Fluorescence Emission Data Research Articles

Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (FImage) of freshly excised cucumber ( Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-band (300-400 nm) excitation source centered at 360 nm. Means of relative fluorescence intensities (RFI) from these spectral fluorescence images were compared with spectral fluorescence emission data obtained from excitation wavelengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of dimethyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (FImage, 280EX, 380EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea (EDU or ethylenediurea) as a chemical protectant against UV-B damage. Plants exhibited well known foliar growth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower chlorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone had no effect on foliar variables, there was no evidence that EDU afforded protection against UV-B. Instead, EDU augmented some UV-B effects when provided in conjunction with UV-B irradiation (e.g., reductions in the chlorophyll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b content).Relative fluorescence intensities (RFI) in the longer visible wavelengths (green, red, and far-red) were uncorrelated for comparisons between the FImage and 380EX data sets. However, blue and green RFI were significantly correlated (0.8≫r≫0.6; P ≤0.002) for comparisons between FImage and 280EX data sets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both images and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and FImage stimulates processes that produce excess blue fluorescence. The molecules that produce the excess blue fluorescence in both the 280EX and the Fimage data are different electron transfer agents that operate in parallel. For FImage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissues (as occurs for the extracts of leaf discs), whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer. UV-B irradiated leaves showed much greater heteorgeneity of RFI in both the green (F550 FImag ) and the red (F680 FImag ) bands than unirradiated leaves; this was true irrespective of EDU treatment.Although qualitative responses in individual bands differed between FImage and 380EX data, similar results were obtained in the detection of UV-B induced effects when the red/green and blue/far-red fluorescence ratios of these data were compared. The red/green ratio (either F680/F550 FImage or F675/F525 380EX ) was lower for UV-B exposed plants in both images and 380EX data. UV-B exposure also significantly enhanced the blue/far-red ratio of images (F450/F740 FImage ) and the comparable 380EX ratio (F450/F730 380EX ) for the combined UV-B/EDU group. The far-red/red ratios were not useful in separating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420 280EX ) was substantially reduced by UV-B exposure and was inversely related to total photosynthetic pigment content. These findings suggest that the red/green ratio (FImage, 380EX) and the UV/blue ratio (280EX) may be as useful as the blue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-B stress damage in plants.

The effects of daunomycin antibiotic on histone H 1: thermal denaturation and fluorescence spectroscopy studies

Using thermal denaturation and fluorescence spectroscopy, we have investigated the interaction of antitumor antibiotic, daunomycin, with calf thymus histone H 1 under several ionic strengths. The results show that daunomycin binds to histone H 1 and increases its melting temperature. Increasing ionic strength elevates this effect. Fluorescence emission data show that the interaction of daunomycin with histone H 1 decreases the emission intensity at 325 nm and induces hyperchromicity in the emission spectrum of the drug. The results suggest that histone H 1 can be considered as a new target for drug action at the chromatin level.

"Axial-Bonding"-Type Hybrid Porphyrin Arrays: Synthesis, Spectroscopy, Electrochemistry, and Singlet State Properties.

A series of phosphorus(V), germanium(IV), and tin(IV) porphyrin-based, "axial-bonding"-type hybrid trimers have been readily constructed by employing a new "building-block" approach. The approach is modular in nature, and it involves simple "inorganic" reactions such as axial bond formation of main group element containing porphyrins and insertion of metal/"metalloid" ions into the porphyrin cavity. The architecture of these arrays is such that, while a phosphorus(V), germanium(IV), or tin(IV) complex of meso-5,10,15,20-(tetratolyl)porphyrin forms the basal scaffolding unit, the free-base, vanadyl, cobalt(II), nickel(II), copper(II), or zinc(II) porphyrins occupy the two axial sites via an aryloxy bridge. Synthesis of an "all-phosphorus" array containing three phosphorus(V) subunits has also been accomplished. Each new porphyrin array investigated in this study has been fully characterized by various physical methods that include mass (FAB), UV-visible, infrared, fluorescence, electron spin resonance (ESR), and (1)H and (31)P nuclear magnetic resonance (NMR; 1D and 2D) spectroscopies and cyclic voltammetry. The UV-visible and ESR spectral parameters and also the redox potential data suggest that there exists no interaction between the pi-planes of the constituent monomeric porphyrins in these arrays. Detailed (1)H NMR investigations carried out with the trimers containing diamagnetic porphyrins reveal characteristic shielding/deshielding effects for the various protons on the axial porphyrin subunits. The ground state data, as probed by the spectroscopic and electrochemical techniques, collectively indicate that there exists a symmetric but nonparallel disposition of the two axial porphyrins with respect to plane of the central porphyrin. Singlet state activity of the photoactive trimers has been probed by the steady state fluorescence method with selective excitation into the bands corresponding to the two constituent monomeric species. Analysis of the fluorescence emission and excitation spectral data suggests the occurrence of electronic energy transfer as well as photoinduced electron transfer reactions in trimers endowed with free-base or zinc(II) porphyrin axial subunits. Efficiencies of the excited state processes of these trimeric arrays are shown to be dependent on the type of metal/metalloid ions present in the porphyrin crevice.

3,5-Diaryl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) Dyes: Synthesis, Spectroscopic, Electrochemical, and Structural Properties

This research was undertaken to obtain new “BODIPY” dyes that fluoresce at relatively long wavelengths. The title compounds 1a−e were prepared via a divergent route involving Suzuki couplings of arylboronic acids to N-tert-butoxycarbonyl-4-bromopyrrole 2, condensation of the products with an acid chloride, and incorporation of the boron difluoride entity. Two alkyl-substituted systems 7a and 7b were also prepared for comparison; the critical difference between structures 1 and 7 is that the former have an aryl group attached to each pyrrole nucleus whereas the latter only have alkyl substituents on that same ring. UV absorption and fluorescence emission data were compared for compounds 1 and 7. Absorption and fluorescence emission maxima for compounds 1 occur at higher wavelengths than for compounds 7, and the Stokes shifts for the aryl-substituted compounds 1 are larger than for the alkyl-substituted compounds 7. Fluorescence quantum yields measured for compounds 1 are less than for compounds 7, and poss...

Synthesis and fluorescence properties of 9,10-bis(phenylethynyl)anthracences

9,10-Bis(phenylethynyl)anthracences (BPEA's) are of great interest as fluorescent emitters for peroxyoxalate chemiluminescence. A series of substituted BPEA's were synthesized by condensing phenylethynyl magnesium bromide with substituted anthraquinones followed by acid hydrolysis and reduction by stannous chloride. Fluorescent quantum yields of 0.72–0.87 were found among most of the compounds, with 1-iodo-BPEA at 0.47 and 1,8-diphenoxy-BPEA at 0.51 as exceptions. Absorption and fluorescent emission data for all compounds are presented.

Evidence for the Formation of a Benzo[a]phenothiazine/2-Hydroxypropyl-β-Cyclodextrin Inclusion Complex Using a Fluorescence Method*

The inclusion complex of benzo[ a]-12H-phenothiazine (BPHT) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) was investigated in an aqueous solution containing 1% ethanol. It was found that the fluorescence emission intensity of the complexed fluorophore is six times larger than that of un complexed species and 1.5 times higher than that in pure ethanol. BPHT forms a 1:1 complex when included in the HP-β-CD. An association constant of the complex of 100±20 M−1 was calculated by using the Benesi–Hildebrand treatment of fluorescence emission data. A discussion based on AM1 semiempirical calculation of BPHT geometry indicates that the inclusion of BPHT in HP-β-CD is partially achieved, probably by the hydrophobic aromatic moiety of the molecule.

All- trans-N-retinylidenetryptamine Schiff base in surfactant solubilized water pools in heptane—a fluorescence study

All- trns-N-retinylidenetryptamine Schiff base was incorporated into aerosol-OT (AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/heptane reverse micelles. This micellar system was used as a model to study the retinal-tryptophan interactions in retinal proteins. The retinylidene Schiff base remains stable in the presence of reverse micelle-solubilized water pools. Partition coefficient and microviscosity measurements show that the Schiff base is located in the micellar interphase. The results are discussed in terms of the interaction between the retinylidene chromophore and the active site environment of rhodopsin and bacteriorhodopsin. In the present model, the quencher and emitting units are covalently attached, and are separated by two carbon spacer units. The fluorescence emission data obtained for the micelle-intercalated Schiff base chromophore are compared with the fluorescence of the native protein and intermediates in the photochemical cycle of bacteriofhodopsin. A comparison of the data obtained for tryptamine and the Schiff base with the results available for bacteriorhodopsin and bacterioopsin reveals that there is a large degree of quenching on intercalation of the retinylidene chromophore in the vicinity of the fluorophore. Evidence provided by this model suggests that energy transfer to retinal can occur from tryptophan residues located in the retinal pocket in the native protein. Thus the retinylidene unit can act as a quencher of the energy of tryptophan, the nature and extent of which may depend on the conformation and relative orientation of the protein-bound fluorophore.

Photochemically induced fluorescence investigation of a?-cyclodextrin: Azure A inclusion complex and determination of analytical parameters

The photooxidation of Azure A and fluorescence properties of Azure A and its photoproduct have been investigated in aqueous media and in the presence ofβ-cyclodextrin (β-CD). The fluorescence intensity of the complex formed between the photoproduct and β-CD was found to be three times higher than that of the uncomplexed Azure A photoproduct. A complex formation constant of 110±40 M−1 was calculated using the Benesi-Hildebrand treatment of the fluorescence emission data. Although the stoichiometry of the Azure A photoproduct: β-CD complex was found to be 1: 1, it seems that the Azure A structure is only partially included. Calibration graphs were plotted for the free Azure A photoproduct and the photogenerated product included in β-CD. The analytical parameters and quantification limits were determined.

Intramolecular excitation energy transfer in anthracene appended porphyrins

Free-base and zinc(II) derivatives of supermolecular porphyrins bearing one or four anthracene subunits have been synthesized and fully characterized. It was observed that, in methylene chloride solutions, fluorescence due to the anthracene subunit of these systems has been quenched in comparison with the fluorescence of free anthracene and that the extent of quenching depends on the number and site of attachment of the anthracerte/s at the porphyrin periphery. Based on the fluorescence emission and excitation spectral data, the observed quenching has been attributed to the singletsinglet energy transfer, from the anthracene donor subunits to the porphyrin. A detailed analysis of the results suggested that the energy transfer process proceeds predominantly through Forster's dipole-dipole mechanism in these systems. Further, in the light of structural and thermodynamic data, other possible mechanisms for the observed quenching were considered. A photoinduced electron transfer reaction has been found-to effectively compete with the energy transfer in polar solvents. The results obtained in this study are discussed in comparison with the results of other porphyrin based energy transfer model systems and also with relevance to the carotene function of the photosynthetic antenna pigments.

Studies on the location of aromatic amino acids in a-crystallin

The locations of tryptophan residues in α-crystallin and homopolymers constructed from the αA- and αB-chains were examined by comparing their fiuorescence emission properties and their accessibilities to quenchers. Two classes of tryptophan could be distinguished on the basis of differences in their spectral characteristics, fluorescence decay lifetimes, quenching with acrylamide and exposure by increasing concentrations of urea. Polarization measurements indicated that the tryptophan residues were associated with flexible segments of the polypeptide chains. The two classes could be assigned, one to Trp-9 (in both A- and B-chains) which is in an hydrophobic environment, and one to Trp-60 (B-chaIn) which appeared to be nearer the surface of the aggregate. No evidence was found for residues inaccessible to the quenchers. An apparent partition coefficient of 40 was obtained for the association of acrylamide with the protein. From temperature-dependence studies, it was concluded that there was a significant energy barrier to the penetration of acrylamide into the protein matrix (E a -- 5.8 kcal/mol) and that entry of the quencher was through channels produced by temporary disruption of the matrix ( Δs = 1.5 eu). The phenolic side-chains of tyrosine residues in several different α-crystallins were found to ionize with p K values above pH 11, indicative of residues highly shielded from the solvent. Comparisons of polypeptide sequences, together with tyrosine fluorescence emission data and the p K values, permitted a tentative assignment of residue locations. All of the data are consistent with a possible micelle-like structure for α-crystallin but not with a layered structure.

Protein: nucleic acid interactions. I. Electronic structures of cytosine, indole, and guanine complexes.

Low singlet transition energies and line strengths were calculated for the cytosine:indole:guanine complex by the INDO/1S-CI method. The chromophores were arranged in three sets of 270 intercalating geometries. Calculations were executed in the supermolecule model with single excited configurations. Errors due to basis set extension and incomplete configuration representation were assessed, for all chromophore pairs, by full BSSE correction calculations and inclusion of double-excited configurations. The intercalation-induced perturbations of the principal transitions are characterized by but not limited to (a) a decrease in strength of [pi*,pi] transitions, (b) increase in strength in [pi*,n] transitions, (c) splitting of [pi*,pi] transitions into components of unequal strength, and (d) energy and strength dependence in mixed transitions on rise and shift movements of the nucleic acid bases. These predictions are in accord with absorption, fluorescence emission, and scattering, and resonance Raman spectroscopic data on oligonucleotides and analogous aromatic complexes. The calculations suggest that major differences in intercalating coordinations are discernible in the near-uv spectroscopic domain of proteins and nucleic acids.

FLUORESCENCE IMAGING AND POINT MEASUREMENTS OF TISSUE: APPLICATIONS TO THE DEMARCATION OF MALIGNANT TUMORS AND ATHEROSCLEROTIC LESIONS FROM NORMAL TISSUE

The possibilities of using laser-induced fluorescence for tissue diagnostics are discussed. The tissue types investigated are malignant tumors and atherosclerotic lesions. Studies with natural autofluorescence as well as with fluorescent tumor markers are included in this paper. Fluorescence emission and decay data are presented for some tissue chromophores contributing to tissue autofluorescence. Optical spectroscopic characteristics of fluorescent malignant tumor markers are analyzed and instrumental designs for clinical applications are discussed. Images recorded with a multicolor fluorescence imaging system developed in Lund are presented.

Synchronous fluorescence spectroscopy of dissolved organic matter in surface waters: Application to airborne remote sensing

Synchronous fluorescence spectroscopy of dissolved organic matter (DOM) in surface waters is investigated as a method of inferring DOM composition. The synchronous fluorescence technique, which has previously been shown to provide structural information for polycyclic hydrocarbons, including crude oils, has been extended to DOM as a method for “fingerprinting” humic substance (HS) types. Fluorescence emission and synchronous fluorescence data were collected for 21 lake water samples. The lake sample synchronous spectra, which are compared with synchronous spectra of lignin and data derived from the literature of other model compounds, provide new insights into the structure of surface water DOM. Further support for this approach is provided by a comparison between the multiple linear regression result found when relating measured pH to fluorescence emission data (R 2 = 0.75) and that found when relating the measured pH to synchronous fluorescence data (R 2 = 0.90). Adapting the synchronous fluorescence spectral technique to a remote sensing context is discussed in terms of using the method for choosing optimal excitation wavelengths for a multiple-wavelength lidar.

Laser-induced fluorescence: Limits to the remote detection of hydrogen ion, aluminum, and dissolved organic matter

Laser-induced fluorescence (LIF) of dissolved organic matter (DOM) in lake water is, to a certain extent, indicative of water quality. DOM fluorescence intensity varies directly with connentration of the fluorescing organic matter; however, the intensity and spectral distribution of fluorescence are also affected by other physical and chemical factors. The chemical factors include concentrations of trace metals, hydrogen ion, and the composition of the DOM itself. An empirical study was conducted seeking specific relationships between the intensity and spectral distribution of DOM fluorescence and the concentration of aluminum and hydrogen ion in lake water. Water samples from 49 lakes in northern Michigan and northern Wisconsin were collected and their fluorescence emission spectra analyzed with respect to the lake water chemistry. Trends that had been apparent in previous measurements using prepared samples were less visible in the lake water data. Observed changes in the fluorescence emission can be produced by one of two general mechanisms: quenching and precipitation. Quenching of DOM fluorescence - a result of cation reactions - is seen as a reduction of the total fluorescence intensity and a shift of the overall emission spectrum to shorter wavelengths. In contrast, preferential precipitation of the high molecular weight DOM by cation reactions appears to shift DOM fluorescence to the longer wavelengths. These nonspecific effects make it difficult, if not impossible, to predict the complicated relationships between fluorescence emission and individual water chemistry parameters. By itself, a simple fluorescence emission spectrum of DOM is not a sufficiently accurate indicator of hydrogen ion or aluminum concentration; however, fluorescence parameters correlate well with dissolved organic carbon (DOC) And with the organic aluminum per unit dissolved organic carbon (Al0 / DOC). DOM fluorescence emission spectra should also be useful as a survey tool in conjunction with limited water chemistry or as a means of monitoring change in a lake whose initial chemistry and fluorescence properties have been defined. Lastly, by augmenting fluorescence emission data with other remote sensing data, e.g., fluorescence excitation spectra, fluorescence decay rates, and Raman spectra, a more detailed chemical analysis should be possible.

Multivariate Analysis of Functional and Structure-Related Properties of Whey-Vegetable Protein Composites

Several multivariate statistical techniques were applied to ultraviolet absorption, fluorescence emission and extrinsic fluorescence data to investigate the relationship between functionality and structure-related properties of whey-potato and whey-pea protein composites over the pH range of 4 to 8. Multiple regression analysis resulted in highly significant (p<0.001) equations predicting solubility, thermocoagulability, foaming capacity and emulsion properties. Cluster and principal component analyses delineated variables into three distinct groups reflecting solubility and/or surface-active properties, surface hydrophobicity and tryptophan exposure. Stepwise linear discriminant analysis led to correct differentiation of individual samples into their respective composites, based solely on significant (p<0.05) contributions to discriminant functions by variables characterizing surface hydrophobicity and solubility and/or surface-active properties. Results from this study support the hypothesis that hydrophobic residues play a key role in functional properties displayed by whey-vegetable protein composites.

A high-resolution, fluorescence-based, semiautomated method for DNA fingerprinting

We describe a fluorescence-based method for the automated analysis of DNA fragments on polyacrylamide gels. A single-stranded oligonucleotide primer (18-mer) with a fluorochrome covalently bound to its 5′-end is annealed to a synthetic oligonucleotide to create a double-stranded oligonucleotide linker with a 5′-overhang complementary to a restriction enzyme site. Cosmid or plasmid DNA is digested with the appropriate restriction enzyme and then ligated to the fluorochrome-labeled linker. The labeled restriction fragments are loaded on a denaturing polyacrylamide gel in a commercially available DNA sequencer. As the restriction fragments migrate through the gel, they intersect a laser beam which excites the fluorochrome-labeled fragment. Fluorescence emission data are captured on a computer in real time and analyzed after the completion of electrophoresis. Fragment length is nearly linearly related to migration time. This method offers very near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases. The fluorochrome-labeling chemistry relies on straightforward enzymatic reactions and can be performed in a single reaction tube. Because four different fluorochromes can be used, each of 16 lanes on the gel can be used to analyze four different digest reactions, one in each color. One of the fluorochromes can be used to label size standards in each lane, eliminating interlane variability and allowing more precise estimates of fragment size. We apply the method to the analysis of overlapping cosmids.

Absolute fluorescence quantum yields by relative fluorescence and photoacoustic measurements of low level luminescence quenching

A method is reported which allows absolute fluorescence quantum yields ΦF to be obtained from relative fluorescence and non-radiative emission (photoacoustic) data. Absolute calibration procedures, external standards, and total fluorescence quenching are eliminated through use of low concentrations of a heavy atom quencher. The relative decrease in fluorescence emission and increase in non-radiative emission as a function of the quencher concentration are related to ΦF through a set of simultaneous equations. Fluorescence quantum yields of ΦF = 0.59 ± 0.03 for cresyl violet perchlorate in EtOH and ΦF = 0.55 ± 0.02 for quinine sulfate in 0.1 N H2SO4 are reported.

Solvent effects on the spectroscopic properties of aflatoxin B 1

1. 1. Solvent-induced changes in the spectral properties of aflatoxin B 1 were investigated using protic and aprotic solvents. 2. 2. The absorption data were less sensitive to solvent effects than the fluorescence emission data. 3. 3. Stokes shifts in protic solvents were greater than those in aprotic solvents indicating hydrogen bond formation between solvent and the excited state of aflatoxin B 1. 4. 4. From the Stokes shift data for aprotic solvents, the dipole moment of aflatoxin B 1 was estimated to increase by 15.7 Debye units upon excitation to the excited singlet state.

Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli

The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.

Non-radiative decay processes in isolated SO 2 molecules excited within the first allowed absorption band (2500–3200 Å)

New and previously published SO 2 fluorescence emission data related to non-radiative decay processes are considered in light of the recent observations of Brus and McDonald. All the present data are consistent with the previous conclusion of Mettee that non-radiative processes in SO 2 singlet photochemistry are unimportant. It appears that any small inefficiency in the emission of light quanta for SO 2 excited at short wavelengths (2650 A) is largely due to the population of a second very short-lived state which is quenched effectively even at pressures down to 1 μ. The very low efficiency of quanta production which we observed at long wavelengths (3020 Å) appears to have its origin not only in the second easily quenched state, but more importantly, in the diffusional loss of the long-lived singlet which for these conditions has a 20-fold greated lifetime than was expected previously.