In situ FLUORESCENCE SPECTROSCOPIC STUDIES ON BOVINE CORNEA

Abstract— The cornea is a transparent ocular tissue and its transparency is thought to be a result of intramolecular interactions and the supramolecular organization of its protein constituents. We have studied the intrinsic fluorescence properties of intact bovine corneas and compared these with that of the opaque sclera. It was observed that with increasing excitation wavelengths the emission maxima shifted toward the red edge exhibiting the phenomenon of red edge excitation shift, which is indicative of immobilization of the constituent fluorophores. The magnitude of the shift increased after photodamage by irradiation at 295 nm. Many of the spectral characteristics of the cornea are shown to be due to its proteoglycans, which show surprisingly significant red edge excitation shift in solution.     

Parallel factor analysis of spider fluorophores

Fluorophores from the hemolymph of yellow sac spiders (Cheiracanthium mildei) have been characterized using excitation emission matrix (EEM) fluorescence spectroscopy. This approach provides characterization of fluorophores present in the organism without having to isolate pure samples. Minimal variation occurs between individual samples and each EEM has two distinct peaks, suggesting two fluorophores may be present in the hemolymph. Parallel factor analysis reveals that three fluorophores (with excitation and emission maxima at 270/319, 330/389, and 350/465 nm) best explains the sample to sample variation. By comparing the spectra of the three individual components to fluorophores found in scorpions it is shown that these spiders possess different fluorophores than scorpions. Furthermore, the fluorescence observed is not consistent with beta-carboline or 4-methyl-7-hydroxycoumarin, two compounds previously described in scorpions.     

Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation-emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250-520 nm, with fluorescence emission spectra collected in the range 260-750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal. com/).     

Spectroscopic studies into the influence of UV radiation on elastin hydrolysates in water solution

An investigation into the influence of UV irradiation on elastin hydrolysates dissolved in water was carried out using UV-Vis spectroscopy and spectrofluorometry. It was found that the absorption of elastin hydrolysates in solution increased during irradiation of the sample. For fluorescence of elastin hydrolysates we observed both, a decrease and increase of this value during irradiation of the sample. After UV irradiation of the elastin solution we observed a minor increase of overall absorption, most notably between 250 nm and 280 nm. Moreover, after UV irradiation a wide peak emerged between 290 nm and 310 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV irradiation of elastin hydrolysates. The fluorescence of elastin hydrolysates was observed at 305 nm and at 380 nm after excitation at 270 nm. UV irradiation caused fluorescence fading at 305 nm and 380 nm. After 30 min of irradiation a new broad weak band of fluorescence, attributable to new photoproducts, emerged in the UV wavelength region with emission maximum between 400 nm and 500 nm.     

The influence of UV radiation on silk fibroin

An investigation into the influence of UV-irradiation on regenerated silk fibroin dissolved in water was carried out using UV-Vis and fluorescence spectroscopy. It was found that the absorption of regenerated silk fibroin in solution increased during UV-irradiation of the sample, most notably between 250 and 400 nm. Moreover, after UV-irradiation a wide peak emerged between 290 and 340 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV-irradiation of regenerated silk fibroin.The fluorescence of regenerated silk fibroin was observed at 305 nm, at 480 nm and at 601 nm after excitation at 275 nm. UV-irradiation caused fluorescence fading at 305 nm and at 601 nm. The increase of fluorescence was observed at 480 nm, probably due to formation of new photoproducts. After excitation at 305 nm the fluorescence of regenerated silk fibroin was observed at 340 nm and at 400 nm. UV-irradiation caused fluorescence fading at 340 nm. FTIR spectroscopy showed that primary structure of regenerated silk fibroin was not significantly affected by UV radiation. SDS-PAGE chromatography showed alterations of molecular weight of silk after UV exposure.     

Photoproduct Formation from a Zinc Benzochlorin Iminium Salt Detected by Fluorescence Microscopy

When examined by fluorescence microscopy, tumor cells loaded with a zinc benzochlorin iminium salt showed a very faint deep-red fluorescence that was rapidly transformed to a substantially brighter red-orange fluorescence. Fourier transfer spectroscopy analysis with a red-sensitive detection system revealed that an initial fluorescence at 770 nm was gradually converted to 640 nm fluorescence during excitation. Image analysis showed that photoproduct formation was accompanied by a change in the site of drug localization from the cytoplasm to the nucleus. These studies illustrate the power of interferometry for the characterization of photoproducts and changes in sensitizer localization during photoproduct formation.     

Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.     

Time-resolved fluorescence spectroscopy of hematoporphyrin, mesoporphyrin, pheophorbide a and chlorin e6 in ethanol and aqueous solution

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.     

A CHARACTERIZATION OF THE FLUORESCENT PROPERTIES OF CIRCULATING HUMAN EOSINOPHILS

Abstract— This paper presents a characterization of the fluorescence properties of human eosinophils isolated from peripheral blood of normal donors over a wide range of excitation and emission wavelengths. Circulating eosinophils possess three fluorescence excitation emission maxima: one at 280 nm excitation, 330 nm emission, attributable to tryptophan fluorescence, and currently unassigned peaks at 360 nm excitation, 440 nm emission and 380 nm excitation, 415 nm emission. Fluorescence microscopy studies show that the Huorescence of eosinophils may be site dependent; specifically, when observed at 365 nm excitation, circulating eosinophil Huorescence appears blue-violet, while the fluorescence of tissue-dwelling eosinophils appears amber-gold. These results should be considered in developing an optical biopsy technique to identify eosinophils in human tissue.     

Insight into the Excitation-Dependent Fluorescence of Carbon Dots

High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few distinct trademarks that make carbon nanodots (CDs) interesting for fundamental research, with potential to replace the prevalent inorganic semiconductor quantum dots. Currently, application and fundamental understanding of CDs are constrained because it is difficult to make a quantitative comparison among different types of CDs simply because their photoluminescence properties are directly linked to their size distribution, the surface functionalization, the carbon core structures (graphitic or amorphous) and the number of defects. Herein, we report a facile one-step synthesis of mono-dispersed and highly fluorescent nanometre size CDs from a ‘family’ of glucose-based sugars. These CDs are stable in aqueous solutions with photoluminescence in the visible range. Our results show several common features in the family of CDs synthesized in that the fluorescence, in the visible region, is due to a weak absorption in the 300–400 nm from a heterogeneous population of fluorophores. Fluorescence quenching experiments suggest the existence of not only surface-exposed fluorophores but more importantly solvent inaccessible fluorophores present within the core of CDs. Interestingly, time-resolved fluorescence anisotropy experiments directly suggest that a fast exchange of excitation energy occurs that results in a homo-FRET based depolarization within 150 ps of excitation.     

Front face fluorescence spectroscopy as a tool for the assessment of egg freshness during storage at a temperature of 12.2 degrees C and 87% relative humidity

The objective of this study was to investigate intrinsic fluorophores of thick albumen and egg yolk in order to assess egg freshness during storage at a temperature of 12.2 °C and 87% relative humidity (RH). A total of 126 intact brown-shelled eggs of the same flock (29 weeks of age) were stored for 1, 6, 8, 12, 15, 20, 22, 26, 29, 33, 40, 47 and 55 days. The emission fluorescence spectra of aromatic amino acids and nucleic acids (AAA + NA) (excitation: 250 nm; emission: 280-450 nm), fluorescent Maillard reaction products (FMRP) (excitation: 360 nm; emission: 380-580 nm) and the excitation spectra of vitamin A (emission: 410 nm; excitation: 270-350 nm) were scanned on thick albumen and egg yolk. Among the intrinsic fluorophores, only the principal component analysis (PCA) applied on the vitamin A fluorescence spectra allowed a good identification of eggs as a function of their storage time. Factorial discriminant analysis (FDA) was then applied on the first five principal components (PCs) of the PCA applied on each spectral data set. Regarding AAA + NA recorded on thick albumen, correct classification of 69.4% and 63.9% was observed for the calibration and validation sets, respectively. Quite similar results were obtained on AAA + NA scanned on egg yolks. The best results were obtained with vitamin A fluorescence spectra since 97.7% and 85.7% of the calibration and validation sets was obtained, respectively. These results showed that vitamin A fluorescence spectra provide useful fingerprints, mainly allow the identification of eggs during storage and could be considered as a powerful intrinsic probe for the evaluation of egg freshness.     

Characterization and Diagnosis of Cancer by Native Fluorescence Spectroscopy of Human Urine

Urine is one of the diagnostically important bio fluids, as it has different metabolites in it, where many of them are native fluorophores. Native fluorescence characteristics of human urine samples were studied using excitation–emission matrices (EEMs) over a range of excitation and emission wavelengths, and emission spectra at 405 nm excitation, to discriminate patients with cancer from the normal subjects. The fluorescence spectra of urine samples of cancer patients exhibit considerable spectral differences in both EEMs and emission spectra with respect to normal subjects. Different ratios were calculated using the fluorescence intensity values of the emission spectra and they were used as input variables for a multiple linear discriminant analysis across different groups. The discriminant analysis classifies 94.7% of the original grouped cases and 94.1% of the cross‐validated grouped cases correctly. Based on the fluorescence emission characteristics of urine and statistical analysis, it may be concluded that the fluorophores nicotinamide adenine dinucleotide (NADH) and flavins may be considered as metabolomic markers of cancer.     

FLUORESCENCE OF MELANIN-DEPENDENCE UPON EXCITATION WAVELENGTH AND CONCENTRATION

Abstract— An introduction to the fundamental characteristics of synthetic melanin fluorescence is presented. The particular difficulties associated with the detection and reduction of the relatively weak signal are discussed and a technique is described for correcting the fluorescence spectra for attenuation of the excitation and emission beams. Spectra are reported for the excitation wavelength range 340–400 nm and an emission range of 360–560 nm. The concentration dependence of the corrected fluorescence signal is examined and is shown to be linear. The variation of the fluorescence spectra with excitation wavelength suggests a two-component fluorescence, for the wavelength range studied. The presence of an isosbestic point in the spectra is used to identify the fluorophores as components of a reaction equilibrium. The possible relationship of this equilibrium to that associated with the melanin photo ESR is discussed     

Two-photon excitation induced fluorescence of a trifluorophore-labeled DNA

Two-photon excitation of a trifluorophore (6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine and cyanine-5 monofunctional dye) labeled DNA, which has a scaffold of 26 nucleotides, was achieved using focused laser light of a Q-switched Nd-YAG laser (1064 nm). The observed fluorescence signature (emission ratio from the three fluorophores) of the labeled DNA after two-photon excitation is very different from the fluorescence signatures produced by one-photon excitation at different wavelength. The additional fluorescence signatures produced by two-photon excitation of the fluorescent oligonucleotides will facilitate their use as combinatorial fluorescence energy transfer tags for multiplex genetic analysis.     

PHOTOCHEMISTRY OF TYPE I ACID-SOLUBLE CALF SKIN COLLAGEN: DEPENDENCE ON EXCITATION WAVELENGTH

Although previous studies have demonstrated that the predominant photochemistry of type I collagen under 254 nm irradiation may be attributed either to direct absorption by tyrosine/phenylalanine or to peptide bonds, direct collagen photochemistry via solar UV wavelengths is much more likely to involve several age- and tissue-related photolabile collagen fluorophores that absorb in the latter region. In this study, we compare and contrast results obtained from irradiation of a commercial preparation of acid-soluble calf skin type I collagen in solution with UVC (primarily 254 nm), UVA (335–400nm) and broad-band solar-simulating radiation (SSR; 290^1–00nm). Excitation spectroscopy and analysis of photochemically induced disappearance of fluorescence (fluorescence fading) indicates that this preparation has at least four photolabile fluorescent chromophores. In addition to tyrosine and L-3,4-dihydroxyphenylalanine, our sample contains two other fluorophores. Chromophore I, with emission maximum at 360 nm, appears to be derived from interacting aromatic moieties in close mutual proximity. Chromophore II, with broad emission at430–435 nm, may be composed of one or more age-related molecules. Collagen fluorescence fading kinetics are sensitive to excitation wavelength and to conformation. Under UVC, chromophore I fluorescence disappears with second-order kinetics, indicating a reaction between two proximal like molecules. Adherence to second-order kinetics is abrogated by prior denaturation of the collagen sample. A new broad, weak fluorescence band at400–420 nm, attributable to dityrosine, forms under UVC, but not under solar radiation. This band is photolabile to UVA and UVB wavelengths. Amino acid analysis indicates significant destruction of aromatic amino acids under UVC, but not under UVA or SSR. When properly understood, collagen fluorescence fading phenomena may act as a sensitive molecular probe of structure, conformation and reactivity.     

Novel fluorophores for single-molecule imaging

Nonlinear optical chromophores based on dicyanodihydrofuran acceptors paired with amine donors have been found to exhibit sufficiently large fluorescence quantum yields and stability to enable single-molecule detection in polymeric hosts. To illustrate the breadth of this class, six fluorophores are presented, spanning the emission range from 505 to 646 nm. In contrast to conventional single-molecule fluorophores, the new molecules feature sensitivity to local rigidity, large ground-state dipole moments, and large polarizability anisotropies, properties that can be used to design new reporter experiments at the single-molecule level.     

INVESTIGATION OF PROTRIPTYLINE PHOTOPRODUCTS WHICH CAUSE CELL MEMBRANE DISRUPTION

Abstract— Protriptyline (PTL; N-methyl-5H-dibenzo[a,d]cycloheptene-5-propylamine) hydrochloride is a skin photosensitizing agent in humans. The fluorescence and photochemical behavior of PTL varies with the solvent. In water, 40% ethanol and ethanol in the hydrochloride salt of PTL has a fluorescence quantum yield of 0.81. The fluorescence quantum yield of PTL free base in selected organic solvents is between 0.41 and 0.17; in ethanol it remains at 0.81. Photolysis of PTL in acidic aqueous solution yields at least five photoproducts which were separated by high-performance liquid chromatography. Three of these photoproducts lysed red blood cells. One of the photoproducts has been identified as a cyclobutyl photodimer of PTL based on its mass spectrum and UV absorption and its ability to undergo photore-versal with 254 nm irradiation. The others were not cyclobutyl dimers. The yield of lytic products decreased as the ethanol content was increased and were not formed from PTL free base in any solvent.     

MODEL STUDIES ON THE PHOTOCHEMICAL PRODUCTION OF LENTICULAR FLUOROPHORES

Abstract With aging, human lens proteins accumulate fluorophores having blue and green emissions. Model studies were undertaken to determine the role of 3-hydroxykynurenine (3-HK) and its glucoside (3-HKG) in the photochemical production of those fluorophores. Experiments were carried out using 10⁻³ M 3-HK solutions in the presence or absence of glycine (1 M ), which was used to mimic the environment of the lens. The solutions were photolyzed (transmission above 295 nm) for various periods of time while the loss of starting material and the formation of fluorescent photoproducts (blue emission at 470 nm, and green emission at 520 nm) were monitored using fluorescence and UV-visible spectroscopy and thin-layer and high-pressure liquid chromatography analysis. Several parameters were varied such as oxygen tension and the addition of the free radical scavenger, penicillamine. The photolytic loss of 3-HK in the absence of glycine occurred approximately 5-10 times faster than in its presence. Conversely, blue and green fluorophores formed in irradiated solutions containing glycine but not with the photolysis of 3-HK alone. The blue fluorophore was formed first and appeared then to be photochemically converted to the green one, with the rate of formation of the latter increasing with an increase in UV dosage or oxidizing conditions. The addition of penicillamine drastically reduced the photochemical formation of both fluorophores.
Both the blue and green fluorophores appear to result from the photochemically induced covalent attachment of 3-HK to glycine. In the human lens, these reactions can explain the age-related loss of 3-HKG with the concomitant formation of fluorophores covalently attached to lens proteins, probably via the amino group of lysine.     

High-resolution colocalization of single molecules within the resolution gap of far-field microscopy.

To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.     

Synthesis of novel rod-shaped and star-shaped fluorescent phosphane oxides--nonlinear optical properties and photophysical properties

The design of a new class of fluorophores is presented. Some push-pull chromophores (D-pi-A) containing polyphenylethynyl units and a phosphane oxide moiety were efficiently prepared from common intermediates. Straightforward syntheses gave novel one-armed, rod-shaped and three-armed, star-shaped fluorophores. The optical properties of the resulting star-shaped derivatives were evaluated, showed high fluorescence quantum yields, and their excitation induces very efficient charge redistribution. Moreover, thanks to their push-pull character, the molecules exhibited significant second-order NLO properties with good transparency, up to 67x10(-30) esu at 1907 nm, with an absorption lambdamax at 369 nm. The effect of the donor group and of the number of phenylethynyl arms have been studied in this work.