Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.     

Immunosuppressive activity of antamanide and some of its analogues

In connection with our discovery of a strong immunosuppressive activity of cyclolinopeptide A (CLA), we investigated immunosuppressive properties of antamanide and a number of its analogues, including symmetrical antamanide, and compared them with the activities of cyclosporin A and CLA. The peptides were investigated by using plaque forming cell (PFC), graft-versus-host (GvH), delayed type hypersensitivity (DTH), and autologous rosette formation cell (ARFC) tests. Antamanide and symmetrical antamanide exhibit an immunosuppressive activity lower than CLA. Linear antamanide fragments are also active. At higher concentrations of the latter peptides, toxic effects occur.     

One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

Oligonucleotide-displaced organic monolayer-protected silver nanoparticles and enhanced luminescence of their salted aggregates

N-(2-mercaptopropionyl)glycine (tiopronin) monolayer-protected silver particles were partially displaced by single-stranded oligonucleotides through ligand exchanges. The oligonucleotide-displaced particles could be hybridized with complementary fluorophore-labeled oligonucleotides. Both the oligonucleotide-displaced and hybridized particles could be aggregated by electrostatic interactions with salt in buffer solution, and the aggregates displayed enhanced luminescence from fluorophores. This result suggests the possible application of surface-enhanced fluorescence from metallic nanoparticle aggregation for DNA detection.     

Ultrabright fluorescein-labeled antibodies near silver metallic surfaces

Fluorescein-labeled antibodies are widely used in clinical assays and fluorescence microscopy. The fluorescent signal per labeled antibody is limited by fluorescein self-quenching, which occurs when the antibody is heavily labeled with multiple fluoresceins. We examined immunoglobulin G (IgG) when labeled with 0.7 to about 30 fluoresceins per antibody molecule. The extent of self-quenching was decreased, and the signal increased, when the labeled antibody was in close proximity to metallic silver particles. Time-resolved measurements showed that the intensity increase was due in part to a silver-induced increase in the radiative decay rate. These results suggest the use of labeled antibodies conjugated to silver particles as ultrabright probes for imaging or analytical applications.     

Fluorescence spectral properties of labeled thiolated oligonucleotides bound to silver particles

We examined the fluorescent spectral properties of fluorescein-labeled DNA oligomers when directly bound to metallic silver particles via a terminal sulfhydryl group. We found a 12-fold increase in fluorescence intensity and 25-fold decrease in lifetime for a fluorescein residue positioned 23 nucleotides from the silver surface compared to labeled oligomers in free solution. Similar results were found for a 23-mer labeled with five fluorescein residues. The absence of long lifetime components in the intensity decays suggests that all labeled oligomers are bound to silver and affected similarly by the metallic surfaces. These results provide the basic knowledge needed to begin use of metal-enhanced fluorescence for the detection of target sequences in simple formats potentially without a washing separation step. The use of metal-enhanced fluorescence provides a generic approach to obtaining a hybridization-dependent increase in fluorescence with most, if not all, commonly used fluorophores.     

Single actomyosin motor interactions in skeletal muscle

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:10⁵. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~ 9 s during muscle contraction.     

Light-induced isomerization of the LHCII-bound xanthophyll neoxanthin: possible implications for photoprotection in plants

Light-harvesting pigment-protein complex of Photosystem II (LHCII) is the largest photosynthetic antenna complex of plants and the most abundant membrane protein in the biosphere. Plant fitness and productivity depend directly on a balance between excitations in the photosynthetic apparatus, generated by captured light quanta, and the rate of photochemical processes. Excess excitation energy leads to oxidative damage of the photosynthetic apparatus and entire organism and therefore the balance between the excitation density and photosynthesis requires precise and efficient regulation, operating also at the level of antenna complexes. We show that illumination of the isolated LHCII leads to isomerization of the protein-bound neoxanthin from conformation 9'-cis to 9',13- and 9',13'-dicis forms. At the same time light-driven excitation quenching is observed, manifested by a decrease in chlorophyll a fluorescence intensity and shortened fluorescence lifetimes. Both processes, the neoxanthin isomerization and the chlorophyll excitation quenching, are reversible in dim light. The results of the 77K florescence measurements of LHCII show that illumination is associated with appearance of the low-energy states, which can serve as energy traps in the pigment-protein complex subjected to excess excitation. Possible sequence of the molecular events is proposed, leading to a protective excess excitation energy quenching: neoxanthin photo-isomerization→formation of LHCII supramolecular structures which potentiate creation of energy traps→excitation quenching.     

Orientation and spectral properties of two stilbazolium merocyanine dyes in stretched and unstretched polyvinyl alcohol films

Spectral properties (anisotropy coefficients calculated for absorption, emission and fluorescence decay time) of two stilbazolium merocyanine dyes have been determined to evaluate the applicability of these dyes as sensitizers in photodynamic therapy. The dyes were embedded in an anisotropic polymer matrix. Analysis of the emission decay components measured in polarized light provides information on the interactions of the dye molecules with the polymer matrix being a model of an anisotropic biological system. Different values of the emission anisotropies obtained from various polarized components of fluorescence decays have shown that the orientations of the dye molecules influence their interactions with the polymer. This means that differently oriented dye molecules located in biological systems should exhibit different interactions with membranes. The chain length and type of side groups attached as well as the salt form of the dye molecule were shown to influence the dye-polymer interactions and should be taken into account before the application of merocyanine dyes in medicine. These dyes seem to be promising optical sensors with spectral properties, including the calculated anisotropy coefficients, sensitive to the molecular environment, useful to study orientation and interaction with neighbouring molecules in biological membranes.     

Play behaviour and active training of Montagu’s harrier (Circus pygargus) offspring in the post-fledging period

Play bouts and active training of juveniles by Montagus harrier (Circus pygargus) adults in the post-fledging period were observed. Fledglings often played with prey and with a variety of inanimate objects such as bits of moss, regurgitated pellets, sticks and a wad of hay. Inanimate objects selected for play were, in length, very similar to the common vole (Microtus arvalis), which is the most common prey of the species during their breeding period. Some recently fledged individuals were trained to capture invertebrate prey by adults demonstrating techniques for the fledglings and thus develop their hunting skills. Training sessions took place only in the foraging areas of the adult birds.