Time-resolved Intensity Decay Research Articles

Spectroscopic Studies of a Phosphonium Ionic Liquid in Supercritical CO2

Fluorescence spectroscopy was used to study a solution comprised of coumarin 153 (C153)+ trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14]+ [Tf2N]−)+ supercritical CO2 (scCO2). We compare the spectroscopy of C153 in neat scCO2 to that of C153/scCO2 with the addition of ionic liquid (IL). Excitation and emission peak frequencies of C153 in scCO2 and in IL/scCO2 diverged at reduced densities (ρr = ρ/ρc) below the CO2 critical density. At low fluid density, spectral changes in the IL/scCO2 solutions showed evidence that C153 experiences a very different microenvironment—one that is unlike neat scCO2. The data show that the presence of IL clearly influences the C153 excitation and emission profiles. Excitation was broadened and red shifted by >2000 cm−1 and the presence of an additional low-energy emission component that was red shifted by ~3000 cm−1 was clearly visible and not observed in neat scCO2. The solution heterogeneity was controlled by changing the scCO2 density and at high fluid density, both the excitation and emission spectra were more similar to those in neat scCO2. Steady-state anisotropy also showed that at low fluid density, the C153 emission was significantly polarized. Aggregation of C153 has been reported in the literature and this led us to hypothesize the possibility that C153 dimer (aggregation) formation may be occurring in scCO2. Another possible explanation is that dye–IL aggregates may dissolve into the scCO2 phase due to C153 acting as a “co-solvent” for the IL. Time-resolved intensity decay measurements yielded only slightly non-exponential decays with accompanying time constants of ~3–4 ns that were significantly shorter than the 5–6 ns time constants in neat scCO2, which are suggestive of C153–IL interactions. However, these data did not conclusively support dimer formation. Pre-exponential factors of the time constants showed that almost all of the emission was due to monomeric C153.

Riboflavin as a Gras Luminescent Probe of Food and Pharmaceutical Quality

Riboflavin fluorescence has been characterized profusely, however little attention has been devoted to riboflavin phosphorescence. We have characterized steady state and time resolved phosphorescence of riboflavin from 77K to 333K in solvent mixtures (water:alcohol, water:glycerol) and amorphous solids (glucose, glucose oligomers, sucrose, dextran) to expand our knowledge on riboflavin photophysical properties and assess its potential use in quality monitoring.When excited at 440nm, the emission spectra of riboflavin exhibited maxima at 515nm (delayed fluorescence) and 620nm (phosphorescence). Deconvolution of the spectra to two individual bands characterized by asymmetric lognormal functions (I(ν)=I0∗exp{-ln(2)(ln[1+2b(ν-νp)/Δ2]/b)} ) facilitated the extraction of parameters and the analysis of their temperature dependence. Time resolved intensity decays were fitted using stretch, multi-exponential and distribution (by MEM) models.Riboflavin exhibited a lifetime of 184ms at 77K in glycerol-water. The lifetime decreased slowly below the solvent mixture's Tg (170K) and abruptly above Tg; e.g., an increase of 20K above Tg reduced the lifetime from 100ms to 10ms. This reduction was associated with molecular motions in the matrix.Molecular mobility was also modulated by changing the composition of the matrix, by using components of different molecular sizes or by adding plasticizers. Riboflavin phosphorescence also exhibited good sensitivity towards molecular mobility changes driven by composition. This suggests its use as a GRAS optical probe for molecular mobility and its potential application to optimize matrix composition in food and pharmaceutical products to enhance stability of micro and bioactive components. Continuous monitoring of riboflavin phosphorescence during heating and cooling cycles revealed differences in the delayed luminescence emission spectra, likely due to a higher rate of irreversible photodegradation of riboflavin at high temperatures. If properly characterized, the thermal dependence of riboflavin photodegradation can potentially be operationalized in sensors for temperature abuse.

Distance-Dependent Metal-Enhanced Intrinsic Fluorescence of Proteins Using Polyelectrolyte Layer-by-Layer Assembly and Aluminum Nanoparticles.

Previously reported studies indicate that aluminum nanostructured substrates can potentially find widespread use in metal-enhanced fluorescence (MEF) applications particularly in the UV or near-UV spectral region toward label-free detection of biomolecules. MEF largely depends on several factors, such as chemical nature, size, shape of the nanostructure and its distance from the fluorophore. A detailed understanding of the MEF and its distance-dependence are important for its potential application in biomedical sensing. Our goal is to utilize intrinsic protein fluorescence for label-free binding assays. This is made possible by the use of metallic nanostructures which provide localized excitation and enhanced fluorescence of UV fluorophores and will also provide a way to separate the surface-bound proteins from the bulk samples. We evaluated varied probe distances from plasmonic nanostructures by the well-established layer-by-layer (LbL) technique. The investigated proteins were adsorbed on different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). Bovine serum albumin (BSA) was electrostatically attached to the positively charged PAH layer, and goat and rabbit IgG were attached to negatively charged PSS layer. We obtained a maximum of a ~ 9 fold increase in fluorescence intensity from BSA at a distance of ~9 nm from the Al nanostructured surface. Approximately 6- and 7- fold increases were observed from goat and rabbit IgG at a distance of ~8 nm, respectively. The minimum lifetimes were about 3-fold shorter than those on bare control quartz slides for all three proteins. The time-resolved intensity decays were analyzed with a lifetime distribution model to understand the distance effect on the metal-fluorophore interaction in detail. The present study indicates the distance dependence nature of metal-enhanced intrinsic fluorescence of proteins and potential of LbL assembly to control the metal-to-fluorophore distance in the UV wavelength region.

Fluorescence lifetime spectroscopy for guided therapy of brain tumors

This study evaluates the potential of time-resolved laser induced fluorescence spectroscopy (TR-LIFS) as intra-operative tool for the delineation of brain tumor from normal brain. Forty two patients undergoing glioma (WHO grade I-IV) surgery were enrolled in this study. A TR-LIFS prototype apparatus (gated detection, fast digitizer) was used to induce in-vivo fluorescence using a pulsed N2 laser (337 nm excitation, 0.7 ns pulse width) and to record the time-resolved spectrum (360 – 550 nm range, 10 nm interval). The sites of TR-LIFS measurement were validated by conventional histopathology (H&E staining). Parameters derived from the TR-LIFS data including intensity values and time-resolved intensity decay features (average fluorescence lifetime and Laguerre coefficients values) were used for tissue characterization and classification. 71 areas of tumor and normal brain were analyzed. Several parameters allowed for the differentiation of distinct tissue types. For example, normal cortex (N = 35) and normal white matter (N = 12) exhibit a longer-lasting fluorescence emission at 390 nm (τ390 = 2.12 ± 0.10 ns) when compared with 460 nm (τ460 = 1.16 ± 0.08 ns). High grade glioma (grades III and IV) samples (N = 17) demonstrate emission peaks at 460 nm, with large variation at 390 nm while low grade glioma (I and II) samples (N = 7) demonstrated a peak fluorescence emission at 460 nm. A linear discriminant algorithm allowed for the classification of low-grade gliomas with 100% sensitivity and 98% specificity. High-grade glioma demonstrated a high degree of heterogeneity thus reducing the discrimination accuracy of these tumors to 47% sensitivity and 94% specificity. Current findings demonstrate that TR-LIFS holds the potential to diagnose brain tumors intra-operatively and to provide a valuable tool for aiding the neurosurgeon–neuropathologist team in to rapidly distinguish between tumor and normal brain during surgery.

Fluorescence Lifetime and Depth Estimation of a Tumor Site for Functional Imaging Purposes

Cancerous cells have irregular environmental conditions, such as temperature and pH, which distinguish them from their surroundings. Fluorescence lifetime imaging using near-IR (NIR) fluorescent probes, whose lifetime value is sensitive to pH and temperature, enables the estimation of these values, and provides functional information about the tumor. The lifetime value, extracted from the time-resolved intensity decay curve, combines the photon time delays, caused by the photon time of flight, and the intrinsic lifetime in which we are interested. In this study, we present a model, based on the diffusion approximation of the radiation transport equation, for extracting both the depth of an NIR fluorescent probe, and its intrinsic lifetime value, from a fluorescence time decay curve. The model was validated for different inclusion depths, fluorescent lifetime values, and scattering coefficients using a time-resolved Monte Carlo simulation. Our reported results are the first step toward performing functional imaging using fluorescence lifetime in vivo measurements.

Optical oxygen sensors based on platinum porphyrin dyes encapsulated in ORMOSILS

A strategic approach to develop an “ideal” sensor platform is addressed. Novel quenchometric O 2 sensing materials with 100-fold adjustable sensitivity ( I N 2 / I O 2 from 3 to over 300), reverse and fast response (1 s), linear calibration, and long-term stability are developed. These materials are spin-casted composite ORMOSILs (organically modified silicates) xerogel films sequestered with luminophore Pt(II) octaethylporphine (PtOEP) and Pt(II) meso-tetra(N-methyl-4-pyridyl)porphyrin tetrachloride (PtTMP). The composite xerogels studied are pentafluorophenylpropyltrimethoxysilane (PFTMOS)/ n-octyltrimethoxysilane (C 8TMOS)/tetramethoxysilane (TMOS) and 3,3,3-trifluoropropyltrimethoxysilane (TFTMOS)/ n-propyltrimethoxysilane (C3TMOS). Luminescence spectroscopy (steady state and time resolved) have been used to investigate their analytical figures of merit for gaseous O 2 sensing and to determine the underlying reasons for the observed performance. Results show that, except for PtTMP-doped PFTMOS/C8TMOS/TMOS, all other xerogel composites show linear Stern–Volmer relationship and the single exponential time-resolved intensity decay, which indicate the homogeneous environment of the luminophore. For PtTMP-doped PFTMOS/C8TMOS/TMOS xerogels, the Stern–Volmer plots are non-linear and the time-resolved intensity decay profiles are best fitted by double exponential decay model, which are resulted from the heterogeneous environment of the luminophore. The sensitivity of the sensing materials can be tuned by adjusting the xerogel composition and luminophore. For PtOEP-doped PFTMOS/C8TMOS/TMOS, while keeping C8TMOS and TMOS at the same mol ratio, sensitivity ( I N 2 / I O 2 ) decreases from 333 to 246 with increased PFTMOS% from 0 to 30%, which is due to the decreased bimolecular quenching constant ( k q ). However, I N 2 / I O 2 = 3 was observed from PtTMP-doped PFTMOS/C8TMOS/TMOS. For PtTMP-doped TFTMOS/C3TMOS, sensitivity increases with increased TFTMOS% from 0 to 50% and decreases with further increased TFTMOS% from 50 to 100%, which is mainly due to corresponding increase and decrease of k q . An optimized composition with high sensitivity is observed at 10 PFTMOS/45C8TMOS/45 C8TMOS with I N 2 / I O 2 = 330 .

Vanillin Phosphorescence as a Probe of Molecular Mobility in Amorphous Sucrose

Changes in molecular mobility are important in defining the stability and quality of amorphous solid foods, pharmaceuticals, and other solid biomaterials. Predictions of stability must consider matrix mobility below and above T(g) (the glass transition temperature); measurement of molecular mobility in amorphous solids over time scales ranging from <10(-9) s to >10(8) s requires specialized methods. This research investigated how the steady-state and time-resolved emission and intensity of phosphorescence from vanillin (4-hydroxy-3-methoxy benzaldehyde), a common flavor compound, can be used to probe molecular mobility when dispersed within amorphous pure sucrose films. Phosphorescence emission spectra and time-resolved intensity decays, measured in sucrose as a function of temperature in the absence of oxygen, were strongly modulated by matrix molecular mobility. Temperature had a significant effect on vanillin phosphorescence peak frequency and bandwidth, intensity, and lifetime both in the glass and in the melt. Time-resolved phosphorescence intensity decays from vanillin were multiexponential both below and above the glass transition temperature, indicating that the pure (single component) amorphous matrix was dynamically heterogeneous on the molecular level. These data show that vanillin is a promising intrinsic probe of molecular mobility and dynamic heterogeneity in amorphous solid foods and perhaps pharmaceuticals.

Behavior of Pyrene End-Labeled Poly(dimethylsiloxane) Polymer Tails in Mixtures of 1-Butyl-3-methylimidazolium Bis(trifluoromethyl)sulfonylimide and Toluene

We report on the steady-state and time-resolved fluorescence of a linear poly(dimethylsiloxane) polymer that is end-labeled with pyrene (Py-PDMS-Py, Mn = 3100, Mw/Mn = 1.07) when it is dissolved at low concentration in an ionic liquid,1-butyl-3-methylimidazolium bis(trifluoromethyl)sulfonylimide ([C4mim][Tf2N]), as a function of temperature and added cosolvent (toluene). Toluene is a good solvent for PDMS. The Py-PDMS-Py behavior in [C4mim][Tf2N]/toluene is reminiscent, in part, of its behavior in liquid MeOH, a poor PDMS solvent. The Py residues are surrounded by a PDMS-rich microenvironment at all toluene loadings (0−50 vol %). No detectable excimer-like emission is seen in pure [C4mim][Tf2N] or [C4mim][Tf2N]/toluene mixtures until 50 vol % toluene. The Py-PDMS-Py ground state is heterogeneous, containing monomers and preformed dimers prior to photoexcitation. The observed excimer-like emission does not arise from a dynamic excimer. The Py-PDMS-Py time-resolved intensity decay data reveal the presence of at least four emitting species. In the [C4mim][Tf2N]/50% toluene mixture the species are (i) a Py monomer in a PDMS-rich microenvironment, (ii) a Py monomer in a microenvironment between pure toluene and pure [C4mim][Tf2N], (iii) a static ground-state dimer that has a geometry akin to a classical dynamically formed excimer, and (iv) a ground-state dimer that has the Py residues misaligned in comparison to (iii). Steady-state fluorescence anisotropy experiments are not consistent with the formation of rigid aggregates (i.e., (Py-PDMS-Py)n); results are consistent with Py residues and PDMS segments of individual Py-PDMS-Py molecules reorienting in a correlated manner.

Molecular Mobility in Amorphous Maltose and Maltitol from Phosphorescence of Erythrosin B

We have used phosphorescence from erythrosin B (tetraiodofluorescein) dispersed in amorphous thin films of maltose and maltitol at mole ratios of 0.8:10(4) dye:sugar to monitor the molecular mobility of these matrixes over the temperature range from -25 to over 110 degrees C. Analysis of the emission peak frequency and bandwidth (full width at half-maximum) and time-resolved intensity decay parameters provided information about thermally activated modes of matrix mobility that enhanced the rate of dipolar relaxation around the triplet state and the rate of intersystem crossing to the ground state (k(TS0)). Detectable dipolar relaxation began in the glassy state about 50 degrees C below T(g) in both maltose and maltitol; the relaxation rate, however, while 3-4 orders of magnitude slower than literature values for the beta relaxation determined from dielectric relaxation, had an activation energy only 2-fold smaller. Dipolar relaxation was further enhanced in the melt above T(g); the dipolar relaxation rates in the melt scaled nearly exactly with rates for the alpha relaxation determined from dielectric relaxation. Intensity decays were well fit using a stretched exponential decay function in which the lifetime (tau) and the stretching exponent (beta) were the physically significant parameters. In maltose, the magnitude of k(TS0) was essentially constant in the glass and increased dramatically at the T(g); in maltitol k(TS0) increased moderately at T(g) = -50 degrees C and more dramatically in the melt at T(g) = +20 degrees C. The value of k(TS0) in maltose:maltitol mixtures was significantly smaller than that seen in pure maltose and maltitol, suggesting that specific interactions decreased the mobility of the mixed sugar matrix; this phenomenon was comparable to the antiplasticization seen in mixtures of small molecule plasticizers with synthetic polymers and starch. The extent of inhomogeneous broadening and dynamic heterogeneity were essentially constant in the glass and increased dramatically in maltose and more gradually in maltitol at the glass transition.

Energy transduction optical sensor in skeletal myosin.

The skeletal myosin cross-bridge in dynamic association with actin is the unitary energy transducer in muscle, converting free energy from ATP hydrolysis into contractile force. Myosin's conserved ATP-sensitive tryptophan (AST) is an energy transduction optical sensor signaling transduction-related transient conformation change by modulating its fluorescence intensity amplitude and relaxation rate. Recently introduced techniques have provided the means of observing the time-resolved intensity decay from this single residue in the native protein to elucidate the mechanism of its ATP sensitivity. AST signal characteristics could be derived from local protein structure by a scenario involving interactions with excited-state tryptophan. This investigation suggests the very different possibility that hypochromism induced in the tryptophan absorption band, a ground-state effect, is a significant structural effector of optical transduction sensing. This possibility makes feasible the interpretation of the transient AST optical signal in terms of dynamical protein structure, thereby raising the empirical signal to the level of a structural determinant. Using the crystallographically based geometry from several myosin structures, the maximum calculated AST hypochromism is <10% to be compared with the value of approximately 30% observed here experimentally. Rationalizing the discrepancy invites further investigation of S1 dynamical structure local to the AST during transduction.

Time-Resolved Spectral Observations of Cadmium-Enriched Cadmium Sulfide Nanoparticles and the Effects of DNA Oligomer Binding

We measured the steady-state and time-resolved fluorescence spectral properties of cadmium-enriched nanoparticles (CdS–Cd2+). These particles displayed two emission maxima, at 460 and 580 nm. The emission spectra were independent of excitation wavelength. Surprisingly, the intensity decays were strongly dependent on the observation wavelength, with longer decay times being observed at longer wavelengths. The mean lifetime increased from 150 to 370 ns as the emission wavelength was increased from 460 to 650 nm. The wavelength-dependent lifetimes were used to construct the time-resolved emission spectra, which showed a growth of the long-wavelength emission at longer times, and decay-associated spectra, which showed the longer wavelength emission associated with the longer decay time. These nanoparticles displayed anisotropy values as high as 0.35, depending on the excitation and emission wavelengths. Such high anisotropies are unexpected for presumably spherical nanoparticles. The anisotropy decayed with two correlation times near 5 and 370 ns, with the larger value probably due to overall rotational diffusion of the nanoparticles. Addition of a 32-base pair oligomer selectively quenched the 460-nm emission, with less quenching being observed at longer wavelengths. The time-resolved intensity decays were minimally affected by the DNA, suggesting a static quenching mechanism. The wavelength-selected quenching shown by the nanoparticles may make them useful for DNA analysis.

Time-Resolved Fluorescence Investigation of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein: Influence of the Binding of Nucleic Acids

Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNA Phe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNA Phe. Indeed, the fluorescence of Trp 37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNA Phe. Despite the multiple binding sites in tRNA Phe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp 37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp 61 only stacked to a small extent with tRNA Phe. The behavior of this residue in the tRNA Phe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar.

Mapping the suramin-binding sites of human neutrophil elastase: investigation by fluorescence resonance energy transfer and molecular modeling.

Neutrophil elastase (NE), a mediator of inflammation, binds with high affinity numerous anionic molecules including suramin, a polysulfated naphthylurea, which inhibits it with a Ki of 0.2 microM and a 4:1 suramin:NE stoichiometry and thus constitutes a potential therapeutic agent. In an attempt to locate the suramin molecules on NE, we investigated the NE-suramin interaction using steady-state and time-resolved fluorescence spectroscopy. The time-resolved intensity decay of NE, a protein with three Trp residues, in positions 27, 141, and 237 (chymotrypsin numbering system) was best described by a three-exponential function with lifetimes ranging from 0.22 to 2.28 ns. Comparison of the accessibility of the three lifetime classes to the fluorescence quenchers acrylamide and iodide with the computed solvent accessibility of the three Trp residues in the crystal structure of NE indicates that the main, if not the sole, contribution to the 2.28 ns lifetime class is brought about by the fully buried Trp 141 residue. The addition of suramin to NE induces a sharp decrease in NE fluorescence and a corresponding increase in suramin fluorescence due to an efficient fluorescence resonance energy transfer (FRET) between the Trp residues of NE, acting as donors, and the naphthalene rings of suramin, behaving as acceptors. From the fate of the longest lifetime class in the presence of variable suramin concentrations, we deduce that two suramins are bound at less than 17 A from Trp 141, whereas the two others are located at least 29 A from Trp 141. Moreover, neither the binding of suramin to NE nor the FRET process was modified when NE was complexed with a peptide chloromethylketone inhibitor, suggesting that suramin does not directly interfere with the substrate binding site of NE. These data were used as constraints to model the NE-suramin complex.

Transient Effects in Fluorescence Quenching Measured by 2-GHz Frequency-Domain Fluorometry.

We used harmonic-content frequency-domain fluorometry to investigate the quenching of indole fluorescence by iodide and acrylamide in aqueous solution. The time-resolved intensity decays were recovered from the frequency response of the fluorescence emission, measured over a frequency range from 10 to 2000 MHz. In the absence of collisional quenching the decay of indole in water is predominately a single exponential. The intensity decays became increasingly nonexponential when quenched by either iodide or acrylamide. We attribute the complex decays to transient effects, as predicted originally by Smoluchowski. At quencher concentrations below 0.1 M the decays are of the form exp(-t/τ - 2bt 1/2), which is known to be an approximate model. At quencher concentrations above 0.1 M this decay law does not account for the data. The data are in better agreement with the radiation model, and the diffusion coefficients and interaction radii are more reasonable. However, in aqueous solution above 0.5 M acrylamide there appear to be deviations from the radiation model. The deviations are less apparent at high iodide concentrations. For comparison with future theoretical developments, the intensity decay laws are reported up to 0.5 M iodide or 0.7 M acrylamide. Evidently, the resolution and sensitivity of the frequency-domain method are adequate to recover the complex subnanosecond decays found at high concentrations of quenchers.