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We have cloned a novel fluorescent protein from the jellyfish Rhacostoma atlantica. The closest known related fluorescent protein is the Phialidium yellow fluorescent protein, with only a 55% amino acid sequence identity. A somewhat unusual alanine-tyrosine-glycine amino acid sequence forms the presumed chromophore of the novel protein. The protein has an absorption peak at 466 nm and a fluorescence emission peak at 498 nm. The fluorescence quantum yield was measured to be 0.77 and the extinction coefficient is 58 200 M(-1) cm(-1) . Several mutations were identified that shift the absorption peak to about 494 nm and the emission peak to between 512 and 514 nm.

Spectroscopic properties of the particle sizing fluorophore Dipole Blue are reported. The probe is cationic in nature, highly water-soluble, and strongly adheres to anionic silica surfaces by electrostatic interactions, as is demonstrated here by Ludox SM 30. The probe has a distinct absorbance band centered at 320 nm, and the fluorescence emission band is Stokes-shifted 100 nm with a peak centered at 426 nm. From time-correlated single-photon counting experiments, the fluorescence lifetime was found to be adequately described by a three-exponential decay model with an intensity-averaged lifetime of 15.6 ns. Perrin graph analysis of steady-state anisotropy shows the presence of silica particles with a radius of (5.44 ± 0.16) nm, which, considering the distribution of particle sizes, is in reasonable agreement with 3.5 nm found from dynamic light scattering experiments.

Marine sponges are major habitat-forming organisms in coastal benthic communities and have an ancient origin in evolution history. Here, we report significant accumulation of polyphosphate (polyP) granules in three common sponge species of the Caribbean coral reef. The identity of the polyP granules was confirmed by energy-dispersive spectroscopy (EDS) and by the fluorescence properties of the granules. Microscopy images revealed that a large proportion of microbial cells associated with sponge hosts contained intracellular polyP granules. Cyanobacterial symbionts cultured from sponges were shown to accumulate polyP. We also amplified polyphosphate kinase (ppk) genes from sponge DNA and confirmed that the gene was expressed. Based on these findings, we propose here a potentially important phosphorus (P) sequestration pathway through symbiotic microorganisms of marine sponges. Considering the widespread sponge population and abundant microbial cells associated with them, this pathway is likely to have a significant impact on the P cycle in benthic ecosystems.

In this Letter we characterize both the photophysical and plasmonic enhanced photophysical properties of carbon nanodots produced by a combustion method. In addition, we have found that max entropy analysis of luminescence decay reveals inhomogeneous distribution of radiative lifetimes. Further, while quantum yields of intrinsic carbon nanodots emission are low [

We report spectroscopic properties of a new nanoparticle sizing fluorophore and demonstrate its applicability to characterize silica particles in colloidal solutions using steady-state anisotropy measurements and Perrin graph analysis. The strong electrostatic interaction between the cationic particle sizing fluorophore and the SiO2 surface is demonstrated by comparing data to that recorded for the anionic fluorophore fluorescein. The Perrin analysis reports a mean particle radius of 6.35 nm, which is larger than the manufacture specified radius of 3.5 nm, possibly indicating a degree of aggregation of the particles. The anionic fluorophore fluorescein shows no interaction with the SiO2 surface, and reports a hydrodynamic radius of 0.40 nm. We thus speculate that fluorescein can be used as an internal microviscosity probe for colloidal silica systems. The intensity averaged fluorescence lifetime of the particle sizing fluorophore and fluorescein are approximately 16 ns and 4 ns, respectively.

A new and perspective addition to traditional fluorescent probes is the Au clusters (8–25 atoms) which can label proteins, rendering them extremely bright and photostable. In this paper, we show that albumins can quickly and effectively be labeled using microwave acceleration, which shortens the time of Au labeling from several hours to

By using the fluorescent dye 6-propionyl-2-(N,N-dimethylamino) naphthalene (PRODAN) to monitor methanol generated during tetramethyl orthosilicate polymerization we have optimised the encapsulation of protein in silica sol-gel monoliths with respect to completion of hydrolysis and distillation in order to remove methanol such that protein can be added without denaturation. A minimum of 24 h at +4 °C was found to be required before hydrolysis is complete and 3-5 min of vacuum distillation at 50 °C and 300 mbar needed to remove methanol before the gel is formed. The biocompatibility of a tetramethyl orthosilicate sol-gel monolith was demonstrated by preserving the trimer protein allophycocyanin (APC) in its native form for up to 500 h. This obviates the previously essential requirement of covalently binding the trimer together in order to prevent dissociation into monomers and has enabled observation of native APC trimer in a sol-gel pore for the first time down to the single molecule level using combined fluorescence spectroscopy and confocal microscopy. The higher stability afforded by the protocol we describe could impact on the application of sol-gel materials to single-molecule studies of wider bearing such as protein folding and aggregation.

With a view to assessing the current boundaries of non-invasive sensing of metabolites we compare the photophysical properties of melanin nanoparticles in vivo, size selected in natural form from Sepia officinalis extracted from cuttlefish, and synthesised from L-dihydoxyphenylalanine (DOPA) in water pools of reverse micelles. The fluorescence emission spectra and excited state decay components correlate well with the particle size. Recent developments in optical technology are discussed in the context of the formidable challenges and opportunities for metabolic sensing presented by endogenous fluorophores.

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.

We have studied the one- and two-photon induced fluorescence anisotropy decay of rhodamine 6G (R6G) during polymerization of tetramethyl orthosilicate (TMOS) approaching the sol-to-gel transition, a time denoted tg, using time-correlated single-photon counting and femtosecond Ti:sapphire laser excitation. A biexponential decay of fluorescence anisotropy is observed at all times. We propose a different interpretation to the widely accepted view, that fluorescence anisotropy reports solely on molecular viscosity in sol-gels. We think our results are consistent with the presence of both free dye and dye bound to nm-size silica particles rather than just the coexistence of different discrete viscosity domains as reported previously. A corollary of our interpretation is that the microviscosity changes very little from that of the initial bulk sol throughout the sol-gel polymerization. Nanometer-size particles are known from small angle scattering studies to be precursors to gelation in sol-gels over a wide range of conditions and our interpretation might prove to be an important step toward understanding the self-assembly mechanisms of silicon alkoxide based materials at the molecular level. According to our measurements and interpretation, for TMOS at pH 2.3 for example, primary silica particles of 0.8-nm mean radius grow by monomer-monomer or monomer-cluster addition to produce larger structures 1.1-nm mean radius after one month.

This report describes a gated sampling approach for studying the initial formation of sol-gel glasses prepared from sodium silicate solution (water glass) and sulphuric acid. Previously described were how changes in particle size and subsequently how sol-gel formation dynamics can be tracked using time-resolved fluorescence anisotropy, by labeling growing silica nanoparticles with suitable fluorescence probes. One limiting factor of this approach was the ≃2 minute measurement time, which limits this technique for studying the initial sol formation dynamics and limits the measurement precision. Using a continuous flow system and delaying sol flow through different tubing lengths overcomes this problem and allows monitoring of the very early stages of sol formation, second by second after sol preparation, irrespective of the anisotropy measurement time. This technique was applied to studying the initial formation dynamics, within the first 30 seconds, of a 12.01% SiO2 (w/w), pH 0.66 sol-gel, finding that silica particles of ≃1.5 nm mean radius are formed within 10 seconds of mixing the sol-gel.

We have developed a method of measuring the growth of nanoparticles during sol-gel glass formation based on labelling the particle with a fluorescent dye and determining the multiphoton excited decay of fluorescence anisotropy due to Brownian rotation. Multiphoton excitation is shown to give a higher dynamic range of measurement than one-photon excitation. We illustrate the sub-nanometre resolution and stability of our approach by detecting a 0.8-1.1 nm silica particle hydrodynamic mean radius increase in a tetramethylorthosilicate sol at pH 2.3 labelled with rhodamine 6G and observed over ≈4 weeks and also with a stable silica colloid of radius 6 nm, pH 8.9, labelled with a 6-methoxyquinoline-type dye.

Three fluorescent probes have been produced by the quaternisation of 6-methylquinoline with methyl bromide, methyl iodide and 3-bromo-1-propanol. The probes are water-soluble and their fluorescence is quenched by modest concentrations of chloride ions. One of the dyes, dye 1 , has a chloride Stern–Volmer constant of 255 mol −1 dm 3 , which is one of the highest reported. Fluorescence quantum yields of the dyes have been determined using quinine sulphate as a reference and are all found to be similar lying in the range 0.5–0.64. Applications for chloride determination in biological systems using these probes are discussed.

The use of molecular genetics for introducing fluorescent molecules enables the use of donor–donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor–donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C α -atoms of the positions V106C–H185C, H185C–M266C, and M266C–V106C were 60.9, 30.8, and 55.1 Å, respectively. These are in good agreement with the distances of 54 ± 4, 38 ± 3, and 55 ± 3 Å, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C α -atoms physically cannot coincide, there is a reasonable agreement between the methods.

The bichromophoric and monochromophoric probes, 1,32-dihydroxy-dotriacontane-bis(Rhodamine) 101 ester (Rh101C32Rh101) and Rhodamine 101 octadecyl ester (Rh101C18), respectively, were solubilised in unilamellar lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). At molar ratios of less than 1/5000 for Rh101C18/lipid and Rh101C32Rh101/lipid, intermolecular electronic energy migration is negligible, contrary to the intramolecular donor–donor energy migration (DDEM) within the Rh101C32Rh101 molecules. The time-resolved fluorescence anisotropy determined for Rh101C18 and Rh101C32Rh101 were globally analysed by using a recently developed model that accounts for reorientational motions, as well as the rate of DDEM(L. B-A. Johansson, F. Bergstrom, P. Edman, I. V. Grechishnikova and J. G. Molotkovsky, J. Chem. Soc., FaradayTrans., 1996, 92,1563). It was found that the rate of intramolecular DDEM within Rh101C32Rh101 is typically about 3×109 s-1. In vesicles formed by DOPC and POPC, the distance between the Rhodamine groups of Rh101C32Rh101 is found to be about 35 A, while it is about 32 A in DPPC vesicles. These values are in excellent agreement with reported thicknesses of the lipid bilayers. Thus, the present work strongly suggests that the Rh101C32Rh101 molecules are spanning across the lipid bilayer, that is, the Rhodamine residues are located on opposite sides of the lipid bilayer.

Three perylene derivatives with fluorescent quantum yields of 100% are suggested as new useful standards for the determination of fluorescence quantum yields. The compounds are N,N′-bis(1-hexylheptyl)-3,4:9,10-perylenebis(dicarboximide) and perylene-3,4,9,10-tetracarboxylictetramethyl ester which dissolve in organic solvents, and the water-soluble perylene-3,4,9,10-tetracarboxylic acid tetrapotassium salt. All compounds are easily handled, very photostable, and show only minor quenching by oxygen under normal experimental conditions.

Structural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins.

Fluorescence anisotropy studies of donor-donor electronic energy is complicated by the influence of reorientational motions of the donor (d) molecules. To test theoretical models for analyzing the fluorescence anisotropy, we have studied well-defined bichromophoric molecules, namely; a bis (9- anthryl methyl phosphonate) bisteroid and the P3 Cys-P203 Cys double mutant of the latent plasminogen activator inhibitor 1 (PAI-11, labelled with sulphydryl specific derivatives BODIPY. A procedure is presented for extracting the rate of energy transfer, as well as the d-d distance from the fluorescence anisotropy.

By using site-specific mutagenesis it is possible to prepare a protein molecule that can be labeled with two identical fluorescent probes at different positions.(1,2) To calculate intramolecular distances between the two fluorescent donors in a protein, a recently developed DDEM model can be applied.(3,4) Here we have studied the influence of incomplete donor labeling on the calculated distances. For this purpose, the previous model has been extended and compared with experiments performed on three mutants (V106C, M266C, and V106C-M266C) of plasminogen activator inhibitor type 1 (PAI-1) labeled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl) iodo-acetamide (SBDY). The Cα of the residues to which the SBDYs are covalently linked are separated by 55.1 A, as determined by X-ray diffraction on the wild-type PAI-1. To examine the reliability of the extracted parameters, synthetic data were generated and reanalyzed with the same model as used to analyze real experiments. It is concluded that, even for a low degree of labeled double mutant (≈60%), a distance of 54 ± 3 A is found for both models.

Distance dependent singlet and triplet metal-enhanced emission of eosin from silica coated silver island films (SiFs) has been studied by steady-state and time resolved fluorescence techniques, along with theoretical finite difference time domain (FDTD) numerical simulations, to understand how the thickness of the dielectric coating surrounding silver nanoparticles fundamentally affects luminescence enhancement. Our findings suggest that the distance dependence of metal-enhanced phenomena such as fluorescence, phosphorescence and delayed fluorescence is underpinned by the decay of the electric near-field, and depending on the actual silver silica sample embodiment, one can see either decreased or enhanced luminescence. These results not only expand our current MEF thinking but also suggest that one may well be able to approximate plasmon-enhanced luminescence values.

In this contribution we show that the Metal-Enhanced Fluorescence (MEF) Excitation Volumetric Effect (EVE), has a profound effect on the formation of Reactive Oxygen Species (ROS), such as singlet oxygen ((1)O2) and superoxide anion radical (O2(-)*), when sensitizers are placed in close proximity to plasmon supporting nanoparticulate substrates. In particular, when the singlet oxygen sensitizer rose bengal is placed on a SiFs surface, i.e. on a silver island film, the (1)O2 response to power is non-linear, and at 100 mW excitation power (535 nm) it is about 5 times higher, as compared to glass control samples, measured with the commercially available (1)O2 probe Sensor Green™. We also report a similar power dependence of superoxide generation for acridine on SiFs surfaces, but using the dihydroethidium O2(-)* probe (DHE). Our findings are consistent with our previously postulated Metal-Enhanced Fluorescence (MEF) and EVE models.

Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

Different optically active and inactive derivatives of the perylene dye, perylene-3,4:9,10-tetracarboxylic bisimide, solubilized in unilamellar lipid vesicles of 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) have been studied by means of polarized light spectroscopic methods. Absorption and fluorescence spectra become distorted at molar ratios greater than one dye molecule per 1000 molecules of DOPC. These observations are compatible with the formation of ground-state aggregates. Circular dichroism (CD) was not detectable for any perylene dye dissolved in liquid solutions, or diluted in lipid vesicles. However, dyes with optically active substituents reveal CD spectra at dye: lipid ratios of greater than 1:1000. For dyes with R and S enantiomers, CD spectra are mirror images, while no CD of racemic mixtures, or optically inactive perylene dyes could be detected. The observed absorption and CD spectra are qualitatively explained by exciton coupling within dimers and their molecular symmetry.

In a previous report by us [1], it was demonstrated that the metal enhanced fluorescence (MEF) signal from fluorophores close to metal nanoparticles increased non-linearly with the far-field excitation power (irradiance). The effect was interpreted as an increase in the near-field excitation volume surrounding the metallic nanoparticles and was subsequently referred to as an excitation volumetric effect (EVE), where the technique would potentially allow for the near-field tuning of fluorescence enhancement factors, when observed from the far-field.In this contribution we show that the EVE phenomenon also has a profound effect on the formation of Reactive Oxygen Species (ROS), such as singlet oxygen (1O2) and superoxide anion radical (O2-∗), when sensitizers are placed in close proximity to plasmon supporting substrates. In particular, when the singlet oxygen sensitizer Rose Bengal is placed on a SiFs surface, i.e. on a silver island film [2], the 1O2 response to power is non-linear, and at 100 mW excitation power (535 nm) it is about 5 times higher, as compared to glass reference samples, measured with the commercially available 1O2 probe Sensor Green (SG). We also report a similar power dependence of superoxide generation for Acridine on SiFs surfaces, but using the dihydroethidium O2-∗probe (DHE). We additionally report the influence of various solvents where the singlet oxygen lifetime varies, i.e., water and deuterium oxide.[1] A. I. Dragan and C. D. Geddes, “Excitation volumetric effects (EVE) in metal-enhanced fluorescence,” Phys. Chem. Chem. Phys., vol. 13, no. 9, pp. 3831-3838, 2011.[2] SiFs paper reference here With rod.

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

In recent years our laboratory and others have demonstrated many examples and concepts in Metal-Enhanced Fluorescence1 (MEF), a surface plasmon phenomenon, which amplifies both fluorescence and luminescence signatures in the near-field, i.e. less than one wavelength of light away from a metallic object1. In all of these examples of MEF, and for over a decade, the fluorescence spectra has simply been reported as being enhanced, i.e. the emission is greater from a plasmonic substrate as compared to a suitable control sample.However, in this paper we will show that Metal-Enhanced Fluorescence from both a variety of plasmonic substrates and using a range of different fluorophores, often results in fluorophore spectral distortion2. More often than not, the red edge of the fluorescence spectra is observed to be distorted, as compared to the emission spectra of a fluorophore observed in the far-field and distal from plasmonic interactions. In addition, a significant MEF effect often results in notable changes in the spectrum full width at half maximum (FWHM). We discuss these new effects in terms of the mechanism of plasmonic enhancement.1Metal-Enhanced Fluorescence, Edited by Geddes, C.D., John Wiley and Sons, New Jersey, June 2010, 625 pgs, ISBN: 978-0-470-22838-8.2 Spectral Shifts in Metal-Enhanced Fluorescence, Karolin, J. and Geddes, C.D., (2014), Applied Physics Letters, 105, 063102.

A new fluorescent molecule 1,7-diazaperylene (DP) has been investigated by means of time-resolved and steady-state polarized fluorescence spectroscopy, as well as X-ray spectroscopy. Absorption and fluorescence spectra of DP in solution are similar to those of perylene. However, absorption and fluorescence spectra of 2,8-dimethoxy DP and 2,8-dipentyloxy DP in solution are red-shifted by ca. 55 nm relative to perylene. The fluorescence decay of DP is exponential with a lifetime of 5.1 ns in ethanol, 4.9 ns in glycerol and 4.3 ns in paraffin oil. The radiative lifetime in ethanol was calculated to be 6.3 ns for DP, 8.0 ns for 2,8-dimethoxy DP and 7.6 ns for 2,8-dipentyloxy DP. The calculated fluorescence quantum yields of 0.8 for DP and its alkoxy derivatives in ethanol, are in good agreement with those obtained from measurements. The calculated Forster radius is 37.2 ± 1 A for DP and 41.9 ± 1 A for its alkoxy derivatives in ethanol. Examining the S0↔ S1 transition, we obtain a limiting fluorescence anisotropy of r0≈ 0.38 for DP and its alkoxy derivatives. The rotational rates of DP in paraffin oil and glycerol were compared to that of perylene. In paraffin oil both molecules show an almost identical biexponential decay of the fluorescence anisotropy, which is compatible with a rotational motion like an oblate ellipsoid. The fluorescence anisotropy is monoexponential for DP in glycerol, and DP appears to rotate like a spherical particle while perylene in glycerol appears to rotate like an oblate ellipsoid. Moreover, the rotational diffusion constant, corresponding to rotation about an axis in the aromatic plane (D⊥), is the same for both DP and perylene in glycerol.

Hydrated nanopores offer a unique environment for studying biological molecules under controlled conditions and fabricating sensors using fluorescence. Silica nanopores for example are non-toxic, biologically and optically compatible with protein, and can be easily synthesized to entrap protein and exclude potentially interfering macromolecules, while transmitting analytes of interest. A well known problem when polymerizing orthosilicates to fabricate silica sol-gel nanopores is the release of alcohol, which denatures proteins. We will describe how using the fluorescence of PRODAN (6-propionyl-2-(N,N-dimethylamino) naphthalene) to monitor methanol generated during polymerization has helped define a protocol with enhanced biocompatibility. The improved biocompatibility of sol-gel nanopores synthesized using tetramethyl orthosilicate (TMOS) has been demonstrated by preserving the unstable native trimer form of allophycocyanin (APC) for up to 500 Hrs without the need to covalently binding the subunits together. This has enabled the observation of native APC trimer by means of its fluorescence in a pore down to the single molecule level. In this paper we demonstrate how PRODAN and another polarity sensitive dye, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, Nile red (NR) report on pore polarity and successfully extend protein encapsulation to nano-channels of alumina (Al2O3). Improved biocompatibility of nanopores has potential impact in nanomedicine where the ability to study single biomolecules is a primary goal as it underpins our understanding of disease pathology and therapeutics at the most fundamental level. In sensing also the advantages of nanopore isolation of metabolite-specific protein for detecting non-fluorescent metabolites has been demonstrated. Similar approaches can in principle be developed for both single-molecules and lab-on-a-chip sensors.

We review the different approaches available to materials science using time-resolved fluorescence depolarization. Performance, limitations and challenges in this emerging area of nanometrology are discussed using the exemplars of sol–gel nanoparticle growth and protein dynamics.

Metal-enhanced fluorescence enhancement factors up to 7-fold have been observed for Basic Fuchsin (BF) in close proximity to Zinc nano particulate substrates. In addition, the emission spectra of BF close-to Zinc as compared to a control sample are heavily distorted, particularly on the red-edge, giving systematic trends in enhancement, anywhere from 3- to 7-fold. We discuss these remarkable wavelength dependent effects with regard to the mechanism of metal-enhanced fluorescence.

In past several years, there has been significant investigation into the various synthetic routes of carbon nanodots along with their associated photophysical properties [1-3]. Carbon nanodots are naturally fluorescing nanometer-sized particles with interesting and unique photophysical properties, which make them highly applicable for various applications in the life sciences [2-3]. Our lab has been investigating these particles produced by various combustion routes for many years, studying both the photophysical and plasmon-enhanced photophysical properties [1]. In order to fully understand the photophysical properties of carbon nanodots, in this poster we have examined the both the internal and external structure of these particles in an attempt to ascertain the origins of the fluorescence signature/s, using a combination of differently charged ions; which ultimately results in both static and dynamic quenching processes being observed. Our results reveal significant vibronic structure of the nanodots’ chromophore, which can readily be quenched by non-charged ions (acrylamide), suggesting a buried fluorescent chromophore center.[1] Y. Zhang, H. Goncalves, J. C. G. Esteves Da Silva, and C. D. Geddes, “Metal-enhanced photoluminescence from carbon nanodots,” Chem. Commun. 47, 5313-5315 (2011).[2] S. Baker and G. Baker, “Luminescent Carbon Nanodots: Emergent Nanolights,” Angew, Chem. Int. Ed. 49, 6726-6744 (2010).[3] H. Li, Z. Kang, Y. Liu, and S-T. Lee, “Carbon nanodots synthesis, properties and applications,” J. Mater. Chem, 22, 24230-24253 (2010).

We describe recent fluorescence studies of the formation dynamics and structure of sol-gel glasses from nanometre particles composed of silica clusters in sols to nanometre pores in silica gels. The “kinetic life-history” of silica produced under both acidic and alkaline conditions from sodium silicate in a hydrogel and from an alkoxide in an alcogel is now starting to be revealed by fluorescence techniques and the influence of key parameters such as pH and silica concentration quantified at the molecular level. Through careful choice of fluoro-probe, anisotropy decay has been shown to provide particle size as well as viscosity information and offer advantages over traditional techniques for silica particle sizing based on small angle neutron, X-ray or light scattering. Fluorescence resonance energy transfer (FRET) can now be used to determine the donor-acceptor spatial distribution function without making any a-priori assumptions as to its form. This in turn promises to make FRET a better means of monitoring pore morphology in the wet gel during drying and ageing, offering distinct advantages over dry gel techniques such as mercury porosimetry and nitrogen adsorption. The insight into sol-gel processes 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.

We report a 2 nm red shift in the fluorescence spectra observed for Rhodamine 800 dissolved in glycerol on copper substrates as compared to glass reference samples, suggesting a wavelength dependence of metal enhanced fluorescence. The full width half maximum of the blue-red spectra is about 1 nm narrower as compared to the reference sample. We speculate that the observation correlates with a specific interaction mechanism between the Rhodamine 800 transition dipole, the enhanced electric field, and subsequent plasmon coupling, an observation not yet reported.

We demonstrate a new application of multiphoton excitation of fluorescence; the measurement of silica particle growth during sol-gel polymerization. Recently we have reported the use of fluorescence anisotropy as an alternative approach to nm particle sizing using conventional techniques such as small angle light, neutron or x-ray scattering. Advantages of our approach include near angstrom resolution, minimal interference from the gel network as well as the additional benefits of providing microviscosity and hydrodynamic information. Fluorescence anisotropy applied to particle sizing in colloids in general is still in its infancy, but in this paper we show using probes fluorescein and rhodamine 6G, under conditions unsuitable for one-photon excitation, can be successful with multiphoton excitation.

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

Application of donor-donor energy migration (DDEM) for examining protein structure and function

We describe the auto-oxidation of 3, 4-dihydroxy-L-phenylalanine (L-DOPA) in the synthesis of eumelanin to spontaneously produce fibrils upon drying. The self-assembled fibrils are of characteristic diameter ∼1 to 2 μm, composed of filaments, and are unidirectional, apart from branches that are formed at typically an angle of 20 to 22 deg. The fibrils are characterized using fluorescence spectroscopy, fluorescence decay times, scanning electron microscopy, atomic force microscopy, and fluorescence lifetime imaging microscopy. The fibrils mimic natural melanin in consisting of core eumelanin with efficient nonradiative properties, but they also display pockets of electronically isolated species with higher radiative rates on the nanosecond timescale. Eumelanin fibrils formed occasionally in solution are tentatively attributed to a scaffold of bacteria or fungus. Fabricating and characterizing novel synthetic eumelanin structures such as fibrils are of interest in helping to reveal a functional structure for eumelanin, in understanding its photophysics, in learning more about L-DOPA as it is used in the treatment of Parkinson's disease, and in producing novel materials which might embody some of the diverse properties of eumelanin.

We compare determining the size of silica nanoparticles using the time-resolved fluorescence anisotropy decay of dye molecules when electrostatically and covalently bound to stable silica nanoparticles. Covalent labelling is shown to offer advantages by simplifying the dye rotational kinetics and the appropriateness of various kinetic models is discussed. Silica nanoparticles produced using Stober synthesis of tetraethylorthosilicate (TEOS) are found to be controllable between ∼3.1 and 3.8 nm radius by adjusting the relative water:TEOS concentration. Covalent labelling with fluorescein 5(6)-isothiocyanate (FITC) bound to (3-aminopropyl) trimethoxysilane (FITC-APS) predicts a larger particle than electrostatically labelling with rhodamine 6G. The difference is attributed to the presence of an additional depolarization mechanism to Brownian rotation of the nanoparticle and dye wobbling with electrostatic labelling in the form of dye diffusion on the surface of the nanoparticle.

Melanins are common pigments with a non-repeating primary structure that is generally accepted to be composed of dihydroxyindoles. However, despite intensive research the secondary structure defining the minimum functional unit (protomolecule) remains elusive. We have revisited eumelanin formation in-situ during the non-enzymatic auto-oxidation of 3,4-dihydroxy-L-phenylalanine by using the fluorescence of thioflavin T; an extrinsic probe known to report on sheet structure. This approach obviates the complex intrinsic fluorescence and reveals a sigmoidal temporal dependence of assembly that is consistent with protomolecule formation and assembly into a stacked sheet structure rather than a randomized heteropolymer formed by monomer addition.

We report on the properties of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester (Rh101C32Rh101) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and in liquid solvents. The results are compared with those of rhodamine 101 octadecanyl ester (Rh101C18). Both molecules are solubilized in the lipid bilayer and the Rh101 moieties are anchored in the lipid-water interface, so that the electronic transition dipole moments (S 0 ↔S 1) are oriented preferentially in the plane of the bilayer. At low concentrations of the dyes in lipid bilayers of DOPC, the fluorescence relaxation is single exponential with a lifetime of τ=4.9±0.2 ns. The relative fluorescence quantum yield of ΦC32/ΦC18 ≈ 0.95 in DOPC vesicles. These results strongly suggest that only a small fraction of the Rh101C32Rh101 molecules are quenched, by, for example, intra- or intermolecular dimers in the ground state at mole fractions of less than 0.1% in the lipid bilayers. For Rh101C32Rh101 in lipid vesicles, the steady-state and time-resolved fluorescence anisotropies are compatible with efficient intramolecular electronic energy transfer. It is concluded that nearly every Rh101C32Rh101 molecule is spanning across the lipid bilayer of DOPC.

We demonstrate nanoparticle size measurement using time-resolved fluorescence anisotropy decay in relation to establishing a nanometrology standard. The rotational correlation time equivalent to the isotropic Brownian rotation of a fluorescent 6-methoxyquinolinium dye attached to amorphous silica nanoparticles was determined in three different LUDOX* colloids from the complex fluorescence anisotropy decay observed. Once competing depolarization and nanoparticle aggregation had been taken into account, good agreement was found of 4.0 ± 0.4 nm, 6.4 ± 0.5 nm and 11.0 ± 1.6 nm corresponding to the manufacturer's reported particle radii of 3.5 nm, 6 nm and 11 nm, for LUDOX SM30, AM30 and AS40 respectively. We describe the measurement science required for acquisition and interpretation of fluorescence anisotropy decay data in order to determine nanoparticle size while highlighting the limitations and useful range of measurement.

Single molecule level detection of the near-infrared fluorescent protein allophycocyanin (APC) has been achieved using surface enhanced resonance Raman scattering (SERRS). The detection limit using the peak height of the 440 cm(-1) band was 1 x 10(-13) mol l(-1), compared to 2 x 10(-12) mol l(-1) for the fluorescence peak at 660 nm.

The authors show that pH increase, due to removal by condensation of silicic acid, correlates with nanoparticle growth during the initial stages of silica hydrogel formation and becomes constant at a time tpH, the point when other particle growth mechanisms dominate. Absorption of common phthalein indicators is shown to allow effectively instantaneous tracking of the pH and nanoparticle size in alkaline and acidic hydrogels. Particle sizes are calibrated using the hydrodynamic radius determined from the fluorescence anisotropy decay. Tracking pH complements fluorescence anisotropy nanometrology by offering a lower cost, speedier, and simpler method of studying particle growth during silica hydrogel fabrication.