Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

Global analysis of steady-state polarized fluorescence spectra using trilinear curve resolution.

Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.     

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.     

Analysis of fluorescence decay curves by means of the Laplace transformation.

A computational procedure is described for the analysis of fluorescence decay data convolved with a lamp flash of finite width. The computer program calculates the ratio of the Laplace transforms of the decay and the lamp flash for different values of s to give the transforms of the impulse response for each value of s. These are set equal to the analytical Laplace transforms of the decay law involved. Solution of the nonlinear simultaneous equations yields the desired decay parameters. The method can be modified to analyze data that contains a component due to scattered light and can also provide essential information regarding transit time changes of the photomultiplier with changes in emission wavelength. The method was tested by the analysis of real and simulated data. The accuracy of the analysis depends on the degree of correlation among the parameters.     

Correction for contaminant fluorescence in frequency-domain fluorometry

We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.     

Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence

Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.     

Sensitive detection of small shifts in fluorescence spectra

Instrumentation is described for the sensitive detection of small shifts in the emission peaks of fluorescence spectra. The method is based on the modulation with time of the wavelength of the detected light at the emission maximum of the starting spectrum. Modulation of the wavelength is achieved by a novel use of a photoelastic light modulator and a specific arrangement of polarizing filters placed at the exit slit of the emission monochromator. A shift in the initial emission spectrum results in an alternating current in the detected light at double the fundamental frequency of the photoelastic modulator, which is detected with a phase-sensitive amplifier. The instrument is sensitive to shifts of 0.2 to 0.5 nm in the wavelength of maximal emission, verified with solutions of 5-dimethylaminonaphthalene-1-sulfonyl- -glutamic acid and reduced β-nicotinamide adenine dinucleotide by following fluorescence shifts resulting from alterations in solvent polarity. Also, the fluorescence of quinine (at micromolar levels) is detectable in the presence of a tenfold higher emission intensity of 9-aminoacridine, although the emission maxima of the two compounds are separated by less than 10 nm. The results of these experiments suggest applications of the technique to problems of biological interest which require sensitive detection of minute changes in fluorescence spectra, changes which are due to a shift in the emission spectrum of the chromophore studied or to the occurrence of a new emitting species which has a slightly shifted fluorescence spectrum.     

In vivo fluorescence excitation and absorption spectra of marine phytoplankton: I. Taxonomic characteristics and responses to photoadaptation

Absorption and fluorescence excitation spectra were measuredfor batch cultures of five species of marine phytoplankton grownunder high and low light. These spectra were examined for propertiescharacteristic of taxonomic position and of photoadaptive response.While regions of absorption and excitation of chlorophyll afluorescence diagnostic of pigment composition were identifiable,photoadaptive response had greater influence on spectral variability.Although reduced growth irradiance caused changes in both theabsorption and fluorescence excitation spectra, the fluorescenceexcitation spectrum appears to be more sensitive to alterationsin the ambient light field for growth than does the absorptionspectrum. For a single species. the fluorescence excitationspectrum for a sample grown at low irradiance showed greaterstructure than that for the sample grown at a high irradiance.Under low light conditions, the excitation of chlorophyll afluorescence by accessory pigments increased relative to theexcitation by chlorophyll a itself The highest fluorescenceyields occur in the blue-green region of the spectrum, correspondingto bands of peak absorption by the accessory pigments. Changesin absorption spectra are less marked, but two features recur.First. in the blue-green region of the spectrum from -500–560nm. absorption is enhanced in the low-light cells relative tothat of the high-light cells. Second, the ratio of absorptionat 435 nm to that at 676 nm was greater for the high-light cells.Correlating changes in pigment concentrations were observed.The influence of photoadaptation on the properties of fluorescenceexcitation spectra is as great or greater than the influenceof pigment complements characteristic of specific algal taxa.     

The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and chlorophyll

1. The fluorescence spectra of the alga Porphyridium have been recorded as energy distribution curves for eleven different incident wave lengths of monochromatic incident light between wave lengths 405 and 546 mµ. 2. In these spectra chlorophyll fluorescence predominates when the incident light is in the blue part of the spectrum which is strongly absorbed by chlorophyll. 3. For blue-green and green light the spectrum excited in Porphyridium contains in addition to chlorophyll fluorescence, the fluorescence bands characteristic of phycoerythrin and of phycocyanin. 4. From these spectra the approximate curves for the fluorescence of the individual pigments phycoerythrin, phycocyanin, and chlorophyll in the living material have been derived and the relative intensity of each of them has been obtained for each of the eleven incident wave lengths. 5. The effectiveness spectrum for the excitation of the fluorescence of these three pigments in vivo has been plotted. 6. From comparisons of the effectiveness spectrum for the excitation of each of these pigments it appears that both phycocyanin and chlorophyll receive energy from light which is absorbed by phycoerythrin. 7. It is suggested that phycocyanin may be an intermediate in the resonance transfer of energy from phycoerythrin to chlorophyll. 8. Since phycoerythrin and phycocyanin transfer energy to chlorophyll, it appears probable that chlorophyll plays a specific chemical role in photosynthesis in addition to acting as a light absorber.     

A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves

In order to examine the transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves, a micro-fluorescence imaging system was devised using a microscope, a CCD camera with an image intensifier, an Ar and a He-Ne laser light source, an image processor, and a microcomputer. A laser light was projected vertically on to the surface of a rice leaf segment at a cut-edge, and scattered light and induced fluorescence were observed at the cut-section from a 90° angle to the axis of the laser beam. The intensity of scattered light showed a maximum at several micrometres depth from the leaf surface and a steep gradient afterwards. Fluorescence reached a maximum crossing with the decline curve of the scattered light. The maximum of fluorescence measured at 741 nm was observed at a greater depth from the leaf surface than that at 687 nm, suggesting that part of the fluorescence of the longer wavelength was emitted due to absorption of fluorescence of the shorter wavelength. Profiles of the scattered light and the chlorophyll fluorescence depended on leaf anatomy.     

Dependence of fluorescence spectra of chloroplasts from the activity of photosystem 2]

By means of high sensitive spectrofluorometer the fluorescence spectra have been measured of normal chloroplasts and those with blocked photosystem 2 activity due to photoinhibition or treatment with 0.6 M tris-buffer. At room temperature fluorescence spectra of inactivated chloroplasts are similar to the spectrum of normal chloroplasts measured at low light intensity. Under excitation by intense light a decrease of intensity at 685 nm is appeared (about 3-4 times) in the fluorescence spectra of inactivated chloroplasts as compared to the spectrum of normal chloroplasts. The sharp intensity decrease of maxima at 685 and 695 nm (3-4 times) and small decrease at 680 and 730 nm (by 30-50%) are observed in low temperature fluorescence spectra of inactivated chloroplasts. Thus, the damage of photosystem 2 reaction centres is not accompanied by the preferential decrease of the only fluorescence band. The similarity of fluorescence difference spectra of chloroplasts distinguished by the state of photosystem 2 reaction centre, and the complex structure of difference spectra indicate that the variable fluorescence of chloroplasts during the induction is due to the emission of bulk chlorophyll alpha of the photosystem 2.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

The use of an internal standard for semiquantitative analysis of low temperature (77°K) fluorescence of photosynthetic cells

A simple method is described which permits quantitative estimation of chlorophyll fluorescence emission intensity relative to the chlorophyll concentration in sample material at 77°K. A fluorescent standard excited by light of a similar wavelength as that of the sample material but emitting at a slightly shorter wavelength is mixed with the sample in known proportions. The fluorescence yield of the various peaks of the sample are normalized to the fluorescence yield of the internal standard. The spectra are recorded using an inexpensive attachment consisting of a Dewar holder, mounted instead of the light source of any double beam or split beam spectrophotometer. A glass rod is immersed in the sample standard mixture and then placed in the liquid nitrogen containing Dewar. Exciting light is provided by a light pipe mounted at 90° to the sample holder, connected with a tungsten-hallogen lamp provided with an appropriate filter. This method has been found to be particularly useful for the quantitation of chlorophyll fluorescence emission at 77°K of photosynthetic cells or chlorophyll containing membrane fractions. For this purpose, phycocyanin was found to be a suitable internal standard.     

植物油的荧光光谱法研究

荧光光谱技术具有灵敏度高,选择性好等优点。植物油中含有各种荧光成分,对食用油中菜籽油、玉米油、芝麻油、葵花仁油、花生油的同步荧光光谱和三维荧光光图谱进行了分析研究,结果表明：利用同步荧光光谱和三维荧光图谱的特征,可以区分植物油的品种,可用于植物油的鉴别和质量监控,该法提供了一种油定性分析的方法,同时也扩展了荧光法在食用油鉴别中的应用。     

Chlorophyll fluorescence emission spectrum inside a leaf.

Chlorophyll a fluorescence can be used as an early stress indicator. Fluorescence is also connected to photosynthesis so it can be proposed for global monitoring of vegetation status from a satellite platform. Nevertheless, the correct interpretation of fluorescence requires accurate physical models. The spectral shape of the leaf fluorescence free of any re-absorption effect plays a key role in the models and is difficult to measure. We present a vegetation fluorescence emission spectrum free of re-absorption based on a combination of measurements and modelling. The suggested spectrum takes into account the photosystem I and II spectra and their relative contribution to fluorescence. This emission spectrum is applicable to describe vegetation fluorescence in biospectroscopy and remote sensing.     

Long-lived fluorescence lifetime from tyrosine in a peptide derived from S-100b.

It was shown in an earlier report (Turner et al., 1989, Biochem. Cell Biol. 67; 179-186) that the anomalous steady-state fluorescence emission spectra observed for the protein S-100b in aqueous solution at pH 7.5 contains a long-lived fluorescence decay component. In this study, a peptide consisting of residues 11 to 27 of the beta-subunit, was investigated. 11Ile-Asp-Val-Phe-His15-Gln-Tyr-Ser-Gly-Arg20-Glu-Gly- Asp-Lys-His25-Lys-Leu27 Fluorescence lifetimes were measured at the emission maximum and in the red edge of the spectrum. At wavelengths greater than 320 nm, the data was best fit with three exponentials. The third exponential gave lifetimes of 13.1 ns and 15.9 ns when the peptide was dissolved in the solvents propane-2-ol and propane-1,2-diol, respectively (lambda EX = 275 nm, lambda EM = 350 nm). These fluorescence lifetimes are similar to that observed for a decay component of native S-100b in the red edge of the emission, suggesting that the 1 degrees and 2 degrees features of a heptadecapeptide from S-100b protein has enough structural information when dissolved in solvents of intermediate polarity provide appropriate conditions for long-lived fluorescence from a tyrosine/tyrosinate species to occur.     

DIFFERENCES BETWEEN IN VIVO ABSORPTION AND FLUORESCENCE EXCITATION SPECTRA IN NATURAL SAMPLES OF PHYTOPLANKTON

The fluorescence excitation spectrum of live phytoplankton cells represents the portion of light absorbed that has been effectively transferred to chlorophyll a of photosystem II, whereas light absorbed by photoprotective pigments will not lead to fluorescence. Therefore, the in vivo fluorescence excitation spectrum of phytoplankton has been used as a proxy for the action spectrum of phytoplankton in computations of primary production in the ocean. The distribution of chlorophyll a between photosystems, as well as variations in the pathway of energy inside the photosynthetic membrane, can also influence the fluorescence excitation spectrum. In this study, we investigated the contribution of photoprotective pigments to the differences found between in vivo absorption and fluorescence excitation spectra of phytoplankton measured during two cruises: one from Las Islas Canarias to Nova Scotia and another in the Labrador Sea. A comparison of normalized fluorescence excitation and absorption spectra showed high variability in the difference between absorption and fluorescence in the blue region of the spectrum for samples from the two cruises. This difference was not entirely correlated with the concentration of photoprotective carotenoids. In this paper, results are interpreted in terms of differences in pigment composition and known patterns of energy distribution in the photosystems of different algal groups.     

The application of fluorescence lifetime readouts in high-throughput screening

Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10(2), but the same accuracy with a 2-component decay requires a peak photon count of 10(3). When a 3rd component is introduced, the minimum peak count increases to 10(4). The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods.     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.     

Spectral analyses of extrinsic fluorescence of the nerve membrane labeled with aminonaphthalene derivatives

A fluorescence spectrophotometer was constructed to determine the emission spectrum of a nerve labeled with various fluorochromes. Using this spectrophotometer, the spectra of 2-p-toluidinylnaphthalene 6-sulfonate (2,6-TNS) and other aminonaphthalene derivatives in squid giant axons were determined at the peak of nerve excitation, as well as in the resting state of the axons. During nerve excitation the fluorescent light deriving from the 2,6-TNS-stained nerve undergoes a transient change in intensity. The spectrum of the light contributing to this change in intensity was found to be much narrower and sharper than the fluorescent spectrum of the light arising from labeled axons at rest. This narrow and sharp spectrum is interpreted as being derived from a transient variation in the polarity of the 2,6-TNS binding sites in the axon. In the Appendix, the results of a physicochemical investigation into the factors affecting the fluorescence of 2,6-TNS in vitro are described.