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.     

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.     

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.     

Reflective Light Properties of Tissue Layers in Beech (Fagus sylvatica L.) Leaves

Abstract— Reflective light properties of various tissue layers (adaxial epidermis, palisade and spongy parenchyma and abaxial epidermis) of beech (Fagus sylvatica L.) leaves were measured using paradermal (parallel to epidermis) and transverse sections in the visible and infrared spectral bands by a high-resolution reflectance cytophotometer. Results showed an increment of reflectances of rest leaves following successive removal of paradermal tissue layers (adaxial epidermis, palisade parenchyma, abaxial epidermis). Reflectances of palisade parenchyma with spongy parenchyma and abaxial epidermis increased linearly in the infrared and green spectral bands with increasing chloroplast count. The enhancement of paradermal reflectances with successive removal of paradermal tissue layers was due to various optical effects of tissue/cell layers, whereby removal of paradermal tissue layers containing light absorbers, e.g. chloroplasts/chlorophyll-protein complexes, is of importance. The parabolic relationships found between lateral reflectances and distances of various tissue/cell layers from adaxial epidermis indicated lateral reflectances of the mesophyll to be governed mainly by selective chlorophyll absorb-ances of light according to Lambert-Beer's law.     

Evidence from action and fluorescence spectra that UV-induced violet-blue-green fluorescence enhances leaf photosynthesis

We assessed the contribution of UV-induced violet-blue-green leaf fluorescence to photosynthesis in Poa annua, Sorghum halepense and Nerium oleander by measuring UV-induced fluorescence spectra (280-380 nm excitation, 400-550 nm emission) from leaf surfaces and determining the monochromatic UV action spectra for leaf photosynthetic O2-evolution. Peak fluorescence emission wavelengths from leaf surfaces ranged from violet (408 nm) to blue (448 nm), while excitation peaks for these maxima ranged from 333 to 344 nm. Action spectra were developed by supplementing monochromatic radiation from 280 to 440 nm, in 20 nm increments, to a visible nonsaturating background of 500 mumol m-2 s-1 photosynthetically active radiation and measuring photosynthetic O2-evolution rates. Photosynthetic rates tended to be higher with the 340 nm supplement than with higher or lower wavelength UV supplements. Comparing photosynthetic rates with the 340 nm supplement to those with the 400 nm supplement, the percentage enhancement in photosynthetic rates at 340 nm ranged from 7.8 to 9.8%. We suspect that 340 nm UV improves photosynthetic rates via fluorescence that provides violet-blue-green photons for photosynthetic energy conversion because (1) the peak excitation wavelength (340 nm) for violet-blue-green fluorescence from leaves was also the most effective UV wavelength at enhancing photosynthetic rates, and (2) the magnitude of photosynthetic enhancements attributable to supplemental 340 nm UV was well correlated (R2 = 0.90) with the apparent intensity of 340 nm UV-induced violet-blue-green fluorescence emission from leaves.     

Two-photon absorption and photoluminescence of europium based emissive probes for bioactive systems

Observation of two-photon excitation (760 nm) and emission of two responsive water soluble europium complexes is reported with cross-sections of up to 2 GM. Two-photon excitation spectra have also been measured, acquisition being achieved by the use of a cavity-dumped mode locked Ti-sapphire laser. Time-gated detection is used to differentiate the ligand fluorescence and metal centred emission in these europium complexes.     

Fluorescence properties of Ca2+-independent discharged obelin and its application prospects

Discharged obelin, a complex of coelenteramide and polypeptide, is a fluorescent protein produced from the photoprotein obelin, which is responsible for bioluminescence of the marine hydroid Obelia longissima. Discharged obelin is stable and nontoxic and its spectra are variable, and this is why it can be used as a fluorescent biomarker of variable color in vivo and in vitro. Here we examined light-induced fluorescence of Ca²⁺-independent discharged obelin (obtained without addition of Ca²⁺). Its emission and excitation spectra were analyzed under variation of the excitation wavelength (260–390 nm) and the emission wavelength (400–700 nm), as well as the 40 °C exposure time. The emission spectra obtained with excitation at 260–300 nm (tryptophan absorption region) included three peaks with maxima at 355, 498, and 660 nm, corresponding to fluorescence of tryptophan, polypeptide-bound coelenteramide, and a hypothetical indole–coelenteramide exciplex, respectively. The emission spectra obtained with excitation at 310–380 nm (coelenteramide absorption region) did not include the 660-nm maximum. The peak in the red spectral region (λ _max?=?660 nm) has not been previously reported. Exposure to 40 °C under excitation at 310–380 nm shifted the obelin fluorescence spectra to the blue, whereas excitation at 260–300 nm shifted them to the red. Hence, red emission and variation of the excitation wavelength form a basis for development of new medical techniques involving obelin as a colored biomarker. The addition of red color to the battery of known (violet to yellow) colors increases the potential of application of obelin.     

Evaluating UV-B effects and EDU protection in soybean leaves using fluorescence

A growth-chamber experiment was conducted to evaluate whether ethylenenediurea (EDU), a chemical shown to be protective against ozone pollution, could ameliorate foliar damage induced by ultraviolet-B (UV-B) radiation exposure in 'Roanoke' soybean (Glycine max L.), a UV-B-sensitive cultivar, and whether these effects could be discriminated using fluorescence (F) observations. The experiment had four treatment groups: control; biologically effective UV-B (18 kJ m(-2) day(-1)); EDU (500 micromol mol(-1)); and both UV-B and EDU (UV/EDU). Measurements included photosynthetic pigments, F image system (FIS) images of adaxial surfaces in four spectral regions (blue, green, red and far-red) and F emission spectra of the pigment extracts produced at two excitation wavelengths, 280 nm (280EX) and 380 nm (380EX). Several F ratios from 280EX, 380EX and the FIS images successfully separated the low UV vs high EDU group responses based on means alone, with intermediate values for controls and the combined UV/EDU groups. A UV-B/blue emission ratio, F315/F420 (280EX), was correlated with chlorophyll content (microg cm(-2))(R = 0.88, P < 0.001), as was a ratio of emissions at two UV-A wavelengths: F330/F385 (280EX) (R = 0.87). These two 280EX ratios were also linearly correlated with emission ratios produced by 380EX, such as the far-red/green ratio, F730/F525 (380EX) (R = 0.92, P < 0.001), and clearly distinguished the UV-B and EDU groups separately, and which bracketed the similar intermediate responses of the UV/EDU and control groups. The FIS images additionally captured the following anatomical spatial patterns across the leaf surfaces: (1) emissions of UV-B-irradiated leaves were more uniform but lower in intensity than those of other groups; and (2) emissions of EDU-treated leaves exhibited the greatest variation in spatial patterns because veins had elevated blue F and leaf edges had enhanced red and far-red F. This experiment supports the hypothesis that EDU substantially ameliorated UV-B damage to foliage, a result that relied on the combined use of FIS images and emission spectra.     

Spectroscopic studies of the human heart conduction system ex vivo: implication for optical visualization

Fluorescence excitation and emission spectra of the heart tissues specimens have been measured ex vivo with the aim of finding out the optical differences characteristic for the human heart conduction system (the His bundle) and ventricular myocardium. The optimal conditions enhancing the spectral differences between the His bundle and myocardium were found by recording the fluorescence signal in the range from 420nm to 465nm under the excitation at wavelengths starting from 320nm to 370nm. In addition, the spectral differences between the His bundle and the connective tissue, which is often present in the heart, could be displayed by comparing the ratios of fluorescence intensities being measured at above 460nm under the preferred excitation of elastin and collagen. The left and right branches of the His bundle were visualized ex vivo in the interventricular septum of the human heart under illumination at 366nm.     

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

Absorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light-induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light-induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.     

Porphyrin bleaching and PDT-induced spectral changes are irradiance dependent in ALA-sensitized normal rat skin in vivo

Photobleaching kinetics of aminolevulinic acid-induced protoporphyrin IX (PpIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for changes in excitation wavelength optical properties using diffuse reflectance measured at the excitation wavelength. Loss of PpIX fluorescence was monitored during photodynamic therapy (PDT) performed using 514 nm irradiation. Bleaching in response to irradiances of 1, 5 and 100 mW cm-2 was evaluated. The results demonstrate an irradiance dependence to the rate of photobleaching vs irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence. The kinetics for the accumulation of the primary fluorescent photoproduct of PpIX also exhibit an irradiance dependence, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction mechanism in vivo, and they provide spectroscopic evidence that PDT delivered at low irradiance deposits greater photodynamic dose for a given irradiation fluence. We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.     

Origin of the anomalous two-photon absorption in fluorescent protein DsRed

The red fluorescent protein DsRed displays a two-photon excitation band around 760 nm which is not accompanied by any feature in the corresponding one-photon spectral region (380 nm). By means of time-dependent density functional theory, we are able to explain such an effect, as arising from an electronic excitation of the DsRed chromophore with ability to couple with a charge-transfer state, through an effective two-photon absorption channel.     

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.     

Upconversion and fluorescence spectral studies of Er3+/Yb3+-codoped Bi2Q3-GeO2-Ga2Q3-Na2O glasses

The absorption spectra and upconversion fluorescence spectra of Er3+/Yb3+-codoped natrium-gallium-germanium-bismuth glasses are measured and investigated. The intense green (533 and 549 nm) and red (672 nm) emission bands were simultaneously observed at room temperature. The quadratic dependence of the green and red emission on excitation power indicates that the two-photon absorption processes occur. The influence of Ga2C3 on upconversion intensity is investigated. The intensity of green emissions increases slowly with increasing Ga2O3 content, while the intensity of red emission increases significantly. The possible upconversion mechanisms for these glasses have also been discussed. The maximum phonon energy of the glasses determined based on the infrared (IR) spectral analysis is as low as 740 cm-1. The studies indicate that Bi2O3-GeO2-Ga2O3-Na2O glasses may be potential materials for developing upconversion optical devices.     

Spectral characterization of chlorophyll fluorescence in barley leaves during linear heating. Analysis of high-temperature fluorescence rise around 60 degrees C

The spectral characteristics of chlorophyll fluorescence and absorption during linear heating of barley leaves within the range 25-75 degreesC (fluorescence temperature curve, FTC) were studied. Leaves with various content of light harvesting complexes (green, Chl b-less chlorina f2 and intermittent light grown) revealing different types of FTC were used. Differential absorption, emission and excitation spectra documented four characteristic phases of the FTC. The initial two FTC phases (a rise in the 46-49 degreesC region and a subsequent decrease to about 55 degreesC) mostly reflected changes in the fluorescence quantum yield peaking at about 685 nm. A steep second fluorescence rise at 55-61 degreesC was found to originate from a short-wavelength Chl a spectral form (emission maximum at 675 nm) causing a gradual blue shift of the emission spectra. In this temperature range, a clear correspondence of the blue shift in the emission and absorption spectra was found. We suggest that the second fluorescence rise in FTC reflects a weakening of the Chl a-protein interaction in the thylakoid membrane.     

Luminescence spectroscopy of matrix-isolated z 6P state atomic manganese

The relaxation of electronically excited atomic manganese isolated in solid rare gas matrices is observed from recorded emission spectra, to be strongly site specific. z 6P state excitation of Mn atoms isolated in the red absorption site in Ar and Kr produces narrow a 4D and a 6D state emissions while blue-site excitation produces z 6P state fluorescence and broadened a 4D and a 6D emissions. MnXe exhibits only a single thermally stable site whose emission at 620 nm is similar to the broad a 6D bands produced with blue-site excitation in Ar and Kr. Thus in Ar(Kr), excitation of the red site at 393 (400) nm produces narrow line emissions at 427.5 (427.8) and 590 (585.7) nm. From their spectral positions, linewidths, and long decay times, these emission bands are assigned to the a 4D72 and a 6D92 states, respectively. Excitation of the blue site at 380 (385.5) nm produces broad emission at 413 (416) nm which, because of its nanosecond radiative lifetime, is assigned to resonance z 6P --> a 6S fluorescence. Emission bands at 438 (440) and 625 (626.8) nm, also produced with blue-site excitation, are broader than their red-site equivalents at 427.5 and 590 nm (427.8 and 585.7 nm in Kr) but from their millisecond and microsecond decay times are assigned to the a 4D and a 6D states. The line features observed in high resolution scans of the red-site emission at 427.5 and 427.8 nm in MnAr and MnKr, respectively, have been analyzed with the W(p) optical line shape function and identified as resolved phonon structure originating from very weak (S=0.4) electron-phonon coupling. The presence of considerable hot-phonon emission (even in 12 K spectra) and the existence of crystal field splittings of 35 and 45 cm(-1) on the excited a 4D72 level in Ar and Kr matrices have been identified in W(p) line shape fits. The measured matrix lifetimes for the narrow red-site a 6D state emissions (0.29 and 0.65 ms) in Ar and Kr are much shorter than the calculated (3 s) gas phase value. With the lifetime of the metastable a 6D92 state shortened by four orders of magnitude in the solid rare gases, it is clear that the probability of the "forbidden" a 6D --> a 6S atomic transition is greatly enhanced in the solid state. A novel feature identified in the present work is the large width and shifted 4D and 6D emissions produced for Mn atoms isolated in the blue sites of Ar and Kr. In contrast, these states produce narrow, unshifted (gas-phase-like) 4D and 6D state emissions from the red site.     

INFLUENCE OF CHANGES IN THE PHOTON PROTECTIVE ENERGY DISSIPATION ON RED LIGHT-INDUCED DETRAPPING OF THE THERMOLUMINESCENCE Z-BAND

-Thermoluminescence emission at 110 K (Z-band) was markedly diminished when thylakoid membranes were exposed to red light during or after Z-band charging with blue light. Analysis of this phenomenon showed that deactivation of Z-band-emitting chlorophyll species occurred preferentially on the low temperature side of the glow curve, and red light of670–680 nm was most efficient in the deactivation. In order to test our hypothesis that this detrapping is related to local heating effects caused by dissipation of absorbed energy, we measured thermoluminescence glow curves and Z-band emission spectra from spinach leaf discs and thylakoid membranes during induction of nonphotochemical chlorophyll fluorescence quenching. Pretreatment of the plant material was designed to achieve different levels of (1) de-epoxidized xanthophylls in the photosynthetic apparatus and (2) the proton concentration in the thylakoid lumen. In comparison, measurements were performed in aggregated and trimeric light-harvesting pigment-protein complexes of photosystem II. We observed on all three levels of organization that a higher capacity of excitation energy dissipation was accompanied by a stronger red light-induced detrapping of Z-band thermoluminescence.     

ANALYSIS OF LASER-INDUCED FLUORESCENCE LINE SHAPE OF INTACT LEAVES: APPLICATION TO UV STRESS DETECTION

Abstract— In vivo laser-induced fluorescence spectra of intact leaves of healthy and UV-irradiated Salvia splendens plants excited at 337 nm by a nitrogen laser were recorded using an optical multichannel analyzer system. The spectra showed the typical fluorescence bands centered around 450, 530, 685 and 730 nm. Exposure to UV radiation changed the relative intensity values of these bands and their peak positions. The analysis of the acquired spectra in terms of a linear combination of Gaussian bands was carried out to determine accurately the peak positions and the relative intensity contribution of the various bands to the laser-induced fluorescence spectra on healthy and UV-treated plants of different age.
The results indicate that a curve-fitting analysis of the measured fluorescence spectra is a useful and sensitive method to discriminate the various band contribution to the whole leaf fluorescence spectrum. The comparison among blue-green and red-far-red fluorescence of leaves was also confirmed as an effective indicator of UV stress in plants.     

Probing intramolecular Förster resonance energy transfer in a naphthaleneimide-peryleneimide-terrylenediimide-based dendrimer by ensemble and single-molecule fluorescence spectroscopy

We report on the ensemble and single-molecule (SM) dynamics of F?rster resonance energy transfer (FRET) in a multichromophoric rigid polyphenylenic dendrimer (triad) with spectrally different rylene chromophores featuring distinct absorption and emission spectra which cover the whole visible spectral range: a terrylenediimide (TDI) core, four perylenemonoimides (PMIs) attached at the scaffold, and eight naphthalenemonoimides (NMIs) at the rim. For FRET from PMI to TDI taking place with an efficiency of 99.5%, single triad molecules optically excited at 490 nm show fluorescence exclusively from the TDI side in the beginning of their emission. On 360-nm excitation, NMI chromophores transfer their excitation energy either directly or in a stepwise fashion to the core TDI, the latter case involving scaffold-substituted PMIs as intermediate acceptors. Indeed, SM experiments on 360-nm excitation evidence highly efficient FRET from NMI chromophores to the TDI core since individual triad molecules show fluorescence exclusively either from TDI or from an intermediate (oxidized) species but never from PMI. Because PMI and TDI are chromophores with high fluorescence quantum yields and high resistance to photobleaching compared to NMI, 360-nm excitation of a single triad molecule leads to bleaching of NMI chromophores with no chance for PMI to be observed. The spatial positioning and the spectral properties of the chosen rylene chromophores make this multichromophoric system an efficient light collector, able to capture light over the whole visible spectral range and to transfer it finally to the core TDI, the latter releasing it as red fluorescence.     

Remote Raman and fluorescence studies of mineral samples

In the present study, we investigated remote laser-induced fluorescence (LIF), at a distance of 4.8 m, of a variety of natural minerals and rocks, and Hawaiian Ti (Cordyline terminalis) plant leaves. These minerals included calcite cleavage, calcite onex and calcite travertine, gypsum, fluorapatite, Dover flint and chalk, chalcedony and nephelene syenite, and rubies containing rock. Pulsed laser excitation of the samples at 355 and 266 nm often resulted in strong fluorescence. The LIF bands in the violet-blue region at approximately 413 and approximately 437 nm were observed only in the spectrum of calcite cleavage. The green LIF bands with band maxima in the narrow range of approximately 501-504 nm were observed in the spectra of all the minerals with the exception of the nephelene syenite and ruby rocks. The LIF red bands were observed in the range approximately 685-711 nm in all samples. Excitation with 532 nm wavelength laser gave broad but relatively low fluorescence background in the low-frequency region of the Raman spectra of these minerals. One microsecond signal gating was effective in removing nearly all background fluorescence (with peak at approximately 610 nm) from calcite cleavage Raman spectra, indicating that the fluorescence was probably from long-lifetime inorganic phosphorescence.