Jan Karolin

Spectroscopic properties of new and convenient standards for measuring fluorescence quantum yields

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.

Donor-Donor Energy Migration for Determining Intramolecular Distances in Proteins: I. Application of a Model to the Latent Plasminogen Activator Inhibitor-1 (PAI-1)

A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.

Photophysics, molecular reorientation in solution and X-ray structure of a new fluorescent probe, 1,7-diazaperylene

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.

1,32-Dihydroxy-dotriacontane-bis(Rhodamine) 101 ester A lipid membrane spanning bichromophoric molecule as revealed by intramolecular donor–donor energy migration (DDEM)

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.

Spectral shifts in metal-enhanced fluorescence

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.

Polarized fluorescence and absorption spectroscopy of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester. A new and lipid bilayer-spanning probe

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 (S0 ↔S1) 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.

Donor–Donor Energy Migration (DDEM) for Determining Intramolecular Distances in Proteins. II. the Effect of Partial Labeling

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 Å, 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 Å is found for both models.

Nanoparticle Sizing and Potential Quality Control of Sols Using a Unique Fluorescence Anisotropy Probe and 3D Contour Anisotropy Mapping.

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.

Spectral Distortions in Metal-Enhanced Fluorescence

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.

Probing the Internal and External Structure of Carbon Nanodots through Fluorescence Quenching

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).