Emma M. Newton

Raman spectroscopic analysis of pigments and substrata in prehistoric rock art

Abstract The application of Fourier-transform Raman spectroscopy to the analysis of pigments in samples of prehistoric cave art is reported. Despite the limitations of a restricted colour palette used by the artists, the nondestructive identification of natural mineral pigments is accomplished and the unique information provided by the Raman technique is highlighted. The observed deterioration in cave art sites caused by local environmental, biochemical and geochemical changes is a cause for concern among conservationists and historians; the role of Raman spectroscopy in the identification of chemical products of biodeterioration, in particular, can assist in the elucidation of agencies which may be responsible for site instability.

Pigmentation as a survival strategy for ancient and modern photosynthetic microbes under high ultraviolet stress on planetary surfaces

Solar radiation is the primary energy source for surface planetary life, so that pigments are fundamental components of any surface-dwelling organism. They may therefore have evolved in some form on Mars as they did on Earth. Photosynthetic microbes are major primary producers on Earth, but are concurrently vulnerable to ultraviolet (UV) damage. Using non-intrusive laser Raman spectroscopy to recognize the component parts of biomolecules, we have shown not only the abundance of microbial photosynthetic and photoprotective pigments in situ, but also their spatial distribution within their microhabitat. This essential aspect of their screening or avoidance survival strategies is lost on extraction with solvents. This precise approach is eminently suited to analysis of epilithic (surface) and endolithic (within rocks) communities in Antarctic desert habitats, which are putative analogues of early Mars. Raman spectra for key biomolecules (e.g. the UV screen parietin and the antioxidant b-carotene in epilithic lichens) enable not only the detection of organics in light-stratified habitats, but also the characterization of unknown pigments. Typical biomarkers of astrobiological relevance in our Raman spectral database include scytonemin (a UV screen), chlorophyll (primary photosynthetic pigment), phycocyanin (accessory pigment for shade adaptation) and a hopanoid extracted from 2-5 Gya microbial stromatolite from Australia. This compound dates from the same time period when a wetter Mars could have had a potentially flourishing surface microbial community of its own. Analyses with a laboratory Raman instrument have been extended to a novel miniature Raman spectrometer, operating at the same optimal excitation wavelength (1064 nm) via an In-Ga-As detector. After evaluation in Antarctica, this instrument will be space-qualified for a proposed Mars rover mission to detect biomolecules in the near- surface sediment profile of palaeolakes, using experience with Antarctic biomarkers to interpret alien spectra of fundamental components, without the need for prior knowledge of the identity of the target compounds.

A novel miniature confocal microscope/Raman spectrometer system for biomolecular analysis on future Mars missions after Antarctic trials

Biomolecules, such as the productive and protective pigments of photosynthetic organisms, are good biomarkers in extreme Antarctic deserts as analogues of early Mars. Laser Raman technology at long wavelengths which minimize fluorescence is ideal for remote analysis of biomolecules in situ. We report Raman spectra obtained with a prototype miniature laser-Raman spectrometer/confocal microscope (specification < 1 kg) under development for a Mars lander and evaluation in Antarctic deserts. We compare the efficiency of its 852 nm laser/CCD detector system with an optimal bench-top 1064 nm FT Raman spectrometer which excels with biomolecules. Using a yellow Antarctic lichen, Acarospora chlorophana, we show good correlation between both instruments restricted to the 460–1350 cm−1 wavenumber range.

Vibrational Raman spectroscopic study of scytonemin, the UV-protective cyanobacterial pigment

The Raman spectrum of the photoprotective pigment scytonemin found in cyanobacterial sheaths has been obtained for the first time. Its skeletal structure is extensively conjugated and unique in nature. Detailed molecular vibrational assignments are proposed and a distinctive group of four corroborative vibrational bands have been identified as unique indicators for the compound. These bands, especially a prominent feature at wavenumber 1590 cm(-1), are sufficiently conspicuous to be detectable in the mixed biomolecular pools of undisturbed natural microbial communities. This has been confirmed by demonstrating the Raman spectral bands for scytonemin in a sample of an intact intertidal cyanobacterial mat.

Molecular structural studies of lichen substances II: atranorin, gyrophoric acid, fumarprotocetraric acid, rhizocarpic acid, calycin, pulvinic dilactone and usnic acid

The FT-Raman and infrared vibrational spectra of some important lichen compounds from two metabolic pathways are characterised. Key biomolecular marker bands have been suggested for the spectroscopic identification of atranorin, gyrophoric acid, fumarprotocetraric acid rhizocarpic acid, calycin, pulvinic dilactone and usnic acid. A spectroscopic protocol has been defined for the detection of these molecules in organisms subjected to environmental stresses such as UV-radiation exposure, desiccation and low temperatures. Use of the protocol will be made for the assessment of survival strategies used by stress-tolerant lichens in Antarctic cold deserts.

Raman spectroscopic studies of a 13th century polychrome statue: identification of a ‘forgotten’ pigment

A Raman microscopic analysis of pigments applied to a 13th century polychrome stone statue of Santa Ana in Santa Maria la Real, Sasamon, Spain, has successfully identified the materials used. The spectral analysis reveals that the stone substrate had been treated with gypsum prior to the application of the pigments. The use of cinnabar, mercury(II) sulfide, in admixture with lead(II) lead(IV) oxide (minium), is a feature of interest; the presence of calcite in the cinnabar could point to a local mineralogical source. The presence of organic compounds in the pigments analysed suggests their use as binding agents or surface varnishes for protection or enhancement of the appearance of the statue. The use of ‘mosaic gold’, tin(IV) sulfide, in place of gold on the hem of the cape, identified by Raman spectroscopy and confirmed by SEM points to the use of a forgotten technology. Our results also suggest a spectroscopic protocol for the identification of tin(IV) sulfide in ancient pigment mixtures in the presence of other sulfide pigments such as orpiment, realgar and cinnabar. Copyright

Molecular spectroscopic studies of lichen substances 1: parietin and emodin

The Raman and infrared spectra of the UV-radiation protectant parietin extracted from Xanthoria lichen species are reported and vibrational assignments made of key features which are important for the characterisation of parietin in situ in stresstolerant living organisms. The spectra of parietin are compared with those of the chemically related emodin, which is of pharmaceutical interest. The key molecular vibrational bands of parietin will be used for the analysis of lichen specimens exposed to high UV-radiation stress growing on Leonie Island, Antarctica, under the ‘ozone hole’ to better understand the production of UV-protectants in controlled habitats. q 2003 Published by Elsevier Science B.V.

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.

Minium; FT-Raman non-destructive analysis applied to an historical controversy

The term minium was applied to both cinnabar (HgS) and red lead (Pb3O4) pigments in antiquity; in Roman times, minium was reserved for mercury(II) sulfide but was applied increasingly to lead tetraoxide by the Renaissance. Confusion in the interpretation of ancient recipes for pigment mixtures is inevitable and is compounded by the practice of adulteration of mercury(II) sulfide with lead tetraoxide for economic or artistic reasons. In this study, the Raman spectra of mixtures of cinnabar and red lead were recorded and used to determine the composition of several pigment mixtures from mediaeval artwork. The potential of the method for the non-invasive quantitative analysis of pigment mixtures involving red lead and cinnabar is thereby demonstrated.

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.