Methodology for dyes purification by preparative bidimensional offline reversed-phase high-performance liquid chromatography separation based on mobile phase pH change: Case study of Justicia spicigera.

The extensive characterization of the natural dyes involves the purification of their colored compounds for their structural analysis and stability studies. As most natural compounds being ionizable, herein is presented the optimization of an easy and affordable preparative bidimensional offline reversed-phase high-performance liquid chromatography purification based on mobile phase pH change. At the analytical scale, several combinations of stationary phases and mobile phases at different pH values were investigated first. The orthogonality between the dimensions was quantitatively evaluated using the nearest-neighbor distance approach. The optimized separation method was transferred to the preparative scale for the successful isolation of two colored compounds from Justicia spicigera, a traditional dye plant from Central America. They were identified as perisbivalvine B (2-amino-8-hydroxy-7-methoxy-3H-phenoxazin-3-one) and one of its derivatives, also found in another tinctorial plant species, Peristrophe bivalvis growing in Asia.

Spectral characterisation of a traditional Mesoamerican dye: relationship between in situ identification on the 16th century Codex Borbonicus manuscript and composition of Justicia spicigera plant extract.

Characterisation of vegetal colourants in manuscripts is still a challenging task. Their identification using non-invasive techniques - since sampling is rarely possible - is valid only if there is evidence that the recorded signals are generated by compounds specific to the plant species. Otherwise, more extensive chemical characterisations are required to relate the non-invasive technique signals to the chemical composition of the dye extract and thus avoid misidentification. The case study of a traditional Mesoamerican dye found in the Codex Borbonicus is reported herein. The non-invasive identification of the colourant plant source and chemical characterisation of the coloured molecules were carried out through a multi-analytical technique approach. In situ Raman and UV-Vis emission fluorescence signals suggested the use of Justicia spicigera leaves to produce the brown paint layers of the manuscript. Analysis of the plant extract by liquid chromatography revealed that two compounds are mostly responsible for the colour, and these compounds were successfully isolated. Both contribute to the Raman spectra while only one of them is mainly responsible for the fluorescence recorded on Codex Borbonicus.

Transparency improves concealment in cryptically coloured moths.

Predation is a ubiquitous and strong selective pressure on living organisms. Transparency is a predation defence widespread in water but rare on land. Some Lepidoptera display transparent patches combined with already cryptic opaque patches. A recent study showed that transparency reduced detectability of aposematic prey with conspicuous patches. However, whether transparency has any effect at reducing detectability of already cryptic prey is still unknown. We conducted field predation experiments with free avian predators where we monitored and compared survival of a fully opaque grey artificial form (cryptic), a form including transparent windows and a wingless artificial butterfly body. Survival of the transparent forms was similar to that of wingless bodies and higher than that of fully opaque forms, suggesting a reduction of detectability conferred by transparency. This is the first evidence that transparency decreases detectability in cryptic terrestrial prey. Future studies should explore the organization of transparent and opaque patches in animals and their interplay on survival, as well as the costs and other potential benefits associated with transparency on land.

Spectroscopies and Electron Microscopies Unravel the Origin of the First Colour Photographs.

The first colour photographs were created by a process introduced by Edmond Becquerel in 1848. The nature of these photochromatic images colours motivated a debate between scientists during the XIXth century, which is still not settled. We present the results of chemical analysis (EDX, HAXPES and EXAFS) and morphology studies (SEM, STEM) aiming at explaining the optical properties of the photochromatic images (UV-visible spectroscopy and low loss EELS). We rule out the two hypotheses (pigment and interferences) that have prevailed since 1848, respectively based on variations in the oxidation degree of the compound forming the sensitized layer and periodically spaced photolytic silver planes. A study of the silver nanoparticles dispersions contained in the coloured layers showed specific localizations and sizes distributions of the nanoparticles for each colour. These results allow us to formulate a plasmonic hypothesis on the origin of the photochromatic images colours.

Why has transparency evolved in aposematic butterflies? Insights from the largest radiation of aposematic butterflies, the Ithomiini.

Defended species are often conspicuous and this is thought to be an honest signal of defences, i.e. more toxic prey are more conspicuous. Neotropical butterflies of the large Ithomiini tribe numerically dominate communities of chemically defended butterflies and may thus drive the evolution of mimetic warning patterns. Although many species are brightly coloured, most are transparent to some degree. The evolution of transparency from a warning-coloured ancestor is puzzling as it is generally assumed to be involved in concealment. Here, we show that transparent Ithomiini species are indeed less detectable by avian predators (i.e. concealment). Surprisingly, transparent species are not any less unpalatable, and may in fact be more unpalatable than opaque species, the latter spanning a larger range of unpalatability. We put forth various hypotheses to explain the evolution of weak aposematic signals in these butterflies and other cryptic defended prey. Our study is an important step in determining the selective pressures and constraints that regulate the interaction between conspicuousness and unpalatability.

Higher iridescent-to-pigment optical effect in flowers facilitates learning, memory and generalization in foraging bumblebees.

Iridescence-change of colour with changes in the angle of view or of illumination-is widespread in the living world, but its functions remain poorly understood. The presence of iridescence has been suggested in flowers where diffraction gratings generate iridescent colours. Such colours have been suggested to serve plant-pollinator communication. Here we tested whether a higher iridescence relative to corolla pigmentation would facilitate discrimination, learning and retention of iridescent visual targets. We conditioned bumblebees (Bombus terrestris) to discriminate iridescent from non-iridescent artificial flowers and we varied iridescence detectability by varying target iridescent relative to pigment optical effect. We show that bees rewarded on targets with higher iridescent relative to pigment effect required fewer choices to complete learning, showed faster generalization to novel targets exhibiting the same iridescence-to-pigment level and had better long-term memory retention. Along with optical measurements, behavioural results thus demonstrate that bees can learn iridescence-related cues as bona fide signals for flower reward. They also suggest that floral advertising may be shaped by competition between iridescence and corolla pigmentation, a fact that has important evolutionary implications for pollinators. Optical measurements narrow down the type of cues that bees may have used for learning. Beyond pollinator-plant communication, our experiments help understanding how receivers influence the evolution of iridescence signals generated by gratings.

2D macro-XRF to reveal redacted sections of French queen Marie-Antoinette secret correspondence with Swedish count Axel von Fersen.

During the French Revolution, Marie-Antoinette, queen of France and wife of Louis XVI, maintained a highly secret correspondence with the Swedish count Axel von Fersen, her close friend and rumored lover. An unidentified censor later redacted certain sections of the exchanged letters. This presumably sensitive content has been puzzling historians for almost 150 years. We report on the methodology that successfully unraveled this historical mystery. X-ray fluorescence spectroscopy was used in macroscanning mode on the redacted sections. Specific data processing was applied to improve the legibility of the hidden writings (elemental ratios, statistical data reduction, multimodal images fusion, unmixing procedure, and image treatments). This methodology successfully revealed the redacted contents of eight letters, shedding new light not only on Marie-Antoinette and Fersen relationship but also on the author of the redactions. It will also be of great interest for other historical and forensic cases involving the disentanglement of superimposed multi-elemental materials.

Mimicry can drive convergence in structural and light transmission features of transparent wings in Lepidoptera.

Müllerian mimicry is a positive interspecific interaction, whereby co-occurring defended prey species share a common aposematic signal. In Lepidoptera, aposematic species typically harbour conspicuous opaque wing colour patterns with convergent optical properties among co-mimetic species. Surprisingly, some aposematic mimetic species have partially transparent wings, raising the questions of whether optical properties of transparent patches are also convergent, and of how transparency is achieved. Here, we conducted a comparative study of wing optics, micro and nanostructures in neotropical mimetic clearwing Lepidoptera, using spectrophotometry and microscopy imaging. We show that transparency, as perceived by predators, is convergent among co-mimics in some mimicry rings. Underlying micro- and nanostructures are also sometimes convergent despite a large structural diversity. We reveal that while transparency is primarily produced by microstructure modifications, nanostructures largely influence light transmission, potentially enabling additional fine-tuning in transmission properties. This study shows that transparency might not only enable camouflage but can also be part of aposematic signals.

Quantitative characterization of iridescent colours in biological studies: a novel method using optical theory.

Iridescent colours are colours that change with viewing or illumination geometry. While they are widespread in many living organisms, most evolutionary studies on iridescence do not take into account their full complexity. Few studies try to precisely characterize what makes iridescent colours special: their angular dependency. Yet, it is likely that this angular dependency has biological functions and is therefore submitted to evolutionary pressures. For this reason, evolutionary biologists need a repeatable method to measure iridescent colours as well as variables to precisely quantify the angular dependency. In this study, we use a theoretical approach to propose five variables that allow one to fully describe iridescent colours at every angle combination. Based on the results, we propose a new measurement protocol and statistical method to reliably characterize iridescence while minimizing the required number of time-consuming measurements. We use hummingbird iridescent feathers and butterfly iridescent wings as test cases to demonstrate the strengths of this new method. We show that our method is precise enough to be potentially used at intraspecific level while being also time-efficient enough to encompass large taxonomic scales.

Characterizing the Intrinsic Fluorescence Properties of Historical Painting Materials: The Case Study of a Sixteenth-Century Mesoamerican Manuscript.

Ultraviolet visible (UV-Vis) fluorescence spectroscopy is widely used to study polychrome objects and can help to identify the nature of certain materials when they present specific fluorescent properties. However, given the complexity of the stratified and heterogeneous materials under study, the characterization of an intrinsic fluorescence related to a given constituent (a pigment or a binder composing a paint layer for example) is not straightforward, and the recorded raw data need to be corrected for a number of effects that can influence the detected spectral distribution. The application of standard correction procedures to experimental fluorescence data gathered on the polychromatic surface of the Codex Borbonicus, a 16th-century Aztec manuscript, is described. The results are confronted to an alternate new methodology that is based on the hypothesis of transparent non-scattering paint layers. This second approach allows to establish more clearly the material origin of the detected emission and to discriminate apparent fluorescence (emitted by the substrate and transmitted through the paint layers) from actual intrinsic emission generated by the coloring materials under study. The results show that most of the various emission profiles detected in the paint layers of the manuscript actually originate from a unique fluorophore (composing the substrate) and should not be used to characterize the coloring materials.

Nanoscale investigation by TEM and STEM-EELS of the laser induced yellowing.

Nd-YAG QS laser cleaning of soiled stone at 1064 nm can sometimes result in a more yellow appearance compared to other cleaning techniques. Especially in France, this yellowing effect is still considered as a major aesthetic issue by the architects and conservators. One explanation states that the yellowing is linked to the formation of iron-rich nanophase(s) through the laser beam interaction with black crusts that would re-deposit on the cleaned substrate after irradiation. To characterize these nanophases, a model crust containing hematite was elaborated and laser irradiated using a Nd-YAG QS laser. The color of the sample shifted instantaneously from red to a bright yellow and numerous particles were ablated in a visible smoke. Transmission electron microscopy (TEM) was used to examine the morphology and the crystallinity of the neo-formed compounds, both on the surface of the samples and in the ablated materials. In addition, an investigation of the chemical and structural properties of the nanophases was conducted by X-ray dispersive energy (EDX) and electron energy loss (EELS) spectroscopies. It was found that both the surface of the sample and the ablated materials are covered by crystallized nano-spheres and nano-residues, all containing iron and oxygen, sometimes along with calcium and sulfur. In particular an interfacial area containing the four elements was evidenced between some nanostructures and the substrate. Magnetite Fe3O4 was also identified at the nanoscale. This study demonstrates that the laser yellowing of a model crust is linked to the presence of iron-rich nanophases including CaxFeySzOδ nanostructures and magnetite Fe3O4 at the surface after irradiation.

Model of light scattering that includes polarization effects by multilayered media.

We present a model for calculating the angular distribution of light, including polarization effects from multilayered inhomogeneous media, with an index of refraction mismatch between layers. The model is based on the resolution of the radiative transfer equation by the discrete ordinate method. Comparisons with previous simpler models and examples of simulations are presented.

Comparison of the auxiliary function method and the discrete-ordinate method for solving the radiative transfer equation for light scattering.

Two methods for solving the radiative transfer equation are compared with the aim of computing the angular distribution of the light scattered by a heterogeneous scattering medium composed of a single flat layer or a multilayer. The first method [auxiliary function method (AFM)], recently developed, uses an auxiliary function and leads to an exact solution; the second [discrete-ordinate method (DOM)] is based on the channel concept and needs an angular discretization. The comparison is applied to two different media presenting two typical and extreme scattering behaviors: Rayleigh and Mie scattering with smooth or very anisotropic phase functions, respectively. A very good agreement between the predictions of the two methods is observed in both cases. The larger the number of channels used in the DOM, the better the agreement. The principal advantages and limitations of each method are also listed.

Absorption in multiple-scattering systems of coated spheres.

We derive formulas for rigorous transfer matrix calculations of absorption in multiple-coherent-scattering systems in which the scatterers are multiply coated spheres (not necessarily concentric). Any of the spherical coatings, cores, or host media may be composed of absorbing materials. For a nonabsorbing host media, the total absorption may be deduced from overall energy conservation. A more detailed description of the absorption is obtained through formulas yielding the absorption within individual scatterers and/or coatings. We present some illustrative applications of these formulas to the design of heterogeneous coated-sphere media exhibiting enhanced absorption.