Padé-Laplace method for analysis of fluorescence intensity decay.

This novel approach to the analysis of multiexponential functions is based on the combined use of the Laplace transform and Padé approximants (Yeramian, E., and P. Claverie. 1987. Nature (Lond.). 326:169-174). It is similar in principle to the well-known Isenberg method of moments (Isenberg, I. 1983. Biophys. J. 43:141-148) traditionally applied to the analysis of fluorescence decay. The advantage of the Padé-Laplace method lies in its ability to detect the number of components in a multiexponential function as well as their parameters. In this paper we modified the original method so that it can be applied to the analysis of multifrequency phase/modulation measurements of fluorescence decay. The method was tested first on simulated data. It afforded recovery up to four distinct lifetime components (and their fractional contributions). In the case of simulated data corresponding to continuous lifetime distributions (nonexponential decay), the results of the analysis by the Padé-Laplace method indicated the absence of discrete exponential components. The method was also applied to real phase/modulation data gathered on known fluorophores and their mixtures and on tryptophan fluorescence in phospholipase A2. The lifetime and fraction recoveries were consistent with those obtained from standard methods involving nonlinear least-square fitting.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K I. ΔpH-dependent quenching

The nature of the light-induced ΔpH-dependent decline of chlorophyll a fluorescence in intact and broken spinach chloroplasts was investigated. Fluorescence spectra at 77 K of chloroplasts frozen in the low-fluorescent (high ΔpH) state showed increased ratios of the band peak at 735 nm (Photosystem (PS) I fluorescence) to the peak at 695 nm (PS II fluorescence). The increase in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio at 77 K was related to the extent of fluorescence quenching at room temperature. Normalization of low-temperature spectra with fluorescein as an internal standard revealed a lowering of F₆₉₅ that was not accompanied by an increase in F₇₃₅: preillumination before freezing decreased both F₆₉₅ and, to a lesser extent, F₇₃₅ in the spectra recorded at 77 K. Fluorescence induction of chloroplasts frozen in the low-fluorescent state showed a markedly decreased variable fluorescence (F_v) of PS II, but no concomitant increase in initial fluorescence (F₀) of PS I. Thus, the buildup of a proton gradient at the thylakoid membrane, as reflected by fluorescence quenching at room temperature, affects low-temperature fluorecence emission in a manner entirely different from the effect of removal of Mg²⁺, which is thought to alter the distribution of excitation energy in favor of PS I. The ΔpH-dependent quenching therefore cannot be caused by such change in energy distribution and is suggested to reflect increased thermal deactivation.     

Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

α-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid.The enthalpy changes associated with the interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (ΔH) are not only larger than at physiological pH, but also show a maximum at about 23°C in the ΔH vs. temperature graph. At pH 6 and 7, on the contrary, ΔH increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of α-lactalbumin, while at pH 4 and 5 at 23°C an extra peak appears in the gel-filtration graphs between the pure vesicles and α-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23°C and a blue shift at 37°C, both to 336 nm, are observed for λ_max of the fluorescence emission spectra at pH 4 when α-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure α-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs.The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.     

Photosynthetic activity of variegated leaves of Coleus × hybridus hort. cultivars characterised by chlorophyll fluorescence techniques

Different pigments often occur together and affect photosynthetic characteristics of the respective leaf portions. In this study, photosynthetic activity in variegated leaves of five cultivars of the ornamental and medicinal plant, Coleus × hybridus hort., was estimated by image analysis and point data measurements of major chlorophyll (Chl) fluorescence parameters and related to the amount of photosynthetic pigments measured with a Chl meter or spectrophotometrically in leaf extracts. Significant differences in Chl and carotenoid (Car) contents were noticed among differentially pigmented sectors of a leaf and among the cultivars. Although the higher Chl concentration was noticed in purple parts compared to green parts of the leaves, the values of minimal and maximal fluorescence yield at the dark- and light-adapted state (F₀, F_m, F₀', F_m', respectively) were a little lower than those in the green sectors, indicating photoprotective effects provided by anthocyanins and Car, more abundant in the red parts. The lowest Chl and Car content was detected in creamy-yellow and pink sectors and this contributed to low F₀, F_m, and F_m', maximal quantum yield of PSII photochemistry, and nonphotochemical and photochemical quenching but high PSII maximum efficiency and effective quantum yield of PSII photochemistry. Both methods of Chl fluorescence analysis revealed heterogeneity in capture, transfer, and dissipation of excitation energy but Chl fluorescence imaging was more suitable in examining very narrow pigmented leaf areas.     

Development of 6-arylcoumarins as nonsteroidal progesterone antagonists. Structure–activity relationships and fluorescence properties

Progesterone is involved in multiple physiological processes, including female reproduction, via binding to the progesterone receptor (PR). We have developed 6-arylcoumarins such as 5 and 6 as non-steroidal PR antagonists with receptor-binding-dependent fluorescence. In this study, we investigated the structure–activity relationships and fluorescence properties of coumarin derivatives bearing a heterocyclic aromatic moiety. Among these derivatives, 7c (IC₅₀: 34 nM) and 10b (IC₅₀: 24 nM) showed more potent PR-antagonistic activity than lead compounds 5 (IC₅₀: 500 nM) and 6 (IC₅₀: 65 nM) in alkaline phosphatase (AP) assay. Compound 9b showed solvent-dependent fluorescence intensity, exhibiting strong fluorescence in the presence of PR LBD only in buffer solution. On the other hand, 10b showed a solvent-dependent shift of the fluorescence maximum wavelength in the presence of PR LBD. These results indicate that 6-arylcoumarin will be a useful scaffold for PR antagonists and fluorescent probes targeting PR.     

Non-exponential decay of indole fluorescence — the red-edge effect

The fluorescence of indole in glycerol decays exponentially at both 22° and ?80°C for 280 nm excitation. Under 296 nm excitation the 22°C emission decay is exponential, but at ?80°C, a second shorter lived (1.3 ns) component is also detected. The relevance of this result to the determination of protein fluorescence lifetime is described.     

Laser scanning fluorescence microscopic measurement of the movement of cleaving egg surface of Rana Amurensis

By laser scanning fluorescence microscopy for quantitative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate during cleavage furrow extending forward,it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape,indicating dark stripe appeared in that place.Then the fluorescence intensity increased at the place where the bottom of ∨ shape had located,and the scanning curve turned to ∧ shape,indicating single stripe was formed.While enhanced fluorescence appeared on the borders of ∧ shape,an M shape curve was found,showing double stripe occurred.During the distance between two borders of M shape incresing from 50μm to 100μm,a fluorescence peak came to sight in the middle of the M shape,which being the cleavge furrow bottom.The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane.At that time the curve became W shape.By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage,showing the stretching of the egg surface being increased.With our[31,33]and others[32] reports that polylysine could induce the appearance of nascent membrane and phytohemagglutinins could decrease or prevent the appearance of nascent membrane,we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stresslay to the outsides of cleavage furrow.     

Observation of two quenchers of chlorophyll fluorescence in chloroplasts at −196°C

Fluorescence induction at ?196°C has been monitored in chloroplasts rapidly frozen after poising at different redox potentials at room temperature. It was found that, as at room temperature, the initial level of fluorescence observed upon shutter opening (F_o), relative to the final level observed after 10 seconds of illumination (F_m) increased as the redox potential of the chloroplasts was lowered. Redox titration revealed the presence of two quenching components with E_m,7.8 at ?70 mV and ?275 mV accounting for approx. 75% and 25% of the variable fluorescence (F_v). Parallel observation of fluorescence yield at room temperature similarly gave two components, with E_m,7.8 at ?95 mV and ?290 mV, also accounting for approx. 75% and 25%. Simultaneous measurement of fluorescence emission at ?196°C at 695 nm and 735 nm indicated that both emissions are quenched by the same redox components.     

Does fluorescence of ANS reflect its binding to PAMAM dendrimer?

The analysis of binding between cationic PAMAM G5 dendrimer and anionic fluorescent probe using fluorescence and equilibrium dialysis has been made. It was found that at low concentrations of ANS the double fluorimetric titration technique can be successfully used for quantitative analysis of binding of ANS to dendrimer. Based on fluorescence and dialysis data the constants of binding and the number of binding centers were calculated for binding of ANS to PAMAM G5 dendrimer: K(b) is approx. (0.5-1)x10(5)M(-1) and n is (0.5-0.7).     

Identification of assembly precursors to photosystems emitting fluorescence at 683 nm and 687 nm by cryogenic fluorescence microspectroscopy

Photosystem I (PSI) and photosystem II (PSII) play key roles in photoinduced electron-transfer reaction in oxygenic photosynthesis. Assemblies of these PSs can be initiated by illumination of the etiolated seedlings (greening). The study aimed to identify specific fluorescence spectral components relevant to PSI and PSII assembly intermediates emerging in greening seedlings of Zea mays, a typical C4 plant. The different PSII contents between the bundle sheath (BS) and mesophyll (M) cells were utilized to spectrally isolate the precursors to PSI and PSII. The greening Zea mays leaf thin sections were observed with the cryogenic microscope combined with a spectrometer. With the aid of the singular-value decomposition analysis, we could identify four independent fluorescent species, SAS677, SAS685, SAS683, and SAS687, named after their fluorescence peak wavelengths. SAS677 and SAS685 are the dominant components after the 30-minute greening, and the distributions of these components showed no clear differences between M and BS cells, indicating immature cell differentiation in this developing stage. On the other hand, the 1-hour greening resulted in reduced distributions of SAS683 in BS cells leading us to assign this species to PSII precursors. The 2-hour greening induced the enrichment of SAS687 in BS cells suggesting its PSI relevance. Similarity in the peak wavelengths of SAS683 and the reported reaction center of PSII implied their connection. SAS687 showed an intense sub-band at around 740 nm, which can be assigned to the emission from the red chlorophylls specific to the mature PSI.     

The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Contribution of each Trp residue toward the intrinsic fluorescence of the Giα1 protein

G_iα1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of G_iα1 bound to GDP•AMF or GTPγS with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the α-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP•AMF or GTPγS, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTPγS, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a π-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic π-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of G_iα1.     

Quantitation of poly-β-hydroxybutyrate by fluorescence of bacteria and granules stained with Nile blue A

Summary Fluorescence from poly--hydroxybutyrate (PHB) inclusions inside Azotobacter vinelandii UWD cells stained with Nile blue A was shown to be proportional to PHB concentration. The intensity of the fluorescence was greatest in native, fluid inclusions and was the least in extracted, crystallized granules. However, isolated air-dried PHB granules also were proportionally stained with Nile blue A. The results show that Nile blue A can be used in the quantitative determination of PHB in a variety of cells.