Fluorescence Intensity Decay Research Articles

Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: Prevention by thiol group protecting agents

Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca 2+-induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20 nmol/mg protein) induced Ca 2+-dependent mitochondrial swelling, depolarization of membrane potential (Δ Ψ), Ca 2+ release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40 nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the Δ Ψ, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H 2O 2 generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl- l-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca 2+-induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to H +; (3) does not significantly affect H 2O 2 generation by mitochondria; (4) its mitochondrial damaging effects are protected by thiol group protecting agents. Based on these conclusions, it is possible to hypothesise that small changes on the redox-status of thiol groups, affecting membrane permeability to cations (Ca 2+ and H +) underlie CisPt-induced liver mitochondrial damage, putatively responsible for its hepatotoxicity. Therefore, we propose that CisPt-induced mitochondrial damage and consequent hepatotoxicity could be prevented by using thiol group protecting agents as therapeutic adjuvants.

Determination of the Förster Distance in Polymer Films by Fluorescence Decay for Donor Dyes with a Nonexponential Decay Profile

Fluorescence resonance energy transfer (FRET) experiments were carried out on three pairs of donor-acceptor dyes in polymer films in which the donor dyes had absorption maxima in the range of 350-450 nm. Two of the donors, a coumarin dye and a naphthalimide dye covalently bound to polystyrene, gave nonexponential decays in the absence of acceptors. The decay profiles could be fitted to a stretched exponential form with a beta value on the order of 0.9. We developed equations for analyzing donor fluorescence intensity decay profiles for donor-acceptor mixtures in rigid matrices for the case of donors showing relatively small deviations from exponentiality. To test these equations, we calculate values of the Förster radius (R0(FR)) from the decay profile data and compare these values to the Förster radius R0(SO) determined by the traditional spectral overlap method. Agreement between these values validates the methodology developed here for the use of such donor dyes in FRET studies of more complex polymer systems.

CREATINE KINASE INHIBITOR IODOACETAMIDE ANTAGONIZES CALCIUM‐STIMULATED INOTROPY IN CARDIOMYOCYTES

1. Inhibition of creatine kinase is known to suppress cardiac contractile reserve in intact hearts, although the underlying mechanism has not been elucidated. 2. The present study was designed to examine whether cardiac depression induced by creatine kinase inhibition was due to action at the level of the essential contractile element, namely cardiomyocytes. Adult rat cardiomyocytes were perfused with the creatine kinase inhibitor iodoacetamide (90 micromol/L) for 90 min. Mechanical and intracellular Ca(2+) properties were evaluated using edge-detection and fluorescence microscopy, respectively. Myocytes were superfused with normal (1.3 mmol/L) or high (3.3 mmol/L) extracellular Ca(2+) contractile buffer. Mechanical function was examined, including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time to 90% PS (TPS(90)), time to 90% relengthening (TR(90)) and integration of shortening/relengthening (normalized to PS). Intracellular Ca(2+) transients were evaluated using the following indices: resting and rise of fura-2 fluorescence intensity (Delta FFI) and intracellular Ca(2+) decay time constant. 3. The results indicate that elevated extracellular Ca(2+) stimulated cardiomyocyte positive inotrope, manifested as increased PS, +/-dL/dt, area of shortening, resting FFI and Delta FFI associated with a shortened TR(90) and intracellular Ca(2+) decay time constant. High extracellular Ca(2+) did not affect TPS(90) and area of relengthening. Iodoacetamide ablated high Ca(2+)-induced increases in PS, +/-dL/dt, area of shortening, resting FFI, Delta FFI and shortened TR(90) and intracellular Ca(2+) decay time constant. Iodoacetamide itself significantly enhanced the area of relengthening and TR(90) without affecting other indices. 4. Collectively, these data demonstrate that inhibition of creatine kinase blunts high extracellular Ca(2+)-induced increases in cardiomyocyte contractile response (i.e. cardiac contractile reserve).

Effect of quinone on the fluorescence decay dynamics of endogenous flavin bound to bacterial luciferase

The interaction of quinone with luciferase from Photobacterium leiognathi was studied based on the fluorescence decay measurements of the endogenous flavin bound to the enzyme. Homologous 1,4-quinones, 1,4-benzoquinone, methyl-1,4-benzoquinone, 2-methyl-5-isopropyl-1,4-benzoquine and 1,4-naphthoquinone, were investigated. In the absence of quinone, the fluorescence intensity and anisotropy decays of the endogenous flavin exhibited two intensity decay lifetimes (~ 1 and 5 ns) and two anisotropy decay lifetimes (~ 0.2 and 20 ns), suggesting a heterogeneous quenching and a rotational mobility microenvironment of the active site of the luciferase, respectively. In the presence of quinone, the intensity decay heterogeneity was largely maintained, whereas the fraction of the short anisotropy decay component and the averaged rotational rate of FMN increased with the increasing hydrophobicity of the quinone. We hypothesize that the hydrophobicity of the quinone plays a role in the non-specific inhibition mechanism of xenobiotic molecules in the bacterial bioluminescence system via altering the rotational mobility of the endogenous flavin in the luciferase.

Use of liposomes as membrane models to evaluate the contribution of drug–membrane interactions to antioxidant properties of etodolac

This work stresses the need to combine antioxidant assays and drug–membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO•) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO•) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties – fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.

Synthesis of a Fluorescent Xanthenic Derivative Useful for Labeling Amine Residues

The 2,5-dioxopyrrolidin-1-yl-4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)-3-methylbenzoate has been synthesized as an amine-reactive derivative able to yield stable covalently labeled biopolymers. The new derivative has been used to label polyribocytidilic acid (5'), poly(C), amine residues. TG-II-poly(C) exhibits monoexponential decay at the physiological pH range. In addition, both steady-state fluorescence intensity and fluorescence decay are also sensitive to solution pH. The large decrease in steady-state fluorescence upon hybridization allows it to be used as a nucleic acid probe in a homogeneous assay format. In summary, we report an efficient synthesis to obtain labeled RNA from commercially available materials in excellent yields.

Nonradiative long range energy transfer in donor–acceptor systems with excluded volume

Long range multistep energy transport has been studied for donor–acceptor systems with excluded volume for donor or acceptor. It has been found that the fluorescence intensity decay of primarily excited donors is slower compared to that of randomly distributed fluorophores with any possible spatial location. This effect is especially strong if acceptors are entrapped in a certain volume inaccessible to donors. The analysis based on Monte–Carlo simulations concerns also other fluorescence characteristics.

Investigation on the photo-induced de-oxygenation process of myoglobin in aqueous solution by use of fluorescence spectroscopy

A photo-induced de-oxygenation process of myoglobin (Mb) in aqueous solution was investigated by use of fluorescence spectroscopy. The spectra are characterized by the fluorescence intensity declining gradually after each scan, and the decay of fluorescence intensity being significant in each scan, which is assigned to the release of oxygen from the opening of the heme-pockets induced by illumination. More illumination will cause more release of oxygen; if the temperature of an Mb solution is increased when it is illuminated, the rate of de-oxygenation will be higher. It was found that ligand-oxygen in Fe-porphyrin could be removed from Mb by nitrogen. This indicates that the interaction between oxy-Mb and other different gases can be tested by the method of fluorescence spectroscopy. In addition, fluorescence spectroscopy can be employed to probe the energy transfer between Fe-porphyrin and tryptophan or tyrosine in Mb molecules.

Noninvasive probing of aqueous Triton X-100 with steady-state and frequency-domain fluorometry

Most fluorescence spectroscopic methods that investigate the properties of surfactant-based systems are invasive in nature as they rely on information afforded by an externally-introduced probe. External probes may adversely affect the colloidal property of interest. Information from chromophoric surfactants as endogenous probes of micellization process is presented. Fluorescence from phenyl moiety of a common nonionic surfactant Triton X-100 in water is demonstrated to be rich in information on surfactant aggregation. While Triton X-100 concentration-dependent fluorescence emission and excitation can be effectively used to monitor the presence of micelles, frequency-domain excited-state fluorescence intensity decay data affords key dynamic parameters characterizing Triton X-100 micelles.

Complexity of Lipid Domains and Rafts in Giant Unilamellar Vesicles Revealed by Combining Imaging and Microscopic and Macroscopic Time-Resolved Fluorescence

The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (lo)/liquid-disordered (ld) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of lo raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the lo phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.

Fluorescence Lifetime Imaging System for in Vivo Studies

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.

Use of liposomes to evaluate the role of membrane interactions on antioxidant activity

In this study the possibility of using liposomes as membrane mimetic systems was evaluated to estimate the antioxidant properties of oxicams and establish a relationship between the interactions of the drugs with the membrane and their consequent antioxidant activity. Different experiments were performed covering the study of the protective effect of oxicams in lipid peroxidation induced by the peroxyl radical (ROO ) derived from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and using two fluorescence probes with distinct lipophilic properties. Lipid peroxidation using the hydrophilic probe fluorescein was evaluated in lipid and aqueous media. Lipid systems labelled with the fluorescent probe diphenylhexatriene propionic acid (DPH-PA) were used to assess the effects of the drugs on membrane peroxidation simultaneously by fluorescence intensity decay and changes in membrane fluidity by steady-state anisotropy measurements. The use of different probes and liposomes as membrane mimetic systems allowed to conclude that membrane lipoperoxidation is related not only to the scavenging characteristics of the antioxidants but also to their ability to interact with the lipid bilayers.

On the Behavior of Indole-Containing Species Sequestered within Reverse Micelles at Sub-Zero Temperatures

We report on the effects of temperature (+30 to -100 degrees C) on the fluorescence from N-acetyl tryptophanamide (NATA) and human serum albumin (HSA) sequestered within Aerosol-OT (AOT) reversed micelles. NATA reports simultaneously from the polar and non-polar side of the reverse micelle interface. As the sample temperature decreases, the relative fraction of NATA molecules associated with the polar side increases. This redistribution process is characterized by DeltaH = -14.8 +/- 0.6 kJ/mol and DeltaS = -54 +/- 2 J/(K mol). The activation energy for thermal quenching (E(a,TQ)) associated with the polar side NATA molecules is 6.7 kJ/mol before the micelles have shed water and 1.0 kJ/mol after water shedding (below approximately -20 degrees C). The time-resolved fluorescence intensity decay for tryptophan-214 in HSA is triple exponential. We suggest that these lifetimes arise from three indole residue conformations in equilibrium. Cooling the sample causes a freezing-in of the least quenched conformer; the other conformers are frozen out. The E(a,TQ) value for the shortest lifetime component is 6 kJ/mol. The E(a,TQ) for the long and intermediate lifetime components are equivalent (approximately 1.5 kJ/mol).

Two‐photon lifetime imaging of fluorescent probes in intact blood vessels: A window to sub‐cellular structural information and binding status

Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.

Flavin mononucleotide fluorescence intensity decay in concentrated aqueous solutions

Time-resolved fluorescence and steady-state absorption measurements of FMN aqueous solutions were carried out over very broad dye concentration range. Efficient formation of FMN dimers with simultaneous absence of higher order aggregates even at highest concentrations was found from absorption spectra measurements. It was found that fluorescence intensity decays are strongly accelerated in the presence of dimers due to excitation energy trapping and become nonexponential. Mean localization time of excitation energy and mean number of its jumps between FMN molecules were calculated as a function of concentration.

Early Aggregation in Prion Peptide Nanostructures Investigated by Nonlinear and Ultrafast Time-Resolved Fluorescence Spectroscopy

We report the characterization of early aggregates in the self-assembly of prion peptides using nonlinear and ultrafast time-resolved fluorescence spectroscopy. The dye-labeled peptide and dye/peptide guest-host systems were used to demonstrate the feasibility of the new approach. By measuring the two-photon absorption cross-section, small aggregates of the dye labeled peptide were characterized. Ultrafast time-resolved fluorescence anisotropy spectroscopy reveals the packing state (microenvironment) of the probes to be tightly associated with aggregates and associated with aggregation progression of the peptides. Fluorescence intensity decay shows a correlation with growth of aggregates having a high level of structured beta-sheet content. A new binding ligand Cascade Yellow shows promise for beta-sheet recognition of prion peptide nanostructures. These findings may have implications for in vivo studies of neurotoxic aggregates targeting with fluorescence markers. Also, these results may provide insight into molecular design of peptide-based nanomaterials.

Mechanical Properties of the Cell Nucleus and the Effect of Emerin Deficiency

Nuclear structure and mechanics are gaining recognition as important factors that affect gene expression, development, and differentiation in normal function and disease, yet the physical mechanisms that govern nuclear mechanical stability remain unclear. Here we examined the physical properties of the cell nucleus by imaging fluorescently labeled components of the inner nucleus (chromatin and nucleoli) and the nuclear envelope (lamins and membranes) in nuclei deformed by micropipette aspiration (confocal imaged microdeformation). We investigated nuclei, both isolated and in intact, living cells, and found that nuclear volume significantly decreased by 60–70% during aspiration. While nuclear membranes exhibited blebbing and fluid characteristics during aspiration, the nuclear lamina exhibited behavior of a solid-elastic shell. Under large deformations of GFP-lamin A-labeled nuclei, we observed a decay of fluorescence intensity into the tip of the deformed tongue that we interpreted in terms of nonlinear, two-dimensional elasticity theory. Here we applied this method to study nuclear envelope stability in disease and found that mouse embryo fibroblasts lacking the inner nuclear membrane protein, emerin, had a significantly decreased ratio of the area expansion to shear moduli ( K/ μ) compared to wild-type cells (2.1 ± 0.2 versus 5.1 ± 1.3). These data suggest that altered nuclear envelope elasticity caused by loss of emerin could contribute to increased nuclear fragility in Emery-Dreifuss muscular dystrophy patients with mutations in the emerin gene. Based on our experimental results and theoretical considerations, we present a model describing how the nucleus is stabilized in the pipette. Such a model is essential for interpreting the results of any micropipette study of the nucleus and porous materials in general.

Fluorescence resonance energy transfer by quencher adsorption into hydrogels containing fluorophores

AbstractWe synthesized anionic hydrogels containing fluorophores and investigated the adsorption of a cationic quencher having an amino group into hydrogels by fluorescence resonance energy transfer (FRET). FRET from the fluorophore to the quencher in hydrogels was examined by fluorescence intensity and fluorescence decay using a fluorescence spectrophotometer and femtosecond laser spectroscopy. The fluorescence intensity of the fluorophore‐containing hydrogels decreased rapidly with increasing amounts of adsorbed cationic quencher. The fluorescence emission of the fluorophore in the quencher‐adsorbed hydrogels containing fluorophores decayed more rapidly than that of the original hydrogels. The aforementioned result indicates that the fluorescence of the fluorophore‐containing hydrogels is quenched due to FRET from the fluorophore to the quencher as the cationic quenchers can approach the fluorophores in hydrogels by electrostatic interactions. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3245–3252, 2006

Determination of energetics and kinetics from single-particle intermittency and ensemble-averaged fluorescence intensity decay of quantum dots

Quantification of energetics and kinetics for the band-edge exciton states of quantum dots and the long-lived dark state is important for better understanding of the underlying mechanism for single-particle intermittency and ensemble fluorescence intensity decay. Based on a multistate diffusion-reaction model by extending our previous studies, we analyze experimental data from ensemble measurements and fluorescence intermittency of single quantum dots and determine important molecular-based quantities such as Stokes shift, free energy gap, activation energy, reorganization energy, and other kinetic parameters.

A new approach to interpretation of heterogeneity of fluorescence decay: Effect of induced tautomeric shift and enzyme → ligand fluorescence resonance energy transfer

Fluorescence decays in protein–ligand complexes are described by a new efficient model of continuous distribution of fluorescence lifetimes, and compared with multi-exponential models. Resulted analytical power-like decay function provides good fits to highly complex fluorescence kinetics. Moreover, this is a manifestation of so-called Tsallis q-exponential function, which is suitable for description of the systems with long-range interactions, memory effect, as well as with fluctuations of the characteristic lifetime of fluorescence. The proposed decay function was used to study effect of the interaction of E. coli purine nucleoside phosphorylase (PNP-I, the product of the deoD gene) with its specific inhibitor, viz. formycin A (FA), on fluorescence decays of ligand and enzyme tyrosine residues, in the presence of orthophosphate (P i, a natural co-substrate). The power-like function provides new information about enzyme–ligand complex formation based on the excited state mean lifetime, heterogeneity parameter ( q) and a number ( N) of decay channels obtained from the variance of gamma distribution of fluorescence decay rates. With FA, which exists as a 85:15 mixture of the N(1)–H and N(2)–H tautomeric forms in aqueous solution, fluorescence intensity decay ( λ exc / λ em 270/335 nm) is described by q ∼ 1 and N ∼ 200. Consequently power-like decay function converges to the single-exponential form, and lifetime distribution to the Dirac delta function. In contrast, selective excitation of the N(2)–H tautomer at higher wavelength led to a highly heterogenic fluorescence decay characterized by q > 1 and 10-fold lower number of decay channels. Heterogeneity of fluorescence decays of both PNP-I and FA is enhanced by PNP–FA–P i complex formation, reflecting a shift of the tautomeric equilibrium of FA in favor of the N(2)–H species, and fluorescence resonance energy transfer (FRET) from protein tyrosine residue (Tyr160) to the bound N(2)–H tautomer. Moreover, proposed model is simple, and objectively describes heterogeneous nature of studied systems.