Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Characterization of a confocal microscope for time-resolved photon counting fluorescence

A confocal microscope setup is developed for time-resolved fluorescence measurements. It is added to a traditional cuvette time-resolved setup, with a pumped Ti-Sa light source. The temporal resolution of 37 ps (FWHM) is not degraded, in comparison with the cuvette setup also described. These setups allow both decay lifetime and anisotropy relaxation time determination. Fluorescence correlation spectroscopy (FCS) is used to determine the observation point size. When associated with the calcium probe calcium green, calcium concentration in single cells can be determined in 10 ms by simultaneous acquisition of early and late fluorescence photons.     

Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation

We examined the steady-state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for OPE- and TPE. The anisotropy decay measurements of Hoechst 33342 in ethanol revealed the same correlation times for TPE as observed for OPE. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for TPE than for OPE. The anisotropy spectra of Hoechst 33342 were examined in glycerol at ?20°C, revealing limiting values close to the theoretical limits for OPE (0.4) and TPE (0.57). The steady-state anisotropy for OPE decreases in the shorter-wavelength region (R6G dye laser, 280–315 nm), but the two-photon anisotropy for 560 to 630-nm excitation remains as high as in the long-wavelength region (690–770 nm). This result suggests that one-photon absorption is due to two electronic, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm. Our demonstration of these favorable two-photon properties for Hoechst 33342, and the high photostability of the dye reported by other laboratories, suggests that this dye will be valuable for time-resolved studies of DNA with TPE and for two-photon fluorescence microscopy.     

Spectral imaging of time-resolved anisotropy: theory and experiment

Time-resolved fluorescence anisotropy on the nanosecond time scale is useful for the study of the rapid rotation of macromolecules. A system combining the capabilities of fluorescence spectral imaging with time-resolved fluorescence anisotropy and enabling the wide-field measurement of the spectroscopic parameters of fluorophores is discussed. The phasor approach is used to quantitatively analyze the time-resolved fluorescence anisotropy by transforming the polarized parallel and perpendicular components to the phasor space in the frequency domain, respectively, and a unique way to calculate the fluorescence rotational correlation time is put forward. Experimental results prove that the phasor approach is a proper model for the time-resolved fluorescence anisotropy.     

Energy transfer between the flavin chromophores of electron-transferring flavoprotein fromMegasphaera elsdenii as inferred from time-resolved red-edge and blue-edge fluorescence spectroscopy

Both a mode-locked argon-ion laser and synchrotron radiation were used as excitation sources to obtain time-resolved polarized fluorescence of the two FAD cofactors in electron transferring flavoprotein fromMegasphaera elsdenii. Red-edge excited and blue-edge detected fluorescence anisotropy decay curves did not contain a fast relaxation process which was observed upon mainband excitation and detection. This relaxation was assigned to homo-energy transfer between the two FAD cofactors. Failure of energy transfer as observed with edge spectroscopy on this protein excludes restricted reorientational motion of the flavins as a possible mechanism of depolarization. From the global analysis of the fluorescence anisotropy decay surface obtained at multiple excitation and detection wavelengths, the distance between and the relative orientation of the flavins could be estimated. The methodology described has general applicability in other multichromophoric biopolymers and has the potential to acquire accurate geometrical parameters in these systems.     

Fluorescence probes of viscosity: A comparative study of the fluorescence anisotropy decay of perylene and 3,9-dibromoperylene in glycerol

The authors compare the results of fluorescence anisotropy decay measurements for glycerol solutions of perylene with those of 3,9-dibromoperylene (DBP). For both molecules a good linear dependence is observed between the glycerol viscosity (varied by temperature) and the longer rotational correlation time obtained as a result of a global (using data obtained at 256- and 430-nm excitation wavelengths) biexponential analysis of the fluorescence anisotropy decay, at least in the range of 7–60 P for perylene and 4–60 P for DBP. This significantly extends the reported range of 0.5 to 150 cP investigated by Williams and Ben-Amotz [1] with the probe BTBP.     

Domain Formation in DODAB–Cholesterol Mixed Systems Monitored via Nile Red Anisotropy

The effect of the cholesterol (ch) on liposomes composed of the cationic lipid dioctadecyldimethylammonium bromide (DODAB) was assessed by studying both the steady-state and time-resolved fluorescence anisotropy of the dye Nile Red. The information obtained combined with analysis of the steady-state emission and fluorescence lifetime of Nile Red (NR) for different cholesterol concentrations (5–50%) elucidated the presence of “condensed complexes” and cholesterol-rich domains in these mixed systems. The steady-state fluorescence spectra were decomposed into the sum of two lognormal emissions, emanating from two different states, and the effect of temperature on the anisotropy decay of Nile Red for different cholesterol concentrations was observed. At room temperature, the time-resolved anisotropy decays are indicative of NR being relatively immobile (manifest by a high r _∞ value). At higher temperature, rotational times ca. 1 ns were obtained throughout and a trend in increasing hindrance was seen with increase of Ch content.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

1 and 2-Photon Fluorescence Anisotropy Decay to Probe the Kinetic and Structural Evolution of Sol-Gel Glasses: A Summary

We review recent 1- and 2-photon fluorescence studies of the formation dynamics and structure of sol-gel glasses, from nanometre-sized particles to clusters, prepared from both aqueous silicates and tetramethylorthosilicate (TMOS), over a broad pH range. Through the careful choice of a fluorescent probe, anisotropy decay has been shown to provide both silica particle size and viscosity information and offers advantages over traditional techniques for silica particle sizing based on small-angle neutron, Xray, or light scattering. Subsequently, we are now able to observe the self-assembly mechanisms (or recently termed kinetic life history) of silica, produced under both acidic and alkaline conditions from sodium silicate solution (water glass) in the case of hydrogels and from alkoxides in the case of alcogels. The controlled preparation of hydrogels, often deemed a blackart, is also discussed in some detail, as are the potential applications and benefits of fluorescence anisotropy decay to industrial sol-gel systems. The insight into the sol-gel process provided by these new interpretations of fluorescence decay data, promises to have implications for both our fundamental understanding and the production of sol-gel systems in general.     

Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb

The steady-state and time-resolved fluorescece spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

The interaction between a free-base, anionic water-soluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a bi-exponential decay is obtained where a short rotational correlation time (phi (int) = 1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin J-aggregates is induced, which can be detected by time-resolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.     

Continuous Fluorescence Depletion Anisotropy Measurement of Protein Rotation

Protein rotation in viscous environments can be measured by fluorescence depletion anisotropy (FDA) which combines long lifetimes of chromophore triplet states with the sensitivity of fluorescence excitation and detection. FDA achieves sensitivity well beyond that attainable by the more common technique of time-resolved phosphorescence anisotropy (TPA). We have now combined benefits of both time-domain and frequency-domain FDA into a single continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repeating waveform. Fluorescence signals excited from triplet-forming fluorescent probes are digitized over recurring waveform periods by a high-speed signal averager. CFDA experiments typically involve substantial ground state depletion. Thus signals, unlike those of TPA, are not linear in the exciting light intensity and simple data analysis based on such linearity is not appropriate. An exact solution of the coupled diffusion and triplet production/decay equation describing CFDA within individual data points has been combined with simulated annealing optimization to extract triplet and anisotropy decay kinetics from experimental data. Related calculations compare possible excitation waveforms with respect to rotational information provided per fluorescence photon. We present CFDA results for the model system of eosin conjugates of carbonic anhydrase, BSA and immunoglobulin G in 90% glycerol at various temperatures and initial cellular results on eosin-IgE bound to 2H3 cell Type I Fcε receptors. We explore how CFDA reflects rotational parameters of heterogeneous systems and discuss challenges of extending this method to single cell microscopic measurements.     

Paramagnetic relaxation of radical cations in alkane solutions as measured by time-resolved magnetic field effects

Spin-lattice relaxation times of radical cations were measured by the decay of the time-resolved magnetic field effect in the recombination fluorescence of radical ion pairs in liquid hydrocarbons at high concentrations of solvent holes acceptor. The dependences of spin-lattice relaxation time on the magnetic field strength and solvent viscosity were studied. The results could not be explained in terms of the model of ion-molecular charge transfer involving monomeric or dimeric radical cations. The paramagnetic relaxation observed in a weak magnetic field is attributed to internal reorganizations of aggregates that originate from radical cations at high acceptor concentration.     

Probing the Sol-Gel Transition in SiO2 Hydrogels—A New Application of Near-Infrared Fluorescence

Picosecond time-resolved fluorescence spectroscopy has enabled us to use a near-infrared fluorescent dye to probe the sol-gel transition in SiO₂ hydrogels, polymerized from sulfuric acid and sodium silicate solution, for the first time. We compare the microviscosity surrounding the probe during the sol-to-gel transition as predicted by two alternative models which both describe the decay of fluorescence anisotropy well. The results for one rotational time and a residual anisotropy imply that macrogelation of the sol leads to relatively small changes in the mobility of the fluorophore caused by small changes in microviscosity, but after much longer times, e.g., 1500 min, the mobility of the fluorophore decreases, reflecting a rapid increase in microviscosity of over several orders in magnitude. In sharp contrast, analysis of the anisotropy in terms of two rotational times predicts little change in microviscosity over the whole polymerization process.     

Confinement of 4,4-Diaminodiphenyl Sulfone by γ –CD in Micellar Environment: A Spectroscopic Investigation

This paper reports the double confinement of 4,4-diaminodiphenyl sulfone (Dapsone) inside γ–cyclodextrin (CD) in presence of surfactants (cationic, anionic and nonionic) using steady-state and time-resolved fluorescence spectroscopy. Interpretation of fluorescence spectra, fluorescence anisotropy and time resolved fluorescence decay of the γ-CD?Dapsone?micellar system hints at lesser microviscosity and the partial release of the probe molecule from the supramolecular host–guest complex in ionic micelles, of which greater in cationic micelles, but due to greater restriction and rigidity in presence of non-ionic micelle makes the probe more rigidly inside CD. Changes in computed rotational decay also corroborate the above findings.

Kinetics study of ultrafast electron transfer from sensitized dyes to silver halide microcrystals

Spectral sensitization micromechanism of cyanine dyes J-aggregate adsorbed on the tabular and cubic AgBr microcrystals with different dye concentrations is studied by using picosecond time-resolved fluorescence spectroscopy, and the dependences of electron transfer and spectral efficiency sensitization on different conditions are analysed in detail. With the steady spectroscopy, the wavelengths of absorption and fluorescence of J-aggregate adsorbed on AgBr microcrystals are found to shift to red relative to dye monomer. The spectrum of fluorescence has a red shift relative to the absorption peak. With the time-resolved fluorescence spectroscopy, the fluorescence decay curves of cyanine dyes J-aggregate adsorbed on the tabular and cubic AgBr grains are found to be fitted well by a double-exponential decay function. The fitting curves consist of a fast and a slow component. Because of the large amplitude of the fast component, this fast decay should be attributable mainly to the electron transfer from J-aggregate of dye to a conduction band of AgBr.     

Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

Comparison Between Fluorescence Correlation Spectroscopy and Time-Resolved Fluorescence Anisotropy as Illustrated with a Fluorescent Dextran Conjugate

The motional properties of rhodamine green alone and conjugated to 10-kDa dextran have been studied by fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence anisotropy (TRFA). With FCS the translational diffusion times of the fluorescent particles can be determined, which are directly proportional to the shear viscosity as shown in aqueous solutions of different sucrose concentrations. With TRFA the rotational correlation times of the fluorescent particles can be determined. TRFA experiments in the case of fluorescent dextran reveal a distinct restricted internal motion of the fluorescent probe independent of the slower overall rotation of the polysaccharide. The fast depolarization is most likely due to internal motion and not to energy transfer between different rhodamine green molecules in the same dextran, since a higher viscosity of the solvent increases the correlation time for internal motion proportionally. FCS and TRFA yield complementary information in the sense that the correlation time for overall dextran rotation can be accurately determined from the translational diffusion coefficient.