ON THE INTERPRETATION OF ABSORPTION SPECTRA OF LEAVES–I. INTRODUCTION and THE CORRECTION OF LEAF SPECTRA FOR SURFACE REFLECTION

Abstract— Reflectance and transmittance spectra of leaves and their sum can be corrected to relate only to the light actually entering the leaf, if the reflectance of the epidermal surface is known. The latter is found if the leaf reflectances at several wavelengths near the transmittance minimum in the red are plotted vs the transmittances of a homogeneous suspension of the native pigment-proteins at the same chlorophyll content per unit area and at the same wavelengths. With non-senescent leaves, the relation is linear and the extrapolation of the pigment transmittance to zero gives the value for the surface reflection. Surface reflectance data (both adaxial and abaxial) are given for the leaves of a number of trees and a few herbs, plus examples of the raw and corrected spectra. With normal, glaucous leaves, the adaxial reflectance averaged 4.5% of the incident light ( n = 23, range = 3.7 −5.9, standard deviation = 0.4). The reflectances of the abaxial surfaces ranged between 7 and 13% since additional near-surface reflection occurred at the inside of the epidermis and in the spongy mesophyll. Reflectance and transmittance data demonstrated strong absorption in the epidermis below 480 nm.     

Flavonoid distribution in tissues of Phillyrea latifolia L. leaves as estimated by microspectrofluorometry and multispectral fluorescence microimaging

A new method for detecting the tissue-specific distribution of flavonoids has been developed by coupling microspectrofluorometry and multispectral fluorescence microimaging techniques. Fluorescence responses of cross sections taken from 1 year old Phillyrea latifolia leaves exposed to full (sun leaves) or 15% (shade leaves) solar radiation in a coastal area of Southern Tuscany were analyzed. Fluorescence spectra of different tissue layers, each normalized at its fluorescence maximum, that were stained or not stained with Naturstoff reagent A (in ethanol), under excitation with UV light (lambdaexc = 365 nm) or blue light (lambdaexc = 436 nm) were recorded. The shape of the fluorescence spectra of tissue layers from shade and sun leaves differed only under UV excitation. The fluorescence of stained cross sections from sun and shade leaves as well as from different layers of sun leaves received a markedly different contribution from the blue (470 nm) and the yellow-red (580 nm) wavebands. Such changes in tissue fluorescence signatures were related to light-induced changes of extractable caffeic acid derivatives and flavonoid glycosides, namely quercetin 3-O-rutinoside and luteolin 7-O-glucoside. Wall-bound phenolics, i.e. hydroxycinnamic acids (p-coumaric, ferulic and caffeic acid) and flavonoids (apigenin and luteolin derivatives), did not substantially differ between sun and shade leaves. A Gaussian deconvolution analysis of fluorescence spectra was subsequently performed to estimate the contribution of flavonoids (emitting at 600 nm, F600 [red fluorescence contribution = signal integrated over a Gaussian band centered at about 600 nm]) relative to the tissue fluorescence (Ftot [total fluorescence = signal integrated over the whole fluorescence spectrum]). The F600/ Ftot ratios sharply differed between analogous tissues of sun and shade leaves, as well as among tissue layers within each leaf type. A highly resolved picture of the tissue flavonoid distribution was finally provided through a fluorescence microimaging technique by acquiring fluorescence images at the blue (fluorescence at about 470 nm [F470]) and yellow-red (fluorescence at about 580 nm [F580]) wavelengths and correcting the F580 image for the contribution of nonflavonoids to the fluorescence at 580 nm. Monochrome images were elaborated by adequate computing functions to visualize the exclusive accumulation of flavonoids in different layers of P. latifolia leaves. Our data show that in shade leaves flavonoids almost exclusively occurred in the adaxial epidermal layer. In sun leaves flavonoids largely accumulated in the adaxial epidermal and subepidermal cells and followed a steep gradient passing from the adaxial epidermis to the inner spongy layers. Flavonoids also largely occurred in the abaxial epidermal cells and constituted the exclusive class of phenylpropanoids synthesized by the cells of glandular trichomes. The proposed method also allowed for the discrimination of the relative abundance of hydroxycinnamic derivatives and flavonoids in different layers of the P. latifolia leaves.     

Quantitative study of fluorescence excitation and emission spectra of bean leaves

A quantitative and comprehensive knowledge of leaf fluorescence is required for the interpretation of fluorescence signals at the canopy level and also for the modelling of leaf and canopy fluorescence. In this work we present full range fluorescence excitation and emission spectra of intact leaves, expressed in units of apparent spectral fluorescence yield, from both the adaxial and the abaxial sides of the leaves, and for both front-side and back-side geometries. Emission spectra were measured for incident radiations in the blue and the green spectral range. The red/far-red fluorescence ratio depended on the measurement geometry and on the excitation wavelength. Excitation spectra were measured for emissions at 687 and 760 nm. When the abaxial side was illuminated, the measured spectra always had a larger intensity compared to adaxial side that is explained by the higher scattering of the spongy tissues. At 760 nm, the spectra had the same shape for front-side and back-side geometry, indicating that scattering predominated. At 687 nm, the shape of the spectra was very different for front-side and back-side geometry due to re-absorption of red fluorescence within the leaf. The comparison of excitation spectra measured from the adaxial or the abaxial side revealed differences in carotenoid absorption.     

Responses of epidermal phenolic compounds to light acclimation: in vivo qualitative and quantitative assessment using chlorophyll fluorescence excitation spectra in leaves of three woody species

Chlorophyll fluorescence (ChlF) excitation spectra were measured to assess the UV-sunscreen compounds accumulated in fully expanded leaves of three woody species belonging to different chemotaxons, (i.e. Morus nigra L., Prunus mahaleb L. and Lagerstroemia indica L.), grown in different light microclimates. The logarithm of the ratio of ChlF excitation spectra (logFER) between two leaves acclimated to different light microclimates was used to assess the difference in epidermal absorbance (EAbs). EAbs increased with increasing solar irradiance intercepted for the three species. This epidermal localisation of UV-absorbers was confirmed by the removal of the epidermis. It was possible to simulate EAbs as a linear combination of major phenolic compounds (Phen) identified in leaf methanol extracts by HPLC-DAD. Under UV-free radiation conditions, shaded leaves of M. nigra accumulated chlorogenic acid. Hydroxybenzoic acid (HBA) derivatives and hydroxycinnamic acid (HCA) derivatives greatly increased with increasing PAR irradiance under the low UV-B conditions found in the greenhouse. These traits were also observed for the HCA of the two other species. Flavonoid (FLAV) accumulation started under low UV-A irradiance, and became maximal in the adaxial epidermis of sun-exposed leaves outdoors. A decrease in the amount of HCA was observed concomitantly to the intense accumulation of FLAV for both leaf sides of the three species. Judging from the logFER, under low UV-B conditions, larger amounts of HCA are present in the epidermis in comparison to FLAV for the three species. Upon transition from the greenhouse to full sunlight outdoors, there was a decrease in leaf-soluble HCA that paralleled FLAV accumulation in reaction to increasing solar UV-B radiation in the three species. In M. nigra, that contains large amounts of HCA, the logFER analysis showed that this decrease occurred in the adaxial epidermis, whereas the abaxial epidermis, which is protected from direct UV-B radiation, continued to accumulate large amounts of HCA.     

霸王鞭和麒麟掌叶片的表面微结构及超疏水性

霸王鞭(Euphorbia antiquorum)和麒麟掌(Euphorbia neriifolia var. cristata)是2种特殊的叶片, 正面不疏水而叶片背面超疏水的沙漠植物. 本文通过接触角测试仪、 电子显微镜和表面张力测试仪分别对叶片的超疏水性、 表面微观形貌和表面黏附力进行了测试和表征. 采用模板法, 以聚乙烯醇为模板、 以聚苯乙烯为基底制备仿叶片背面结构的聚苯乙烯薄膜, 并对薄膜表面的超疏水性、 表面微观形貌和表面黏附力进行了测试和表征, 发现这2种叶片背面的平均间距为1~3 μm的层片状微观结构可以构建出具有超疏水高黏附力特性的表面.     

Optics of a Bifacial Leaf: 1. A Novel Combined Procedure for Deriving the Optical Parameters

Abstract— A novel experimental-theoretical procedure, deriving optical parameters of a bifacial leaf, is presented and analyzed. Its theoretical basis is the four-flux approximation of the radiative transfer theory. Its experimental basis is three-dimensional measurements of internal radiances at different depths and in different directions within a leaf by using optical microprobes. The intrinsic instrumental error of the microprobe measurements is accounted for by a special correcting theory. The treatment yields the spectral curves for attenuation, scattering and absorption coefficients as well as for the asymmetry of scattering in both palisade and spongy tissue layers. The values of obtained optical parameters were verified by comparing calculated transmission and remission of a leaf with those measured with the integrating sphere. It is concluded that the treatment provides a sound basis for the analysis of the light microenvironment as a function of leaf structure and the nature of incident light.     

Botanical Characterization of Guiera senegalensis Leaves

Guiera senegalensis J. F. Gmel. (Combretaceae) is one of the most important West African medicinal plants, often used to treat a variety of microbial infections. The most frequently used plant part is the leaf, its medicinal use being corroborated by several in vitro antimicrobial activity studies. However, quality criteria for pharmaceutical use, including botanical identification, are not yet determined. Aiming the establishment of such criteria, the present work deepens today's knowledge on G. senegalensis leaf morphology and anatomy, the anatomical characters of both leaf transversal section and powdered leaves being hereby presented for the first time. The most useful characters for identification purposes are leaf isobilateral organization, with similar upper and lower surfaces and a palisade parenchyma on both surfaces; trichome insertion points on both epidermis, surrounded by polar arrangements of cells; tomentose indumentum on the upper epidermis consisting of compartmented and uniseriated trichomes with long, curved, or straight terminal cells; brown scales, isolated or inserted into the epidermis, formed by radially arranged cells surrounding a central cell, with a more or less circular form and a conspicuous base; calcium oxalate cluster crystals, isolated or inserted into the palisade parenchyma and absent in lateral veins.     

RETARDATION OF ULTRAVIOLET LIGHT ACCELERATED CHLOROSIS BY VISIBLE LIGHT OR BY BENZYLADENINE IN NICOTIAN A GLUTINOSA LEAVES: CHANGES IN AMINO ACID CONTENT AND CHLOROPLAST ULTRASTRUCTURE*

Abstract— Low doses (180–720 Jm^-2) of ultraviolet light (254 nm) are known to accelerate the chlorosis of detached leaves in darkness. The development of such chlorosis is prevented by a photoreactivation treatment. However, we found that delayed light exposure or benzyladenine treatments (which were not effective in photorepair of UV-induced thymine dimers in cell DNA) were also effective in retarding the UV-accelerated chlorosis. Small drops of benzyladenine solution placed on the UV irradiated leaf formed green islands which acted as strong sinks for the accumulation of free amino acids during dark incubation. To a lesser degree, non–irradiated green tissues surrounded by irradiated yellow leaf tissue also acted as sinks for amino acid accumulation. The accelerated chlorophyll loss in UV-irradiated leaves was correlated with degradation of chloroplast ultrastructure. Visible light or benzyladenine retarded this chloroplast degradation. The accelerated senescence of UV irradiated leaf tissue, therefore, is ultrastructurally and physiologically similar to normal senescence of detached dark-incubated leaves, but progresses at a faster rate. When the lower leaf surface was irradiated with high UV doses (3600–10,800 Jm^-2), the chloroplast ultrastructure of the spongy cells (except the envelope) was preserved for 3 days after dark incubation. However, the chloroplasts of the palisade cells were in a late stage of senescence. Since the spongy cells were dead (plasmalemma, tonoplast and chloroplast envelope disappeared), the maintenance of green color and ultrastructure of chloroplasts could have been due to inhibition of degrading enzymes normally associated with senescence.     

Effects of supplemental UV-A on the development, anatomy and metabolite production of Phyllanthus tenellus cultured in vitro

Phyllanthus tenellus is widely used for its antiviral, analgesic and hepatoprotective properties. Although the production of several chemical classes of secondary metabolites is influenced by UV radiation, particularly phenolic compounds, we also know that UV radiation can result in anatomical and developmental damage. However, the morphological, anatomical and phytochemical changes in response to UV-A exposure are generally understudied in the Phyllanthaceae. Therefore, we evaluated the effects of UV-A radiation on plant development and leaf anatomy, as well as the production of secondary metabolites and the contents of carotenoids and chlorophylls a and b, in P. tenellus. To accomplish this, in vitro cultures of P. tenellus were maintained for 60 days under white light (WL) and WL plus UV-A radiation. Results showed different phenotypic responses under additional UV-A, such as high phenolic metabolite production, increasing dimensions of abaxial epidermis and thickness of palisade parenchyma. Compared to plants cultured under WL, UV-A radiation caused damage to plant morphogenesis, including a reduced number of branches and shoots, consequently reducing the rate of proliferation. On the other hand, geraniin, ellagic acid and carotenoid contents increased after UV-A exposure, indicating that this light source is an important resource for inducing phenolic compounds.     

Histological study of some Echium vulgare, Pulmonaria officinalis and Symphytum officinale populations

Plants living in different ecological habitats can show significant variability in their histological and phytochemical characters. The main histological features of various populations of three medicinal plants from the Boraginaceae family were studied. Stems, petioles and leaves were investigated by light microscopy in vertical and transverse sections. The outline of the epidermal cells, as well as the shape and cell number of trichomes was studied in leaf surface casts. Differences were measured among the populations of Echium vulgare in the width and height of epidermis cells in the stem, petiole and leaf, as well as in the size of palisade cells in the leaves. Among the populations of Pulmonaria officinalis significant differences were found in the length of trichomes and in the slightly or strongly wavy outline of epidermal radial cell walls. Populations of Symphytum officinale showed variance in the height of epidermal cells in leaves and stems, length of palisade cells and number of intercellular spaces in leaves, and the size of the central cavity in the stem. Boraginaceae bristles were found to be longer in plants in windy/shady habitats as opposed to sunny habitats, both in the leaves and stems ofP. officinalis and S. officinale, which might be connected to varying levels of exposure to wind. Longer epidermal cells were detected in the leaves and stems of both E. vulgare and S. officinale plants living in shady habitats, compared with shorter cells in sunny habitats. Leaf mesophyll cells were shorter in shady habitats as opposed to longer cells in sunny habitats, both in E. vulgare and S. officinale. This combination of histological characters may contribute to the plant's adaptation to various amounts of sunshine. The reported data prove the polymorphism of the studied taxa, as well as their ability to adapt to various ecological circumstances.     

ULTRAVIOLET-RADIATION-ACCELERATED LEAF CHLOROSIS: PREVENTION OF CHLOROSIS BY REMOVAL OF EPIDERMIS OR BY FLOATING LEAF DISCS ON WATER*

Abstract— Low doses (1800–7200 ergs/mm²) of ultraviolet (U V) radiation accelerated chlorosis in the mesophyll of Nicotiana glutinosa leaves when the lower epidermis of the leaves was irradiated. This occurred in either a subsequent light or dark incubation. However, within 12 h after irradiation, peeling of the lower epidermis prevented this accelerated chlorosis. The accelerated chlorosis was also prevented by floating irradiated leaf discs on water during the incubation period without removal of the epidermis. These results suggest that accelerated chlorosis in mesophyll tissue caused by low doses of UV is due to an indirect effect of the UV-damaged epidermis, possibly mediated by some toxic substance released from the epidermal cells. High UV doses (36,000–108,000 ergs/mm²) prevented normal yellowing of the leaf. The irradiated portion of the leaf remained green, while the nonirradiated area turned yellow upon dark incubation. However, if the irradiated leaf was incubated in continuous light, bleaching of the irradiated area took place, and the irradiated area became yellow faster than the nonirradiated area. Peeling of the epidermis did not affect the outcome of these experiments. These results suggest that high UV doses directly damage the mesophyll tissue.     

PHYTOCHROME MACRO-DISTRIBUTION, LOCAL PHOTOCONVERSION AND INTERNAL PHOTON FLUENCE RATE FOR Cucurbita pepo L. COTYLEDONS

Abstract— Using 7-day-oId cotyledons of Cucurbita pepo L., local phytochrome photoconversions could be measured for blue, red and far-red light. For this purpose, after nonsaturating irradiation, cotyledons were sliced into discs 0.3 to 0.5 mm thick and signals measured. This method also yielded the internal phytochrome distribution of the cotyledons with maximal concentration near the adaxial surface, dropping to about 50% in the center and reaching again about 90% at the abaxial surface. Local phytochrome conversion rates were used to calculate internal fluence rates across the cotyledons. Relative internal fluence rates were also derived from measured reflectances and transmittance according to the Kubelka-Munk theory. The general shape of the internal fluence distribution calculated on the basis of these two methods coincided well. It was observed that the internal local photoconversion is proportional to the penetration depth over a wide range of incident fluences and for all wavelengths tested, showing in addition that reciprocity holds. A method to calculate internal fluence rates by a simplified procedure assuming either linear or exponential functions is described.     

Natural UV-Screening Mechanisms of Norway Spruce (Picea abies [L.] Karst.) Neediest

Abstract— Ultraviolet-light screening potential of Norway spruce (Picea abies [L.] Karst.) needles was investigated by UV-spectroscopic, microscopic, fluorescence spectroscopic techniques as well as by HPLC, mass spectrometry and NMR spectroscopy. Results showed four potential barriers of UV screening by Norway spruce needles: (1) UV-light screening via reflectance of UV/violet light by epidermis, (2) UV-light screening via reduction of transmission of UV light by special anatomical arrangement of the epidermal cells containing the UV-screening allomelanins as well as by the light-reflecting hyaline hypodermal cells, (3) conversion of UV light by epidermis into photosynthetically active radiation (PAR; blue and red spectral bands) via fluorescence and (4) UV-light screening by absorption of UV light by UV-screening substances contained in the epidermis, whereby the latter was found to be the most important UV-screening mechanism. Staining of needle cross sections with Naturstoffreagenz A showed the localization of bound flavonoids and its derivatives in the cell walls of the outer epidermal cell layer as revealed by confocal laser scanning microscopy. By fluorescence spectroscopy and confocal laser scanning microscopy, the conversion of UVA light into PAR in the epidermis was related to various UV-screening substances contained in the epidermis. The methanol-soluble UV-absorbing substances were found to create novel UV-screening barrier zones: UVC, >200–253 nm; UVC/UVB, >253–300/303 nm; and UVB/UVA, >300–362/368 nm in epidermis as well as in mesophyll (±vascular bundles) tissues, suggesting the protective functions of epidermis for the underlying mesophyll as well as of mesophyll for the underlying vascular bundles. The following sequence of efficiency of UV-screening barrier zones of the methanol-soluble extracts of the needle epidermis and mesophyll (± vascular bundles) for various UV-spectral bands was detected: UVC screening at less than 265 nm > UVC screening at 265–280 nm > UVB screening at 280–320 nm > UVA screening at 280–320 nm, whereby the UV screening at 280–320 nm was suggested as the most relevant barrier against enhanced UVB radiation. A blend of various UV-screening substances occurred in the methanol-soluble fractions of needle epidermis, whereby p-hydroxybenzoic acid 4-O-β-D-glucopyranoside, picein, (+)-catechin, p-hydroxyacetophenone, benzoic acid and astragalin were identified as UVC/UVB-screening substances; picein, (+)-catechin, astringin, p-hydroxyacetophenone and astragalin(s) as UVB-screening substances and astragalin(s) as UVA/B-screening substances. Alkaline hydrolysis of methanol-insoluble epidermal cell wall fractions released p-coumaric acid, ferulic acid and as-tragalin(s) as major UVB-screening substances. Loss of vitality of Norway spruce trees (forest decline disease) led to a significant reduction of UVB (315 nm)-screening ability of methanol-soluble fractions from epidermis, mesophyll (±vascular bundles) and whole needles. The HPLC analysis showed that the loss of vitality is due to a reduction in accumulation of UVB-absorbing substances, mainly picein, (+)-catechin, isorhapontin and astragalin(s) in the epidermis of needles from the second needle year in accordance with the detected loss of UVB-screening ability. It is concluded that the natural UV-screening mechanisms of Norway spruce needles are highly complex but mainly actively mediated by the ability of methanol-soluble UV-absorbing substances to form variable UVB-AJVA-screening barrier zones and passively by the ability of epidermal cell wall-bound UV-screening substances to screen UV light, whereby in the epidermis a conversion of excess UV light into PAR takes place.     

Crisp and soft multivariate methods visualize individual cell nuclei in Raman images of liver tissue sections

Raman and infrared spectroscopy have been recognized to be promising tools in clinical diagnostics because they provide molecular contrast without external stains. Here, vertex component analysis (VCA) was applied to Raman and Fourier transform infrared (FTIR) images of liver tissue sections and the results were compared with K-means cluster analysis, fuzzy C-means cluster analysis and principal component analysis. The main components of VCA from three Raman images were assigned to the central vein, periportal vein, cell nuclei, liver parenchyma and bile duct. After resonant Mie scattering correction, VCA of FTIR images identified veins, liver parenchyma, cracks, but no cell nuclei. The advantages of VCA in the context of tissue characterization by vibrational spectroscopic imaging are that the tissue architecture is visualized and the spectral information is reconstructed. Composite images were constructed that revealed a high molecular contrast and that can be interpreted in a similar way like hematoxylin and eosin stained tissue sections.     

Early Diagnosis of Mercury Stress of Wheat Seedlings Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy

The aim of the study was the non-destructive and rapid evaluation of the phytotoxicity of different concentrations of mercury (0.2, 0.4, 0.6, 0.8, and 1?mM) on the biochemical profile of wheat seedlings using attenuated total reflectance–Fourier transform infrared (ATR–FTIR) spectroscopy in combination with principal component analysis. Infrared spectra of the leaves of control and mercury-treated wheat seedlings were recorded in the spectral region from 4000 to 485?cm^?1. The preprocessing of the recorded spectra by second derivation enhanced the specificity of the infrared bands. The area of the bands estimated by curve fitting was used for the relative quantitative estimation of the biochemical changes involved. The principal component analysis of the acquired spectral measurements discriminated between the biochemicals of the control and mercury-treated wheat seedlings. The result showed that mercury (0.2–1?mM) significantly enhanced the content of the cell wall polysaccharides, amino acids, β-sheet component of proteins, and lipids in the leaves accompanied by reduction in the amount of α-helix. The change in β-sheet/α-helix ratio indicated mercury induced structural changes in the secondary structure of proteins. The increase in the levels of methylene bands and carboxyl bands indicates changes in lipid configuration and peroxidative damage caused by mercury. The results of this study demonstrate the potential of ATR–FTIR in combination with principal component analysis as an environmental monitoring tool for the identification of plant populations affected by environmental factors like heavy metal stress prior to the appearance of visual signs of toxicity.     

电镜-能谱仪在柳树叶重金属污染研究中的应用

采用自然干燥、化学处理、冷冻处理3种方法制备待测样本,运用扫描电镜和透射电镜结合X射线能谱仪研究柳树叶肉组织对重金属的累积状况,并定位到栅栏组织的细胞壁,为定性定量以及定位分析组织细胞固定、转移重金属污染探索合适的制样方法.结果表明:(1)重金属Pb、Cu、Cd、Zn在柳树叶肉组织被检测到,并已进入到细胞壁内,同时定量结果显示污染越严重,重金属累积量越多,证明了柳树叶对重金属污染的吸收能力;(2)通过综合比较,认为冷冻处理的样本比较适合进行植物组织微区的元素定性定量以及定位分析,因此推测冷冻制样加冷冻电镜的方法将会最为理想.     

Cellulose microfibril orientation in onion (Allium cepa L.) epidermis studied by atomic force microscopy (AFM) and vibrational sum frequency generation (SFG) spectroscopy

Cellulose microfibril orientation in plant cell walls changes during cell expansion and development. The cellulose microfibril orientation in the abaxial epidermis of onion scales was studied by atomic force microscopy (AFM) and sum frequency generation (SFG) vibrational spectroscopy. Onion epidermal cells in all scales are elongated along the onion bulb axis. AFM images showed that cellulose microfibrils exposed at the innermost surface of the abaxial epidermis are oriented perpendicular to the bulb axis in the outer scales and more dispersed in the inner scales of onion bulb. SFG analyses can determine the orientation of cellulose microfibrils averaged over the entire thickness of the cell wall. We found that the average orientation of cellulose microfibrils inside onion abaxial epidermal cell walls as revealed by SFG is similar to the orientation observed at the innermost cell wall surface by AFM. The capability to determine the average orientation of cellulose microfibrils in intact cell walls will be useful to study how cellulose microfibril orientation is related to biomechanical properties and the growth mechanism of plant cell walls.     

Isolation of C-glycosyl xanthones from Coffea pseudozanguebariae and their location

The biochemical composition of leaves from Coffea pseudozanguebariae, a wild caffeine-free coffee species, was determined. Two phenolic compounds were extracted from leaves, separated and characterized. Their structures were elucidated by mass spectrometry, and 1D and 2D NMR spectroscopy and were shown to be mangiferin (1) and isomangiferin (2), which were the main polyphenol products. Multiphoton fluorescence imaging was performed to visualize polyphenol distribution in leaf cross sections. Consistent biochemical analysis cell imaging techniques on leaves revealed yellow fluorescence in the epidermis and parenchyma cells corresponding to xanthone compounds.     

Characterization of the structure of human skin substitutes by infrared microspectroscopy

The skin acts mainly as a protective barrier from the external environment, thanks to the stratum corneum which is the outermost layer of the skin. As in vitro tests on skin are essential to elaborate new drugs, the development of skin models closer to reality becomes essential. It is now possible to produce in vitro human skin substitutes through tissue engineering by using the self-assembly method developed by the Laboratoire d’Organogénèse Expérimentale. In the present work, infrared microspectroscopy imaging analyses were performed to get in-depth morpho-spectral characterization of the three characteristic layers of human skin substitutes and normal human skin, namely the stratum corneum, living epidermis, and dermis. An infrared spectral analysis of the skin is a powerful tool to gain information on the order and conformation of the lipid chains and the secondary structure of proteins. On one hand, the symmetric stretching mode of the lipid methylene groups (2,850 cm^?1) is sensitive to the acyl chain conformational order. The evolution profile of the frequency of this vibrational mode throughout the epidermis suggests that lipids in the stratum corneum are more ordered than those in the living epidermis. On the other hand, the frequencies of the infrared components underneath the envelop of the amide I band provide information about the overall protein conformation. The analysis of this mode establishes that the proteins essentially adopt an α-helix conformation in the epidermis, probably associated with the presence of keratin, while modifications of the protein content are observed in the dermis (extracellular matrix made of collagen). Finally, the lipid organization, as well as the protein composition in the different layers, is similar for human skin substitutes and normal human skin, confirming that the substitutes reproduce essential features of real skin and are appropriate biomimetics.     

Assessing carotenoid content in plant leaves with reflectance spectroscopy

Spectral reflectance of maple, chestnut and beech leaves in a wide range of pigment content and composition was investigated to devise a nondestructive technique for total carotenoid (Car) content estimation in higher plant leaves. Reciprocal reflectance in the range 510 to 550 nm was found to be closely related to the total pigment content in leaves. The sensitivity of reciprocal reflectance to Car content was maximal in a spectral range around 510 nm; however, chlorophylls (Chl) also affect reflectance in this spectral range. To remove the Chl effect on the reciprocal reflectance at 510 nm, a reciprocal reflectance at either 550 or 700 nm was used, which was linearly proportional to the Chl content. Indices for nondestructive estimation of Car content in leaves were devised and validated. Reflectances in three spectral bands, 510+/-5 nm, either 550+/-15 nm or 700+/-7.5 nm and the near infrared range above 750 nm are sufficient to estimate total Car content in plant leaves nondestructively with a root mean square error of less than 1.75 nmol/cm2.