Fluorescence Excitation Spectroscopy Research Articles

Degradation of Rhodamine B Dye by Cactus Polysaccharide‐Synthesized Silver Nanoparticles Monitored by Fluorescence Excitation‐Emission Matrix (FEEM) Spectroscopy

The degradation of Rhodamine B dye (RhB) by polysaccharide‐synthesized silver nanoparticles (PE‐AgNPs) has been studied by fluorescence excitation (λex) and emission (λem) matrices (FEEM) spectroscopy. This study reports the behavior of RhB under the influence of PE‐AgNPs using the FEEM technique. The observed 3D excitation and emission maps reveal that, at λex (400 nm)/λem (580 nm), fluorescence quenching occurs. The 3D maps/peaks show that this type of quenching occurs in the presence of PE‐AgNPs and is indicated by the decrease in the intensity of spectral peaks. At pH 8, the maximum quenching of these peaks occurrs. Further investigations show that temperature and contact time influence RhB quenching significantly. The trends observed shows that higher temperatures leads to reduction of RhB fluorescence intensity increases with increase in temperature and decrease with time. This study suggests that the fluorescence quenching of RhB, can be due to its adsorption on the conductive surface of PE‐AgNPs. The results presented in this study give experimental evidence and insights that PE‐mediated green synthesized AgNPs can be a promising RhB degrading agent. Besides, the interactions of RhB with PE‐AgNPs, can prevent leaching and thus minimize dye toxicity in water.

The solid-state synchronous vs. conventional fluorescence spectroscopy and complementary methods to study the interactions of aluminum metal-organic framework Basolite A100 with dimethyl sulfoxide

Abstract We report, for the first time, an experimental study of interaction of metal-organic framework (MOF) with dimethyl sulfoxide (DMSO). Adsorption of DMSO on aluminum MOF Basolite A100 results in formation of the stoichiometric complex [A100]2[DMSO]3. The solid-state synchronous fluorescence spectroscopy allows observation of the following major excitation/emission transitions in Basolite A100 and its adsorption complex [A100]2[DMSO]3 which are not well-resolved in “conventional” fluorescence spectra: 1) narrow resonance of the isolated BDC linker, 2) excitation across optical bandgap, and 3) excimers. In the adsorption complex [A100]2[DMSO]3, the DMSO molecules interact with the μ2-OH group of Basolite A100 and with its BDC linker through the π-π system. We present, for the first time, the new variety of the solid-state synchronous fluorescence spectroscopy, which we denote as the solid-state enhanced-resolution synchronous fluorescence (excitation) spectroscopy. The accurate measurements of small changes in the optical bandgap of the adsorption complex [A100]2[DMSO]3 and Basolite A100 are described by the solid-state enhanced-resolution synchronous fluorescence (excitation) spectroscopy.

Differentiation between milk from low-input biodynamic, intermediate-input organic and high-input conventional farming systems using fluorescence excitation spectroscopy (FES) and fatty acids

ABSTRACTThis study evaluated the ability of fluorescence excitation spectroscopy (FES) to differentiate milk samples from different origins. Three different farming systems were chosen: D-samples originating from low-input biodynamic farms (cows fed on hay or pasture); O-samples from intermediate-input organic farms (cows fed mainly on grass silage); and C-samples from high-input conventional farms (indoor housing, cows fed on maize and grass silage). Milk samples were collected every second month between July 2015 and June 2016 from 12 farms (four farms per system), and a total of 70 samples were obtained. Fat-, protein- and urea-concentrations, somatic-cell count and fatty acid levels (FA) were determined. FES-measurements were performed by exciting the sample with light of different wavelengths and detecting delayed luminescence. Differences between farming systems in each season were checked by ANOVA. Factors of season, system and breed were evaluated in a linear regression model. By linear-discriminant analysis, variables contributing to correct classification were analysed. Milk FAs, especially the concentration of omega-3 (n3) and omega-6 (n6) FAs, were different between farming systems, while conjugated linoleic acid (CLA) and C18:1t11 (tVA)-concentration was mainly influenced by season (pasture). FES-parameters showed slight seasonal variations, but strong farming-system impacts. Differentiation between the three farming systems was possible for 81% of the samples by using FAs as variables. FES-parameters discriminated up to 86% of the samples, and, in combination, 93% of the samples were classified correctly. These results indicated that FES-results contributed to correct discrimination and that FES-results may be linked with qualities different to the FA profile.

Measuring delayed luminescence by FES to evaluate special quality aspects of food samples – an overview

Abstract The measuring devices which are used for measuring delayed luminescence by Fluorescence Excitation Spectroscopy (FES) are described. Results are given to show the measured emission of food samples in relation to farming practices (biodynamic, organic, or conventional) and growing conditions. This overview points to the special food-quality aspects which could be evaluated by FES. A relation to molecular structures, the physiological status, or possible physiological processes are discussed as important for the characteristic delayed luminescence after colour- (wavelength-) specific excitation.

Nonlinear optical spectroscopy and two-photon excited fluorescence spectroscopy reveal the excited states of fluorophores embedded in a beetle's elytra.

Upon illumination by ultraviolet light, many animal species emit light through fluorescence processes arising from fluorophores embedded within their biological tissues. Fluorescence studies in living organisms are however relatively scarce and so far limited to the linear regime. Multiphoton excitation fluorescence analyses as well as nonlinear optical techniques offer unique possibilities to investigate the effects of the local environment on the excited states of fluorophores. Herein, these techniques are applied for the first time to study of the naturally controlled fluorescence in insects. The case of the male Hoplia coerulea beetle is investigated because the scales covering the beetle's elytra are known to possess an internal photonic structure with embedded fluorophores, which controls both the beetle's coloration and the fluorescence emission. An intense two-photon excitation fluorescence signal is observed, the intensity of which changes upon contact with water. A third-harmonic generation signal is also detected, the intensity of which depends on the light polarization state. The analysis of these nonlinear optical and fluorescent responses unveils the multi-excited states character of the fluorophore molecules embedded in the beetle's elytra. The role of form anisotropy in the photonic structure, which causes additional tailoring of the beetle's optical responses, is demonstrated by circularly polarized light and nonlinear optical measurements.

Low-dose X-ray irradiation of wheat and bean samples detectable by fluorescence excitation spectroscopy (FES)

ABSTRACTIonizing irradiation is used to preserve foodstuffs and improve food safety, but it is not permitted for organic products, with the exception for using low doses (less than 500 mGy) for detection purposes (e.g. the presence of foreign objects). Analytical verification of irradiation is possible by detection of 2-alkylcyclobutanones (2-ACBs) in products irradiated with 10 Gy or more. Effects of lower doses of radiation on foodstuffs have not been investigated yet, but in blood, irradiation is detectable even at intensities of 1 mGy.The aim of this preliminary study was to determine whether fluorescence excitation spectroscopy (FES), a method which is very sensitive to small changes in the product, could differentiate between bean seeds and wheat grains exposed to X-rays of different intensities. The bean and wheat grain samples were irradiated at 500 mGy (dose limit value for detection purposes), 50 mGy (very low dose) and 0 mGy (control). Measuring the delayed luminescence by FES, effects of irradiation were found in the wheat samples in the FES-parameter Mw1-r, with the highest values for samples irradiated with 500 mGy, intermediate values for 50 mGy and lowest values for 0 mGy (control) (p < 0.001). The effects were less apparent in bean seeds, with a significant difference (p < 0.05) in the parameter Mw1-r only when comparing irradiation at 500 mGy with the control. The parameter differentiating the irradiation intensities was the same for both seeds and changed in the same way. Thus, systematic dose-specific effects were observed for low-dose irradiation intensities.

Single-Cell and Bulk Fluorescence Excitation Signatures of Seven Phytoplankton Species During Nitrogen Depletion and Resupply.

Phytoplankton play a vital role as primary producers in aquatic ecosystems. One common approach to classifying phytoplankton is fluorescence excitation spectroscopy, which leverages the variation in types and concentrations of pigments among different phytoplankton taxonomic groups. Here, we used a fluorescence imaging photometer to measure excitation ratios ("signatures") of single cells and bulk cultures of seven differently pigmented phytoplankton species as they progressed from nitrogen N-replete to N-depleted conditions. Our objective was to determine whether N depletion alters the fluorescence excitation signature of each species and, if so, how quickly they recover when N (as nitrate) was resupplied, because these factors affect our ability to classify the species correctly. Of the seven species studied, only Proteomonas sulcata, a marine cryptophyte, showed measurable changes in single-cell fluorescence excitation ratios and bulk fluorescence excitation spectra. These changes were likely due to decreases in the cellular concentration of phycoerythrin, a N-rich pigment, as N became scarce. Within 3 h of resupply of N, fluorescence signatures began returning to pre-depletion values and were indistinguishable from N-replete cells by 80 h after resupply. These data suggest that our classification approach is robust for non-PE containing phytoplankton. PE-containing phytoplankton might exhibit systematic changes in their signatures depending on their level of N depletion, but this could be detected and the phytoplankton re-classified following a few hours of incubation in N replete conditions.

Correlation between photochemical reaction and structural feature of benzyliden derivatives

The benzyliden derivatives with tree photo active centers were synthesized and their physicochemical properties are determined by NMR, absorption, fluorescence excitation and emission spectroscopies. The semiempirical calculations carried out according with the theory of the PM3/CI level has shown, that at an irradiating of their solutions by UV-light give no possibilities of the photo-Fries reaction. The calculated values are in a good accordance with the experimental ones.

Photochemical activities of polymers with aurone fragment

ABSTRACTIn this work we report preliminary results obtained for methacrylic polymers incorporating aurone side-group as photochemical active molecule. The aurone polymers were obtained by a three-step synthetic approach: (2Z)-6-hydroxy-2-(4-R-benzyliden)-1-benzofuran-3(2H)-ones were synthesized by using condensation reation of 6-hydroxybenzofuran-3-one with benzaldehydes; then methacrylate monomers were synthesized by reaction the alcohols with methacryloyl chloride; and the polymers were synthesized by free radical polymerization. Polymers were characterized by 1HNMR spectroscopy and DSC techniques. Their physicochemical properties are determined by absorption, fluorescence excitation and emission spectroscopies.

Comparison of Extraction Methods of Extracellular Polymeric Substances from Activated Sludge

Extracellular polymeric substances (EPSs) are biosynthetic polymers of microbial origin in the sludge activation process and crucially affect the properties of sludge in biological wastewater treatment reactors, such as the formation of sludge flocs, stabilization of sludge structure, and protection of microbes against noxious environmental conditions. However, the EPS extraction efficiency differs significantly according to the extraction method used. In this study, soluble EPSs and loosely bound EPSs can be extracted by centrifugation first and tightly bound EPSs in activated sludge require additional eight treatments for extraction, respectively. Three physical methods (centrifugation, sonication, and heating) and five chemical methods (cation exchange resin, NaOH, formaldehyde+NaOH, EDTA, and formaldehyde+EDTA) were tested, and the content and composition of TB-EPS were analyzed. Meanwhile, the functional groups and elements in TB-EPS were investigated. Results showed that the heating method did not introduce exogenous substances during the EPS extraction process and that the destruction of cells from this method was relatively slight. Heating was shown to be a gentle and efficient method in this study. The three-dimensional excitation and emission matrix (EEM) fluorescence spectroscopy showed that the cation exchange resin method had good extraction effect on humic-like and protein-like substances. As to fulvic-acid-like substances, NaOH was better than the other seven methods. Infrared spectroscopy showed that no notable difference appeared in the functional groups of the TB-EPS extracted by physical methods, whereas chemical methods induced big differences and showed particular bands that did not appear in the TB-EPS extracted by physical methods. Overall, the amounts of TB-EPS elements extracted using chemical methods were greater than those extracted using physical methods. In conclusion, a method must be selected and established for each case, taking into consideration the experimental purpose, and the most appropriate method should be chosen carefully.

Structural Variations in Chlorosomes from Wild-Type and a bchQR Mutant of Chlorobaculum tepidum Revealed by Single-Molecule Spectroscopy.

Green sulfur bacteria can grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. They contain chlorosomes, perhaps the most efficient light-harvesting antenna system found in photosynthetic organisms. Chlorosomes contain supramolecular structures comprising hundreds of thousands of bacteriochlorophyll molecules, which are properly positioned with respect to one another solely by self-assembly and not by using a protein scaffold as a template for directing the mutual arrangement of the monomers. These two features-high efficiency and self-assembly-have attracted considerable attention for developing light-harvesting systems for artificial photosynthesis. However, reflecting the heterogeneity of the natural system, detailed structural information at atomic resolution of the molecular aggregates is not yet available. Here, we compare the results for chlorosomes from the wild type and two mutants of Chlorobaculum tepidum obtained by polarization-resolved, single-particle fluorescence-excitation spectroscopy and theoretical modeling with results previously obtained from nuclear-magnetic resonance spectroscopy and cryo-electron microscopy. Only the combination of information obtained from all of these techniques allows for an unambiguous description of the molecular packing of bacteriochlorophylls within chlorosomes. In contrast to some suggestions in the literature, we find that, for the chlorosomes from the wild type as well as for those from mutants, the dominant secondary structural element features tubular symmetry following a very similar construction principle. Moreover, the results suggest that the various options for methylation of the bacteriochlorophyll molecules, which are a primary source of the structural (and spectral) heterogeneity of wild-type chlorosome samples, are exploited by nature to achieve improved spectral coverage at the level of individual chlorosomes.

Dynamics of Histone H3 Tails in Mononucleosomes Studied by Single-Molecule FRET and MD Simulations

The nucleosome, consisting of two copies of each of the four histone proteins H2A, H2B, H3, H4, and a 147bp long DNA, is the basic unit of chromatin compaction and key to gene regulation. The N-terminal tails of the four core histone proteins and the C-terminal tail of H2A are protruding from the core particle in a disorder manner. Even though, the nucleosome has been widely studied in the field of epigenetics, surprisingly little is known about the actual conformation of the tails and their dynamics. Here we focus on the conformation and dynamics of the H3 N-terminal tail at various NaCl concentrations and linker DNA length in experiments and MD simulations. Salt dependent conformational changes were measured by FRET with reconstitute Widom 601 mononucleosomes with a DNA length of 170 bp, 190 bp or 210 bp. Nucleosomes were labeled on the H3 tail at position K9 and at various positions on the intranucleosomal DNA or linker DNA. Both, bulk experiments using microplate scanning FRET spectroscopy (µpsFRET), and single molecule experiments using alternating laser excitation (ALEX) or multidimensional confocal fluorescence spectroscopy with pulsed interleaved excitation (MFD-PIE) were performed. Our results confirm the previously described binding preference of the H3 tail towards the linker DNA at low salt concentrations as well as an influence of the linker DNA length on the H3 tail:DNA interaction. At higher salt concentrations (> 600 mM), when nucleosome disassembly occurs, interaction of the H3 tail with the linker DNA is disrupted and interaction with the inner DNA gyre is favorized. The designed constructs enable us for the first time to our knowledge to monitor conformational changes of the H3 tail, which underpins the proposed dynamic nature of the H3 tail.

Evaluation of disinfection by-products (DBPs) formation potential in ANAMMOX effluents.

Disinfection by-products (DBPs), major health concerns in the potable reuse of municipal wastewater effluent, are process-related in wastewater treatment systems. Anammox is a promising and increasingly-applied technology for nitrogen removal in wastewater. In this study, the relationship between DBP formation potential and the anammox process has been investigated based on a lab-scale sequencing batch reactor (SBR). Excitation and emission matrix (EEM) fluorescence spectroscopy was employed to identify the compositions of the DBP precursors. The results showed that the effluents from the anammox SBR could yield both carbonaceous and nitrogenous DBPs after chlorination. Trichloromethane (TCM) was the dominant product among all DBPs detected. The anammox effluent has a low specific TCM formation potential of 0.778 μmol/mmol C and a trichloronitromethane (TCNM) formation potential of 0.0725 μmol/mmol C, leading to a TCM and TCNM formation potential ratio of 10.7. We found that substrate utilization of anammox did not enhance DBP yields, and the DBP formation potential decreased after 10 hour starvation. High pH conditions stimulated the production of TCM precursors in the anammox reactor. Humic acid-like and protein-like substances were identified in the EEM spectra of anammox effluents.

Fluorescence Excitation Spectroscopy for Phytoplankton Species Classification Using an All-Pairs Method: Characterization of a System with Unexpectedly Low Rank

An all-pairs method is used to analyze phytoplankton fluorescence excitation spectra. An initial set of nine phytoplankton species is analyzed in pairwise fashion to select two optical filter sets, and then the two filter sets are used to explore variations among a total of 31 species in a single-cell fluorescence imaging photometer. Results are presented in terms of pair analyses; we report that 411 of the 465 possible pairings of the larger group of 31 species can be distinguished using the initial nine-species-based selection of optical filters. A bootstrap analysis based on the larger data set shows that the distribution of possible pair separation results based on a randomly selected nine-species initial calibration set is strongly peaked in the 410-415 pair separation range, consistent with our experimental result. Further, the result for filter selection using all 31 species is also 411 pair separations; The set of phytoplankton fluorescence excitation spectra is intuitively high in rank due to the number and variety of pigments that contribute to the spectrum. However, the results in this report are consistent with an effective rank as determined by a variety of heuristic and statistical methods in the range of 2-3. These results are reviewed in consideration of how consistent the filter selections are from model to model for the data presented here. We discuss the common observation that rank is generally found to be relatively low even in many seemingly complex circumstances, so that it may be productive to assume a low rank from the beginning. If a low-rank hypothesis is valid, then relatively few samples are needed to explore an experimental space. Under very restricted circumstances for uniformly distributed samples, the minimum number for an initial analysis might be as low as 8-11 random samples for 1-3 factors.

Absorption and Fluorescence Features of an Amphiphilic meso-Pyrimidinylcorrole: Experimental Study and Quantum Chemical Calculations.

Corroles are emerging as an important class of macrocycles with numerous applications because of their peculiar photophysical and metal chelating properties. meso-Pyrimidinylcorroles are easily deprotonated in certain solvents, which changes their absorption and emission spectra as well as their accessible supramolecular structures. To enable control over the formation of supramolecular structures, the dominant corrole species, i.e., the deprotonated form or one of the two NH-tautomers, needs to be identified. Therefore, we focus in the present article on the determination of the UV-vis spectroscopic properties of the free-base NH-tautomers and the deprotonated form of a new amphiphilic meso-pyrimidinylcorrole that can assemble to supramolecular structures at heterointerfaces as utilized in the Langmuir-Blodgett and liquid-liquid interface precipitation techniques. After quantification of the polarities of the free-base NH-tautomers and the deprotonated form by means of quantum chemically derived electrostatic potential distributions at the corroles' van der Waals surfaces, the preferential stabilization of (some of) the considered species in solvents of different polarity is identified by means of absorption spectroscopy. For the solutions with complex mixtures of species, we applied fluorescence excitation spectroscopy to estimate the relative weights of the individual corrole species. This technique might also be applied to identify dominating species in molecularly thin films directly on the subphase' surface of Langmuir-Blodgett troughs. Supported by quantum chemical calculations we were able to differentiate between the spectral signatures of the individual NH-tautomers by means of fluorescence excitation spectroscopy.

Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Vibronic Origin of the Qy Absorption Tail of Bacteriochlorophyll a Verified by Fluorescence Excitation Spectroscopy and Quantum Chemical Simulations.

The long-wavelength tail of the lowest-energy Qy singlet absorption band of bacteriochlorophyll a in triethylamine peaking at 768.6 nm was examined by means of fluorescence excitation spectroscopy at ambient temperature of 22 ± 1 °C. The tail, usually considered a Gaussian, does in fact weaken quasi-exponentially, being clearly evident as far as 940 nm, nearly 2400 cm-1 (∼12 kBT) away from the absorption peak. Quantum chemical simulations identified vibronic transitions from the thermally populated normal modes and their overtones in the ground electronic state as the origin of this tail. Because energy transfer and relaxation processes generally depend on vibronic overlap integrals, these findings may have important implications on the interpretation of numerous photoinduced phenomena that involve bacteriochlorophyll and similar molecules, including photosynthesis.

Effect of Facial Encumbrance on Excimer Formation and Charge Resonance Stabilization in Model Bichromophoric Assemblies

Excimer formation and charge resonance stabilization in covalently linked bichromophoric systems with flexible spacers are important processes relevant to biochemistry and functional materials. Requiring a π-stacked cofacial arrangement of a pair of aromatic molecules at a van der Waals contact, the underlying geometrical reorganization that accompanies these events continues to be debated. Here we use a variety of methods including two-color resonant two-photon ionization spectroscopy (2CR2PI), ion yield measurements, hole-burning spectroscopy (HB), and laser-induced fluorescence (LIF) excitation and emission spectroscopy to compare the gas-phase spectroscopy and dynamics of the van der Waals dimers of fluorene, 9-methylfluorene (MF), and 9,9′-dimethylfluorene (F1). The goal of this work is to probe the influence of methyl substitution on the dynamics of excimer formation and charge resonance (CR) stabilization. The fluorene dimer, (F)2, displays lifetime broadened electronic spectra and the dominance of...

Controls on the distribution of fluorescent dissolved organic matter during an under-ice algal bloom in the western Arctic Ocean.

In this study we used fluorescence excitation and emission matrix spectroscopy, hydrographic data, and a self‐organizing map (SOM) analysis to assess the spatial distribution of labile and refractory fluorescent dissolved organic matter (FDOM) for the Chukchi and Beaufort Seas at the time of a massive under‐ice phytoplankton bloom during early summer 2011. Biogeochemical properties were assessed through decomposition of water property classes and sample classification that employed a SOM neural network‐based analysis which classified 10 clusters from 269 samples and 17 variables. The terrestrial, humic‐like component FDOM (ArC1, 4.98 ± 1.54 Quinine Sulfate Units (QSU)) and protein‐like component FDOM (ArC3, 1.63 ± 0.88 QSU) were found to have elevated fluorescence in the Lower Polar Mixed Layer (LPML) (salinity ~29.56 ± 0.76). In the LPML water mass, the observed contribution of meteoric water fraction was 17%, relative to a 12% contribution from the sea ice melt fraction. The labile ArC3‐protein‐like component (2.01 ± 1.92 QSU) was also observed to be elevated in the Pacific Winter Waters mass, where the under‐ice algal bloom was observed (~40–50 m). We interpreted these relationships to indicate that the accumulation and variable distribution of the protein‐like component on the shelf could be influenced directly by sea ice melt, transport, and mixing processes and indirectly by the in situ algal bloom and microbial activity. ArC5, corresponding to what is commonly considered marine humic FDOM, indicated a bimodal distribution with high values in both the freshest and saltiest waters. The association of ArC5 with deep, dense salty water is consistent with this component as refractory humic‐like FDOM, whereas our evidence of a terrestrial origin challenges this classic paradigm for this component.

The Kollias legacy: Skin autofluorescence and beyond.

In a paper published at the J Invest Dermatol in 1998 Nik Kollias and coworkers described distinct changes in skin native fluorescence associated with skin aging and photoaging, using in vivo fluorescence excitation spectroscopy. The assignment of the 295nm band to tryptophan fluorescence had a profound significance influencing many later studies from multiple groups. The reproducible changes in skin native fluorescence suggested that aging causes predictable alterations in both the epidermis and the dermis, whereas chronic UV exposure induces the appearance of new fluorophores. This seminal, but insufficiently widely appreciated work deserves re-examination as it points to important horizons in future experimental dermatology, such as cancer diagnostics, diabetes, wound healing, and understanding skin aging and photoaging mechanisms.