presented here; in addition, some of their unique characteristics and physiological adaptations to environments of low water activity are reviewed. The issue of extreme long-term microbial survival is considered and its implications for the search for extraterrestrial life. The development of detection methods for subterranean haloarchaea, which might also be applicable to samples from future missions to space, is presented.
Extreme halophiles; Haloarchaea; Life detection; Microbial longevity; Salt mines; Salt sediments; Space missions; Subterranean; Taxonomy of halobacteriaceae
Various effects of microgravity on prokaryotes have been recognized in recent years, with the focus on studies of pathogenic bacteria. No archaea have been investigated yet with respect to their responses to microgravity. For exposure experiments on spacecrafts or on the International Space Station, halophilic archaea (haloarchaea) are usually embedded in halite, where they accumulate in fluid inclusions. In a liquid environment, these cells will experience microgravity in space, which might influence their viability and survival. Two haloarchaeal strains, Haloferax mediterranei and Halococcus dombrowskii, were grown in simulated microgravity (SMG) with the rotary cell culture system (RCCS, Synthecon). Initially, salt precipitation and detachment of the porous aeration membranes in the RCCS were observed, but they were avoided in the remainder of the experiment by using disposable instead of reusable vessels. Several effects were detected, which were ascribed to growth in SMG: Hfx. mediterranei's resistance to the antibiotics bacitracin, erythromycin, and rifampicin increased markedly; differences in pigmentation and whole cell protein composition (proteome) of both strains were noted; cell aggregation of Hcc. dombrowskii was notably reduced. The results suggest profound effects of SMG on haloarchaeal physiology and cellular processes, some of which were easily observable and measurable. This is the first report of archaeal responses to SMG. The molecular mechanisms of the effects induced by SMG on prokaryotes are largely unknown; haloarchaea could be used as nonpathogenic model systems for their elucidation and in addition could provide information about survival during lithopanspermia (interplanetary transport of microbes inside meteorites). Key Words: Haloferax mediterranei—Halococcus dombrowskii—Simulated microgravity—Rotary cell culture system—Antibiotic resistance—Lithopanspermia. Astrobiology 11, 199–205.
Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.
Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (aw). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of aw ≤ 0.75, but exposure to buffered 4 m LiCl (aw ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering aw should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.
Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.
Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003).
When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars.
Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (-SS-) and loss of sulfhydryl (-SH) in the lens nucleus of O2-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90° geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of -SS- (503 cm−1) and -SH (2577 cm−1) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the -SS- and -SH regions. -SS- levels in the O2-treated nucleus were found to have increased by a factor of 2.1 (p=0.0001) and 2.5 (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the -SS- increase was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. -SH levels in the O2-treated nucleus decreased by 13% (p=0.007) and 35% (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in -SS- or loss of -SH was observed in the lens cortex of the O2-treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of -SS- and loss of -SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O2, disulfide-crosslinking of lens crystallins in the nucleus, and nuclear cataract.
Raman spectroscopy; lens; nucleus; hyperbaric oxygen; disulfide; sulfhydryl; nuclear cataract; animal model
The Raman spectroscopic technology can be utilized for the detection of changes occurring at the molecular level during the pathological transformation of the tissue. The potential of its use in urology is still in its infancy and increasing utility of this technology will transform noninvasive tissue diagnosis. The Nobel laureate, Sir C.V. Raman is credited for the discovery of the principles of Raman spectroscopy.
Materials and Methods:
Applications of Raman spectroscopy in the bladder, renal, prostate, and other urological disorders were gathered from Medline and abstracts from recent international urological meetings. Current status and future directions of Raman spectroscopy in urology were also reviewed.
Raman spectroscopic technology is used to interrogate biological tissues. The potential use of this technology in urology has shown encouraging results in the in vitro diagnosis and grading of cancers of the bladder and the prostate. Raman microprobes have been used for the characterization and identification of renal lithiasis. Technology may be available for the urologists to determine the margin status intraoperatively during partial nephrectomy and radical prostatectomy. The future would see the development of optical fiber probes to incorporate them into catheters, endoscopes, and laparoscopes that will enable the urologist to obtain information during the operation.
Raman spectroscopy is an exciting tool for real-time diagnosis and in vivo evaluation of living tissue. The potential applications of Raman spectroscopy may herald a new future in the management of various malignant, premalignant, and other benign conditions in urology.
Applications; Raman spectroscopy; review; urology
In the molecular world, researchers act as detectives working hard to unravel the mysteries surrounding cells. One of the researchers' greatest tools in this endeavor has been Raman spectroscopy. Raman spectroscopy is a spectroscopic technique that measures the unique Raman spectra for every type of biological molecule. As such, Raman spectroscopy has the potential to provide scientists with a library of spectra that can be used to unravel the makeup of an unknown molecule. However, this technique is limited in that it is not able to manipulate particular structures without disturbing their unique environment. Recently, a novel technology that combines Raman spectroscopy with optical tweezers, termed Raman tweezers, evades this problem due to its ability to manipulate a sample without physical contact. As such, Raman tweezers has the potential to become an incredibly effective diagnostic tool for differentially distinguishing tissue, and therefore holds great promise in the field of virology for distinguishing between various virally infected cells. This review provides an introduction for a virologist into the world of spectroscopy and explores many of the potential applications of Raman tweezers in virology.
To support the translation of Raman spectroscopy into clinical applications, synthetic models are needed to accurately test, optimize and validate prototype fiber optic instrumentation. Synthetic models (also called tissue phantoms) are widely used for developing and testing optical instrumentation for diffuse reflectance, fluorescence, and Raman spectroscopies. While existing tissue phantoms accurately model tissue optical scattering and absorption, they do not typically model the anatomic shapes and chemical composition of tissue. Because Raman spectroscopy is sensitive to molecular composition, Raman tissue phantoms should also approximate the bulk tissue composition. We describe the fabrication and characterization of tissue phantoms for Raman tomography and spectroscopy. These phantoms have controlled chemical and optical properties, and also multilayer morphologies which approximate the appropriate anatomic shapes. Tissue phantoms were fabricated to support on-going Raman studies by simulating human wrist and rat leg. Surface meshes (triangle patch models) were generated from computed tomography (CT) images of a human arm and rat leg. Rapid prototyping was used to print mold templates with complex geometric patterns. Plastic casting techniques used for movie special effects were adapted to fabricate molds from the rapid prototypes, and finally to cast multilayer gelatin tissue phantoms. The gelatin base was enriched with additives to model the approximate chemistry and optical properties of individual tissue layers. Additional studies were performed to determine optimal casting conditions, phantom stability, layer delamination and chemical diffusion between layers. Recovery of diffuse reflectance and Raman spectra in tissue phantoms varied with probe placement. These phantoms enable optimization of probe placement for human or rat studies. These multilayer tissue phantoms with complex geometries are shown to be stable, with minimal layer delamination and chemical diffusion.
A profile across 8 layers from a fossil travertine terrace from a low temperature geothermal spring located in Svalbard, Norway has been studied using both Raman spectroscopy and SEM (Scanning Electron Microscopy) techniques to identify minerals and organic life signals.
Calcite, anatase, quartz, haematite, magnetite and graphite as well as scytonemin, three different carotenoids, chlorophyll and a chlorophyll-like compound were identified as geo- and biosignatures respectively, using 785 and/or 514 nm Raman laser excitation wavelengths. No morphological biosignatures representing remnant microbial signals were detected by high-resolution imaging, although spectral analyses indicated the presence of organics. In contrast, in all layers, Raman spectra identified a series of different organic pigments indicating little to no degradation or change of the organic signatures and thus indicating the preservation of fossil biomarker compounds throughout the life time of the springs despite the lack of remnant morphological indicators.
With a view towards planetary exploration we discuss the implications of the differences in Raman band intensities observed when spectra were collected with the different laser excitations. We show that these differences, as well as the different detection capability of the 785 and 514 nm laser, could lead to ambiguous compound identification. We show that the identification of bio and geosignatures, as well as fossil organic pigments, using Raman spectroscopy is possible. These results are relevant since both lasers have been considered for miniaturized Raman spectrometers for planetary exploration.
An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.
The laser, detection system, and methods that enable femtosecond broadband stimulated Raman spectroscopy (FSRS) are presented in detail. FSRS is a unique tool for obtaining high time resolution (<100 fs) vibrational spectra with an instrument response limited frequency resolution of <10 cm–1. A titanium:Sapphire-based laser system produces the three different pulses needed for FSRS: (1) A femtosecond visible actinic pump that initiates the photochemistry, (2) a narrow bandwidth picosecond Raman pump that provides the energy reservoir for amplification of the probe, and (3) a femtosecond continuum probe that is amplified at Raman resonances shifted from the Raman pump. The dependence of the stimulated Raman signal on experimental parameters is explored, demonstrating the expected exponential increase in Raman intensity with concentration, pathlength, and Raman pump power. Raman spectra collected under different electronic resonance conditions using highly fluorescent samples highlight the fluorescence rejection capabilities of FSRS. Data are also presented illustrating our ability: (i) To obtain spectra when there is a large transient absorption change by using a shifted excitation difference technique and (ii) to obtain high time resolution vibrational spectra of transient electronic states.
Raman spectroscopy is an optical imaging modality which analyses the Raman effect in which energy is exchanged between light and matter. Although Raman spectroscopy has been widely used for chemical and molecular analysis, its use in clinical applications has been hindered by the inherently weak nature of the Raman effect. Raman-silica-gold-nanoparticles (R-Si-Au-NPs) overcome this limitation by producing high Raman signals via Surface Enhanced Raman Scattering. Targeted polyethylene glycol (PEG)-ylated R-Si-Au-NPs (e.g. PEG-R-Si-Au-NPs labeled with an affibody which binds specifically to the epidermal growth factor receptor) are currently being designed to detect colorectal cancer after administration into the bowel lumen. With this approach, PEG-R-Si-Au-NPs are not expected to enter the systemic circulation and would be removed from the body via defecation. We examined the acute toxicity and biodistribution of core PEG-R-Si-Au-NPs after different routes of administration in mice. After intravenous administration, PEG-R-Si-Au-NPs were removed from the circulation by marcophages in the liver and spleen (i.e. the reticuloendothelial system). At 24 hours, PEG-R-Si-Au-NPs elicited a mild inflammatory response and an increase in oxidative stress in the liver, which subsided by 2 weeks. No evidence of significant toxicity was observed by measuring clinical, histological, biochemical or cardiovascular parameters for 2 weeks. Notably, after administration per rectum, we observed no significant bowel or systemic toxicity and no PEG-R-Si-Au-NPs were detected systemically. Although additional studies are required to investigate the long-term effects of PEG-R-Si-Au-NPs, these initial results support the idea that they can be safely used in living subjects, especially when administered rectally.
The lipid composition of the extremely halophilic archaeon
Haloquadratum walsbyi was investigated by thin-layer
chromatography and electrospray ionization-mass spectrometry. The
analysis of neutral lipids showed the presence of vitamin MK-8,
squalene, carotene, bacterioruberin and several retinal isomers. The
major polar lipids were phosphatidylglycerophosphate methyl ester,
phosphatidylglycerosulfate, phosphatidylglycerol and sulfated
diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or
dimeric phospholipids, only traces of bisphosphatidylglycerol were
detected. When the cells were exposed to hypotonic medium, no changes
in the membrane lipid composition occurred. Distinguishing it from
other extreme halophiles of the Halobacteriaceae
family, the osmotic stress did not induce the
neo-synthesis of cardiolipins in H. walsbyi. The
difference may depend on the three-laminar structure of the cell wall,
which differs significantly from that of other Haloarchaea.
Archaea; archaeal phospholipids; ether lipids; Halobacteriaceae
Raman spectra, obtained using a Raman microscope, offer an unique and incisive approach to follow interactions and reactions inside a single crystal under soak-in or soak-out conditions. The utility of this approach derives from the finding that the Raman spectra from single macromolecular crystals, under normal (non-resonance) conditions, are extremely stable, with a low “light background,” and provide ideal platforms for Raman difference spectroscopy. In turn, this allows the interrogation of sub-molecular changes in very large and complex macromolecular environments. There is often great synergy with X-ray crystallography, with the Raman spectroscopist providing crystallography colleagues with the best soak-in conditions to generate a targeted intermediate for flash freezing and X-ray analysis. On the other hand, X-ray structures at points along a reaction pathway provide invaluable benchmarks for interpreting the Raman data from populations seen by Raman to be changing in real-time. These principles will be illustrated by two reactions: The first involves a complex, branching reaction pathway underlying the inhibition of β-lactamases by clinically important pharmaceutical compounds, where different combinations of drug and enzyme function in different regions of the pathway. The second shows how temporal data can be derived for several events in the initiation step of RNA synthesis—more specifically, when one GTP molecule is joined to one ATP molecule to form a G•A dimer in the active site of a 115,000 Dalton crystalline RNA polymerase. Finally, we will summarize the extension of Raman microscopy to nucleic acid crystals and the information that has been obtained for RNA-based enzymes.
Eighteen strains of extremely halophilic bacteria and three strains of moderately halophilic bacteria were isolated from four different solar salt environments. Growth tests on carbohydrates, low-molecular-weight carboxylic acids, and complex medium demonstrated that the moderate halophiles and strains of the extreme halophiles Haloarcula and Halococcus grew on most of the substrates tested. Among the Halobacterium isolates were several metabolic groups: strains that grew on a broad range of substrates and strains that were essentially confined to either amino acid (peptone) or carbohydrate oxidation. One strain (WS-4) only grew well on pyruvate and acetate. Most strains of extreme halophiles grew by anaerobic fermentation and possibly by nitrate reduction. Tests of growth potential in natural saltern brines demonstrated that none of the halobacteria grew well in brines which harbor the densest populations of these bacteria in solar salterns. All grew best in brines which were unsaturated with NaCl. The high concentrations of Na+ and Mg2+ found in saltern crystallizer brines limited bacterial growth, but the concentrations of K+ found in these brines had little effect. MgSO4 was relatively more inhibitory to the extreme halophiles than was MgCl2, but the reverse was true for the moderate halophiles.
Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We developed Raman spectroscopy decision algorithms to detect breast microcalcifications, based on fit coefficients (FC) derived by modeling tissue Raman spectra as a linear combination of the Raman spectra of 9 chemical and morphologic components of breast tissue. However, little or no information is available on the precision of such measurements and its effect on the ability of Raman spectroscopy to make predictions for breast microcalcification detection. Here we report the precision, that is, the closeness of agreement between replicate Raman spectral measurements - and the model FC derived from them - obtained ex vivo from fresh breast biopsies from patients undergoing stereotactic breast needle biopsy, using a compact clinical Raman system. The coefficients of variation of the model FC averaged 0.03 for normal breast tissue sites, 0.12 for breast lesions without and 0.22 for breast lesions with microcalcifications. Imprecision in the FC resulted in diagnostic discordance among replicates only for line-sitters, that is, tissue sites with FC values near the decision line or plane. The source of this imprecision and their implications for the use of Raman spectroscopy for guidance of stereotactic breast biopsies for microcalcifications are also discussed. In summary, we conclude that the precision of Raman spectroscopy measurements in breast tissue obtained using our compact clinical system is more than adequate to make accurate and repeatable predictions of microcalcifications in breast tissue using decision algorithms based on model FC. This provides strong evidence of the potential of Raman spectroscopy guidance of stereotactic breast needle biopsies for microcalcifications.
Raman; spectroscopy; precision; breast; microcalcifications
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. Key Words: Atacama Desert—Life detection—Biosensor—Biopolymers—In situ measurement. Astrobiology 11, 969–996.
Background: Raman spectroscopy has the unique potential to detect and quantify cholesterol and calcification in an atherosclerotic plaque in vivo.
Objective: To evaluate the sensitivity and specificity of this technique for detecting cholesterol or calcification in human coronary artery and aorta specimens ex vivo, using a compact clinical fibreoptic based Raman system developed for in vivo applications.
Design: From nine coronary arteries and four aorta specimens, 114 sites were evaluated for the presence of cholesterol and calcification by Raman spectroscopy and standard histology. Raman spectra were acquired and evaluated on-line in around five seconds.
Results: The correlation between Raman spectroscopy and histology was r = 0.68 for cholesterol and r = 0.71 calcification in the plaque (p < 0.0001). Sensitivity and specificity for detecting cholesterol and calcification were excellent: receiver operating characteristic (ROC) analysis for each of the components revealed areas under the curves of > 0.92 (p < 0.0001). At the optimal cut-off values determined by ROC analysis, positive predictive values of > 80% and negative predictive values of > 90% were obtained.
Conclusions: On-line real time catheter based Raman spectroscopy detects accumulation of cholesterol and calcification in atherosclerotic plaque with high sensitivity and specificity.
atherosclerosis; vulnerable plaque; Raman spectroscopy
This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cell’s biochemical composition is collected at a spatial resolution of typically 25 mm. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes.
Epithelial cancers, including those of the skin and cervix, are the most common type of cancers in humans. Many recent studies have attempted to use Raman spectroscopy to diagnose these cancers. In this paper, Raman spectral markers related to the temporal and spatial effects of cervical and skin cancers are examined through four separate but related studies. Results from a clinical cervix study show that previous disease has a significant effect on the Raman signatures of the cervix, which allow for near 100% classification for discriminating previous disease versus a true normal. A Raman microspectroscopy study showed that Raman can detect changes due to adjacent regions of dysplasia or HPV that cannot be detected histologically, while a clinical skin study showed that Raman spectra may be detecting malignancy associated changes in tissues surrounding nonmelanoma skin cancers. Finally, results of an organotypic raft culture study provided support for both the skin and the in vitro cervix results. These studies add to the growing body of evidence that optical spectroscopy, in this case Raman spectral markers, can be used to detect subtle temporal and spatial effects in tissue near cancerous sites that go otherwise undetected by conventional histology.
Raman spectroscopy; optical diagnosis; cervix; dysplasia; skin cancer; malignancy associated changes; field effect; raft cultures; spectral markers