Fluorescence Lifetime Data Research Articles

Quantitating the association of charged molecules with ionic micelles

We have studied micelles comprised of cationic (CTAB) and anionic (SDS) surfactants through the interactions of solution phase anionic disodium fluorescein (DSF) and cationic rhodamine 110 (R110) dyes with perylene sequestered within the micelles. Fluorescence lifetime measurements monitor energy transfer between the nonpolar optical donor within the micelle and ionic probes in the surrounding solution. The efficiency of this process is mediated by the extent to which the ionic dyes interact with the micelle palisade layer, and our fluorescence lifetime data allow us to determine the association constants for acceptor–micelle interactions.

Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure—the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS–protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe–protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS–apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS–apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state U A. At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS–apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S 1,CT of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S 1,np of ANS stabilized by the strong ANS–apomyoglobin interaction. Lowering the pH diminishes the contribution from the S 1,np state. Meanwhile, more protein molecules become populated at the U A state, which exhibits a short lifetime that is not distinguishable from the S 1,np state. At pH 3.4, when the population of the U A becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the U A. Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the U A below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.

Quantitating the Dynamics of NBD Hexanoic Acid in Homogeneous Solution and in Solutions Containing Unilamellar Vesicles

We report here on the motional and fluorescence lifetime dynamics of the chromophore NBDHA (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) in neat solvents and in aqueous solutions containing unilamellar vesicles of varying composition. We measure the transient response of this chromophore by time-correlated single-photon counting, using one- and two-photon excitation to resolve the Cartesian components of the rotational diffusion constant, D. Our experimental data for NBDHA in selected solvents of varying viscosity demonstrate that one- and two-photon excitation probe different components of the rotational diffusion constant and that this chromophore reorients as a prolate rotor with an aspect ratio of approximately 2. For NBDHA in aqueous solutions containing unilamellar vesicles of varying composition, we recover the same reorientation behavior regardless of vesicle composition. Fluorescence lifetime and steady-state fluorescence data show the chromophore to reside in a polar environment that is different from neat water. We understand these data in the context of the chromophore residing in close proximity to the unilamellar vesicle polar headgroups in all cases.

Polar Plot Representation for Frequency-Domain Analysis of Fluorescence Lifetimes

We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for analysis of dielectric relaxation experiments (Cole-Cole plots), which have proved to be exceptionally useful in that field for decades. We compare this analytical tool that is well developed and extensively used in dielectric relaxation and chemical kinetics to fluorescence measurements.

Full correlation from picoseconds to seconds by time-resolved and time-correlated single photon detection

We present an advanced time-correlated single photon counting (TCSPC) technique that delivers traditional fluorescence correlation (FCS) or cross correlation (FCCS) and fluorescence lifetime data simultaneously. Newly developed electronics allow for detection and registration of single photon events over time periods of hours with picoseconds accuracy. Subsequent software-correlation yields correlation curves covering more than 12 orders of magnitude in time. At the same time, the original data, containing all information accessible by single photon counting techniques, can be analyzed conventionally according to common single molecule fluorescence techniques. Potential applications of the new technique using pulsed or cw laser excitation are discussed.

Fluorescence lifetime measurements to determine the core–shell nanostructure of FITC-doped silica nanoparticles: An optical approach to evaluate nanoparticle photostability

In this paper, we described a novel fluorescence lifetime-based approach to determine the core–shell nanostructure of FITC-(fluorescein isothiocyanate, isomer I) doped fluorescent silica nanoparticles (FSNPs). Because of phase homogeneity between the core and the shell, electron microscopic technique could not be used to characterize such core–shell nanostructure. Our optical approach not only revealed the core–shell nanostructure of FSNPs but also evaluated photobleaching of FSNPs both in the solvated and non-solvated (dry) states. The FSNPs were produced via Stöber's method by hydrolysis and co-condensation reaction of tetraethylorthosilicate (TEOS) and fluorescein linked (3-aminopropyl)triethoxysilane (FITC–APTS conjugate) in the presence of ammonium hydroxide catalyst. To obtain a pure silica surface coating, FSNPs were then post-coated with TEOS. The average particle size was 135 nm as determined by TEM (transmission electron microscope) measurements. Fluorescence excitation and emission spectral data demonstrated successful doping of FITC dye molecules in FSNPs. Fluorescence lifetime data revealed that approximately 62% of dye molecules remained in the solvated silica shell, while 38% of dye molecules remained in the non-solvated (dry) silica core. Photobleaching experiments of FSNPs were conducted both in DI water (solution state) and in air (dry state). Severe photobleaching of FSNPs was observed in air. However, FSNPs were moderately photostable in the solution state. Photostability of FSNPs in both solution and dry states was explained on the basis of fluorescence lifetime data.

Effect of ethanol variation on the internal environment of sol–gel bulk and thin films with aging

Sol-gel derived bulk and thin films were prepared from different compositions at low pH ( approximately 2.0) containing varying concentrations of ethanol from 15 to 60% at constant water (H(2)O)/tetraethyl-orthosilicate (TEOS) ratio (R=4). The fluorescence microscopic and spectroscopic measurements on fluorescent probe, Hoechst 33258 (H258) entrapped in these compositions were carried out at different days of storage to monitor the effects of concentration of ethanol on the internal environment of sol-gel materials. Fluorescence microscopic observations on sol-gel thin films, prepared by dip coating technique depicted uniform and cracked surface at withdrawal speed 1cm/min (high speed) and 0.1cm/min (low speed) respectively, which did not change during aging. Fluorescence spectral measurements showed emission maximum of H258 at approximately 535 nm in fresh sols at all concentrations of ethanol which depicted slight blue shift to 512 nm during aging in bulk. No such spectral shift has been observed in sol-gel thin films coated at high speed whereas thin films coated at low speed clearly showed an additional band at approximately 404 nm at 45 and 60% concentration of ethanol after about one month of storage. Analysis of the fluorescence lifetime data indicated single exponential decay (1.6-1.8 ns) in fresh sol and from third day onwards, invariably double exponential decay with a short (tau(1)) and a long (tau(2)) component were observed in sol-gel bulk with a dominant tau(1) at approximately 1.2 ns at all concentrations of ethanol. A double exponential decay consisting of a short component (tau(1)) at approximately 0.2 ns and a long component (tau(2)) at approximately 3.5 ns were observed at all ethanol concentrations in both fresh and aged sol-gel thin films. Further, distribution analysis of lifetimes of H258 showed two mean lifetimes with increased width in aged bulk and thin films. These results are likely to have strong implications in designing the internal environment for applications in biosensors.

Evidence for a Novel Mechanism of Time-Resolved Flavin Fluorescence Depolarization in Glutathione Reductase

Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft. Fluorescence lifetime data, however, have shown a more rigid active-site structure for wild-type GR than for the tyrosine mutants. These results suggest that the rapid depolarization in wild-type GR originates from an interaction with the flavin-shielding tyrosine, and not from restricted reorientational motion of the flavin. A novel mechanism of fluorescence depolarization is proposed that involves a transient charge-transfer complex between the tyrosine and the light-excited flavin, with a concomitant change in the direction of the emission dipole moment of the flavin. This interaction is likely to result from side-chain relaxation of the tyrosine in the minor fraction of enzyme molecules in which this residue is in an unsuitable position for immediate fluorescence quenching at the moment of excitation. Support for this mechanism is provided by binding studies with NADP + and 2′P-5′ADP-ribose that can intercalate between the flavin and tyrosine and/or block the latter. Fluorescence depolarization analyses as a function of temperature and viscosity confirm the dynamic nature of the process. A comparison with fluorescence depolarization effects in a related flavoenzyme indicates that this mechanism of flavin fluorescence depolarization is more generally applicable.

Synthesis and photochemical properties of a novel iron-sulfur-nitrosyl cluster derivatized with the pendant chromophore protoporphyrin IX.

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe(2)(NO)(4)[(mu-S,mu-S')P] (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 +/- 0.7) x 10(-4) and (2.5 +/- 0.5 x 10(-4)) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe(2)(mu-SR)(2)(NO)(4) esters for which R is a simple alkyl group such as CH(3)CH(2)- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe(2)S(2)(NO)(4) cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.

CN spectroscopy and cosmic background radiation measurements

A source of possible error, not previously considered, in the determination of the cosmic background radiation (CBR) temperature Tγ(CN) from interstellar CN absorption is proposed. This concerns the intrinsic assumption of the validity of using Hönl–London rotational line intensity factors to determine the rotational components of the oscillator strengths of the R(0), R(1), and P(1) lines of the CN B2Σ+–X2Σ+(0,0) transition. Published data on interstellar CN absorption shows that Tγ(CN) is slightly greater than the standard cosmological CBR temperature, Tγ(COBE) = 2.725 K, determined by the COBE satellite, even when local excitation effects of CN are taken into account. From this difference, an estimation was made of the maximum corrections, a few percent, to the ratio of the line strengths and to the ratio of the Hönl–London factors. Fluorescence lifetime data were shown to give similar values for the corrections as well as providing evidence for intramolecular coupling between the relevant B2Σ+state rotational levels and close-lying levels of the A2Π state, this being responsible for rendering the standard Hönl–London factors invalid. Key words: CN spectroscopy, cosmic background radiation, Hönl–London factors.

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences.

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.

Laser-induced fluorescence and dispersed fluorescence spectroscopy, isotopic studies, and lifetime measurements for rhodium monoxide

RhO molecules have been generated from laser-ablated rhodium atoms and 16 O 2 or 18 O 2 and studied spectroscopically. Thirty LIF band features in the 500–650 nm region have been examined. Dispersed fluorescence and excited state lifetime data have been recorded. Measurements on vibrational levels v=0 to 5 of the molecular ground state give ω e =807 cm −1 and ω e x e =5 cm −1 . Excited state vibrational parameters have been estimated. Evidence is found for low-lying electronic states at 3744, 5980 and 6477 cm −1.

Dynamics of 7-Azatryptophan and Tryptophan Derivatives in Micellar Media. The Role of Ionic Charge and Substituent Structure

We have investigated the motional and population relaxation dynamics of several derivatives of 7-azatryptophan (7AT) and tryptophan (TRP), and N-acetyl-tryptophanamide (NATA), in anionic, cationic, and neutral surfactant solutions above the surfactant critical micelle concentrations. We chose the specific derivatives of 7AT and TRP used previously, along with NATA, to elucidate the roles of chromophore charge and side group structure in mediating interactions with micelles. Fluorescence lifetime data are sensitive to local environment, revealing significant differences in the ability of different chromophores to interact with the micelles. Reorientation dynamics of these chromophores indicate persistent probe/micelle interactions that are determined by a balance between ionic charge(s) and dispersion interactions.

Thulium cross-relaxation in a low phonon energy crystalline host

We report on the cross-relaxation of ${\mathrm{Tm}}^{3+}$ in a low phonon energy chloride host. The temperature and concentration dependence for ${\mathrm{Tm}}^{3+}$ cross-relaxation in the host ${\mathrm{YCl}}_{3}$ was studied between 300 and 500 K, in crystals of ${\mathrm{YCl}}_{3}$ with concentrations ranging from 0.7 to $7\ifmmode\times\else\texttimes\fi{}{10}^{20}{\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{s}/\mathrm{c}\mathrm{m}}^{3}.$ Crystals were grown from melts of anhydrous ${\mathrm{YCl}}_{3}$ and ${\mathrm{TmCl}}_{3}$ powders under ${\mathrm{Cl}}_{2}$ atmosphere using a self-seeded vertical Bridgeman technique. Fluorescence spectra and lifetimes resulting from quasi-cw pumping with a 0.5-W, 811-nm diode were collected and analyzed for the spectral region 1100--2000 nm versus temperature for three crystals with varying ${\mathrm{Tm}}^{3+}$ densities. Each of the fluorescence spectra contain three broad features centered at 1200, 1490, and 1800 nm that reflect the populations of the first three excited levels of ${\mathrm{Tm}}^{3+}.$ Rates for multiphonon relaxation and cross-relaxation were determined from the fluorescence lifetime data. An increase in 1200-nm fluorescence as the temperature rises is evidence of an endothermic cross-relaxation process. This suggests a fundamentally new mechanism for optical cooling. A macroscopic rate equation model with its temperature and concentration dependence given by theories for phonon-assisted energy transfer is fit to the spectral data.

Fluorescence of cis-1-Amino-2-(3-indolyl)cyclohexane-1-carboxylic Acid: A Single Tryptophan χ1 Rotamer Model

A constrained derivative, cis-1-amino-2-(3-indolyl)cyclohexane-1-carboxylic acid, cis-W3, was designed to test the rotamer model of tryptophan photophysics. The conformational constraint enforces a single chi(1) conformation, analogous to the chi(1) = 60 degrees rotamer of tryptophan. The side-chain torsion angles in the X-ray structure of cis-W3 were chi(1) = 58.5 degrees and chi(2) = -88.7 degrees. Molecular mechanics calculations suggested two chi(2) rotamers for cis-W3 in solution, -100 degrees and 80 degrees, analogous to the chi(2) = +/-90 degrees rotamers of tryptophan. The fluorescence decay of the cis-W3 zwitterion was biexponential with lifetimes of 3.1 and 0.3 ns at 25 degrees C. The relative amplitudes of the lifetime components match the chi(2) rotamer populations predicted by molecular mechanics. The longer lifetime represents the major chi(2) = -100 degrees rotamer. The shorter lifetime represents the minor chi(2) = 80 degrees rotamer having the ammonium group closer to C4 of the indole ring (labeled C5 in the cis-W3 X-ray structure). Intramolecular excited-state proton transfer occurs at indole C4 in the tryptophan zwitterion (Saito, I.; Sugiyama, H.; Yamamoto, A.; Muramatsu, S.; Matsuura,T. J. Am. Chem. Soc. 1984, 106, 4286-4287). Photochemical isotope exchange experiments showed that H-D exchange occurs exclusively at C5 in the cis-W3 zwitterion, consistent with the presence of the chi(2) = 80 degrees rotamer in solution. The rates of two nonradiative processes, excited-state proton and electron transfer, were measured for individual chi(2) rotamers. The excited-state proton-transfer rate was determined from H-D exchange and fluorescence lifetime data. The excited-state electron-transfer rate was determined from the temperature dependence of the fluorescence lifetime. The major quenching process in the -100 degrees rotamer is electron transfer from the excited indole to carboxylate. Electron transfer also occurs in the 80 degrees rotamer, but the major quenching process is intramolecular proton transfer. Both quenching processes are suppressed by deprotonation of the amino group. The results for cis-W3 provide compelling evidence that the complex fluorescence decay of the tryptophan zwitterion originates in ground-state heterogeneity with the different lifetimes primarily reflecting different intramolecular excited-state proton- and electron-transfer rates in various rotamers.

Spectroscopic Studies of Cyclodextrin Complexes with 2,5-Bis-(4-Methylphenyl)Oxazole

The complexation of γ-cyclodextrins (γ-CDs) with the guest molecule 2,5-bis-(4-methylphenyl)oxazole (MPPO) has been studied by use of fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and dynamic light scattering (DLS). The changes in the 1H NMR aromatic signals of MPPO in aqueous γ-CD provide evidence of complexation and inclusion. DLS was used to confirm the existence of a distribution of the aggregates in the nanometer range. Excimer fluorescence was pronounced in the presence of γ-CD. The evidence of the existence of two species was shown through fluorescence lifetime data. The emission intensities of the steady state fluorescence and the fluorescence anisotropies of the linear aggregates revealed a phase transition temperature of 60 °C.

Binding of hemoglobin to red cell membranes with eosin-5-maleimide-labeled band 3: analysis of centrifugation and fluorescence data.

We have studied the binding of hemoglobin to the red cell membrane by centrifugation and fluorescence methods. The intact red cell was labeled with eosin-5-maleimide (EM), which specifically reacts with lysine 430 of band 3. Even though this residue is not part of the cytoplasmic domain of band 3 (cdb3) associated with hemoglobin binding, fluorescence quenching was observed when hemoglobin bound to inside-out vesicles (IOVs). The use of fluorescence quenching to measure band 3 binding was quantitatively compared with the binding determined by centrifugation, which measures binding to band 3 and non-band 3 sites. For the centrifugation it was necessary to include the non-band 3 association constants determined from chymotrypsin-treated IOVs. The binding of hemoglobin to band 3 was interpreted in terms of the binding of two hemoglobin tetramers to each band 3 dimer. An anticooperative interaction associated with the conformational change produced when hemoglobin binds results in a 2.8-fold decrease in the intrinsic constant of (1.54 +/- 0.25) x 10(7) M(-1) for the binding of the second hemoglobin molecule. From the changes in lifetime produced by binding the first and second hemoglobin molecules, it was possible to show that the conformational change associated with binding the second hemoglobin molecule results in a decrease of the heme-eosin distance from 47.90 to 44.78 A. Reaction of cyanate with the alpha-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding of hemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobin molecule to band 3 decreases by a factor of 29 to (5.34 +/- 0.15) x 10(5) M(-1). The anticooperative interaction is greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobin tetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobin is very different with the second HbOCN increasing the heme-eosin distance to 55.99 A. The utilization of eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directly study the binding to band 3. At the same time a sensitive probe of the conformational changes, which occur when hemoglobin binds to band 3, is provided.

Fluorescence lifetime measurements of disulfonated aluminium phthalocyanine in the presence of microbial cells.

The fluorescence lifetimes of disulfonated aluminium phthalocyanine (AlPcS2) in the presence of several different microbial cells are measured using the technique of time correlated single photon counting (TCSPC) employing front-face illumination. The microbes studied are: Escherichia coli, Porphyromonas gingivalis (Gram-negative bacteria), Streptococcus mutans (a Gram-positive bacterium), and the yeast Candida albicans. Complex fluorescence decays are observed when AlPcS2 is in the presence of these microbes; the fluorescence decay data can be fitted to a distribution of exponential lifetimes indicating that the AlPcS2 molecules experience a range of micro-environments The average fluorescence lifetimes of AlPcS2 in the presence of the microbes studied range from 4.85 to 5.95 ns, indicating differences in the cellular localisation of AlPcS2 with each of the microbes studied. These novel data are presented alongside a brief summary of existing AlPcS2 fluorescence lifetime data measured both in solution and in model biological systems.

Characterization of crude oils using fluorescence lifetime data

The average fluorescence lifetimes of nine North Sea crude oils with API gravities of between 20 and 51 were measured using a modular, filter based, instrument developed in-house. Two pulsed light emitting diode (LED) excitation sources (460 and 510 nm) were used to excite fluorescence, the lifetime of which was measured at a range of emission wavelengths. Fluorescence lifetimes were found to vary from 1.8 to 8.2 ns with confidence intervals of ±0.11 ns. The average lifetimes at all emission wavelengths were linearly correlated with API gravity and with aromatic concentration with the best results being obtained with the 460 nm excitation source. Predictive models with an accuracy of ±7.6 API degrees were generated using partial least-squares methods from average fluorescence lifetimes measured at an emission wavelength of 500 nm using 460 nm excitation. A better correlation was found between the aromatic concentration of the oils and the ratio of the average fluorescence lifetimes at measured at 550 and 650 nm using 460 nm excitation. This led to a quantitative model with an accuracy of ±5.4% for aromatic concentration.

Frequency-domain fluorescence microscopy with the LED as a light source.

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz--250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10--15 microm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C(12),with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.