Fluorescenec Emission of SrS:Eu2+ Phosphor-Energy Level Splitting of Eu2+

A method of preparation of strontium sulphide phosphors doped with europium is given. Nitrogen laser excited fluorescence emission spectra of these phosphors in the visible region are recorded. A band with line structure in the region 350-430 nm and a new broad band at 460 nm are observed. The splitting pattern for the 6p levels of Eu 2+ are given.

Synthesis, structural and emission studies of a bis (carbamoyl methyl) sulfone complex of uranyl nitrate

A new tri-functional ligand (Bu2NCOCH2SO2CH2CONBu2)-Bu-i-Bu-i (L) was prepared and characterized. The coordination chemistry of this ligand with uranyl nitrate was studied with IR, (HNMR)-H-1, ES-MS, TG and elemental analysis methods. The structure of the compound [UO2(NO3)(2)L] was determined by single crystal X-ray diffraction techniques. In the structure the uranium(VI) ion is surrounded by eight oxygen atoms in a hexagonal bi-pyramidal geometry. Four oxygen atoms from two nitrate groups and two oxygen atoms from the ligand form a planar hexagon. The ligand acts as a bidentate chelate and bonds through both the carbamoyl groups to the uranyl nitrate. An ES-MS spectrum shows that the complex retains the bonding in solution. The compound displayed vibronically coupled fluorescence emission.

Subdiffraction fluorescence imaging of biomolecular structure and distributions with quantum dots

We introduce semiconductor quantum dot-based fluorescence imaging with approximately 2-fold increased optical resolution in three dimensions as a method that allows both studying cellular structures and spatial organization of biomolecules in membranes and subcellular organelles. Target biomolecules are labelled with quantum dots via immunocytochemistry. The resolution enhancement is achieved by three-photon absorption of quantum dots and subsequent fluorescence emission from a higher-order excitonic state. Different from conventional multiphoton microscopy, this approach can be realized on any confocal microscope without the need for pulsed excitation light. We demonstrate quantum dot triexciton imaging (QDTI) of the microtubule network of U373 cells, 3D imaging of TNF receptor 2 on the plasma membrane of HeLa cells, and multicolor 3D imaging of mitochondrial cytochrome c oxidase and actin in COS-7 cells.

Chlorophyll fluorescence of tropical tree species in a semi-deciduous forest gap

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Upconversion fluorescence spectroscopy of Er3+/Yb3+-doped heavy metal Bi2O3-Na2O-Nb2O5-GeO2 glass

Upconversion fluorescence emission of Er3+/Yb3+-doped Bi2O3-Na2O-Nb2O5-GeO2 heavy metal glass samples excited at 1.06 mu m is experimentally investigated. The results reveal the existence of intense emission bands centered around 520, 545, and 655 nm. The germano-niobate based host glass presents high transparency in the region of 400-2700 nm, the capability of incorporating high dopant concentrations, high melting temperature, and large resistance to atmospheric moisture. The observed intensity of the green fluorescence emission, suggested that the niobium based host glass material plays an important role in the efficiency of the upconversion process. Emission lines centered at 425, 483, 503, 608, and 628 nm were also observed. (C) 1997 American Institute of Physics.

Fluorescence probe study of the interaction between acrylic acid-co-ethyl methacrylate copolymers and sodium dodecylsulfate

The interaction between sodium dodecylsulfate (SDS) and acrylic acid (AA)-ethyl methacrylate (EMA) copolymers has been investigated using steady state fluorescence and conductimetric measurements to assess the effect of the polymer composition on the aggregation process. Micropolarity studies using the ratio between the emission intensities of the vibronic bands of pyrene (I-1/I-3) and the shift of the fluorescence emission of pyrene-3-carboxaldehyde show, that the interaction of SDS with AA-EMA copolymers occurs at surfactant concentrations smaller than that observed for the pure surfactant in water and depends on the copolymer composition. The increase of ethyl methacrylate in the copolymers lowers the critical aggregation concentration (CAC) due to the larger hydrophobic character of the polymer backbone. The formation of aggregates on the macromolecule is induced mainly, by hydrophobic interactions, but the process is also influenced by the ionic strength due to the counter-ions of the polyelectrolyte.

The Role of Wing Pigmentation, UV and Fluorescence as Signals in a Neotropical Damselfly

Pigmentation patterns, ultraviolet reflection and fluorescent emission are often involved in mate recognition and mate quality functions in many animal taxa. We investigated the role of wing ultra-violet reflection, fluorescence emission, and pigmentation on age and sexual signals in the damselfly Mnesarete pudica. In this species, wings are sexually dimorphic in colour and exhibit age dependency: males and females show a smoky black colouration when young, turning red in mature males while it turns brown in females. First, we investigated wing UV patterns through reflectance and emission spectra. Second, behavioural experiments were undertaken to show male and female responses to manipulated wing pigmentation and experimentally reduced UV (UV-). Reflectance spectra of the wings of juvenile and mature males and females were used to show the differences between controls and individuals with manipulated colouration used in the behavioural experiment. UV-reduced, females with wings painted red, and control males and females were tethered and presented to conspecific males and females, and their behavioral responses were recorded. The male red wing pigmentation and females with red wings elicited an aggressive response in territorial males and a sexual response in females. Both males and females showed neutral responses towards individuals with reduced UV. Wing signals of juvenile individuals also provoked neutral responses. These results suggest that UV, together with pigmentation, plays a role during mate recognition in males and females. Other than butterflies and spiders, it seems that fluorescence signals and UV reflectance can also be part of communication in odonates. © 2013 Springer Science+Business Media New York.

Rotational diffusion membrane and fluorescence anisotropy.

Structural properties of model membranes, such as lipid vesicles, may be investigated through the addition of fluorescent probes. After incorporation, the fluorescent molecules are excited with linearly polarized light and the fluorescence emission is depolarized due to translational as well as rotational diffusion during the lifetime of the excited state. The monitoring of emitted light is undertaken through the technique of time-resolved fluorescence: the intensity of the emitted light informs on fluorescence decay times, and the decay of the components of the emitted light yield rotational correlation times which inform on the fluidity of the medium. The fluorescent molecule DPH, of uniaxial symmetry, is rather hydrophobic and has collinear transition and emission moments. It has been used frequently as a probe for the monitoring of the fluidity of the lipid bilayer along the phase transition of the chains. The interpretation of experimental data requires models for localization of fluorescent molecules as well as for possible restrictions on their movement. In this study, we develop calculations for two models for uniaxial diffusion of fluorescent molecules, such as DPH, suggested in several articles in the literature. A zeroth order test model consists of a free randomly rotating dipole in a homogeneous solution, and serves as the basis for the study of the diffusion of models in anisotropic media. In the second model, we consider random rotations of emitting dipoles distributed within cones with their axes perpendicular to the vesicle spherical geometry. In the third model, the dipole rotates in the plane of the of bilayer spherical geometry, within a movement that might occur between the monolayers forming the bilayer. For each of the models analysed, two methods are used by us in order to analyse the rotational diffusion: (I) solution of the corresponding rotational diffusion equation for a single molecule, taking into account the boundary conditions imposed by the models, for the probability of the fluorescent molecule to be found with a given configuration at time t. Considering the distribution of molecules in the geometry proposed, we obtain the analytical expression for the fluorescence anisotropy, except for the cone geometry, for which the solution is obtained numerically; (II) numerical simulations of a restricted rotational random walk in the two geometries corresponding to the two models. The latter method may be very useful in the cases of low-symmetry geometries or of composed geometries.

Grating coupled surface plasmon enhanced fluorescence spectroscopy

Over the last three decades, sensors based on the phenomenon of surface plasmon resonance have proven particularly suitable for real time thin film characterization, gas detection, biomolecular interaction examination and to supplement electrochemical methods. Systems based on prism coupling have been combined with fluorescence detection under the name of surface plasmon fluorescence spectroscopy to increase sensitivity even further. Alternatively, metal gratings can be employed to match photons for plasmon resonance. The real time monitoring of binding reactions not yet been reported in the combination of fluorescence detection and grating coupling. Grating-based systems promise more competitive products, because of reduced operating costs, and offer benefits for device engineering. This thesis is comprised of a comprehensive study of the suitability of grating coupling for fluorescence based analyte detection. Fundamental properties of grating coupled surface plasmon fluorescence spectroscopy are described, as well as issues related to the commercial realization of the method. Several new experimental techniques are introduced and demonstrated in order to optimize performance in certain areas and improve upon capabilities in respect to prism-based systems. Holographically fabricated gratings are characterized by atomic force microscopy and optical methods, aided by simulations and profile parameters responsible for efficient coupling are analyzed. The directional emission of fluorophores immobilized on a grating surface is studied in detail, including the magnitude and geometry of the fluorescence emission pattern for different grating constants and polarizations. Additionally, the separation between the minimum of the reflected intensity and the maximum fluorescence excitation position is examined. One of the key requirements for the commercial feasibility of grating coupling is the cheap and faithful mass production of disposable samples from a given master grating. The replication of gratings is demonstrated by a simple hot embossing method with good reproducibility to address this matter. The in-situ fluorescence detection of analyte immobilization and affinity measurements using grating coupling are described for the first time. The physical factors related to the sensitivity of the technique are assessed and the lower limit of detection of the technique is determined for an exemplary assay. Particular attention is paid to the contribution of bulk fluorophores to the total signal in terms of magnitude and polarization of incident and emitted light. Emission from the bulk can be a limiting factor for experiments with certain assay formats. For that reason, a novel optical method, based on the modulation of both polarization and intensity of the incident beam, is introduced and demonstrated to be capable of eliminating this contribution.

New enhancement strategies for plasmon-enhanced fluorescence biosensors

This thesis investigates metallic nanostructures exhibiting surface plasmon resonance for the amplification of fluorescence signal in sandwich immunoassays. In this approach, an analyte is captured by an antibody immobilized on a plasmonic structure and detected by a subsequently bound fluorophore labeled detection antibody. The highly confined field of surface plasmons originates from collective charge oscillations which are associated with high electromagnetic field enhancements at the metal surface and allow for greatly increased fluorescence signal from the attached fluorophores. This feature allows for improving the signal-to-noise ratio in fluorescence measurements and thus advancing the sensitivity of the sensor platform. In particular, the thesis presents two plasmonic nanostructures that amplify fluorescence signal in devices that rely on epifluorescence geometry, in which the fluorophore absorbs and emits light from the same direction perpendicular to the substrate surface.rnThe first is a crossed relief gold grating that supports propagating surface plasmon polaritons (SPPs) and second, gold nanoparticles embedded in refractive index symmetric environment exhibiting collective localized surface plasmons (cLSPs). Finite-difference time-domain simulations are performed in order to design structures for the optimum amplification of established Cy5 and Alexa Fluor 647 fluorophore labels with the absorption and emission wavelengths in the red region of spectrum. The design takes into account combined effect of surface plasmon-enhanced excitation rate, directional surface plasmon-driven emission and modified quantum yield for characteristic distances in immunoassays. Homebuilt optical instruments are developed for the experimental observation of the surface plasmon mode spectrum, measurements of the angular distribution of surface plasmon-coupled fluorescence light and a setup mimicking commercial fluorescence reading systems in epifluorescence geometry.rnCrossed relief grating structures are prepared by interference lithography and multiple copies are made by UV nanoimprint lithography. The fabricated crossed diffraction gratings were utilized for sandwich immunoassay-based detection of the clinically relevant inflammation marker interleukin 6 (IL-6). The enhancement factor of the crossed grating reached EF=100 when compared to a flat gold substrate. This result is comparable to the highest reported enhancements to date, for fluorophores with relatively high intrinsic quantum yield. The measured enhancement factor excellently agrees with the predictions of the simulations and the mechanisms of the enhancement are explained in detail. Main contributions were the high electric field intensity enhancement (30-fold increase) and the directional fluorescence emission at (4-fold increase) compared to a flat gold substrate.rnCollective localized surface plasmons (cLSPs) hold potential for even stronger fluorescence enhancement of EF=1000, due to higher electric field intensity confinement. cLSPs are established by diffractive coupling of the localized surface plasmon resonance (LSPR) of metallic nanoparticles and result in a narrow resonance. Due to the narrow resonance, it is hard to overlap the cLSPs mode with the absorption and emission bands of the used fluorophore, simultaneously. Therefore, a novel two resonance structure that supports SPP and cLSP modes was proposed. It consists of a 2D array of cylindrical gold nanoparticles above a low refractive index polymer and a silver film. A structure that supports the proposed SPP and cLSP modes was prepared by employing laser interference lithography and the measured mode spectrum was compared to simulation results.rn

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 μs at pH 7 to 45 μs at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 μs and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic segment that mediates binding of MRP to CaM. We show that the effector domain is in a polar environment in free MRP, suggesting exposure to water, in agreement with a rod-shaped structure of the protein. The effector domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties of the myristoylated and unmyristoylated forms of a construct in which a tryptophan residue was introduced at position 4 close to the myristoylated N terminus of MRP suggests that the lipid moiety is also involved in the interaction of MRP with CaM.

Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10−6–10−3-s time range, by using fluorescence correlation spectroscopy at 10−8 M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over ≈6 × 102 to 5 × 106 W/cm2. These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of ≈10−3. Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-μs time range. The independence of these two bright–dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.