Many large double-stranded DNA viruses employ high force-generating ATP-driven molecular motors to package to high density their genomes into empty procapsids. Bacteriophage T4 DNA translocation is driven by a two-component motor consisting of the procapsid portal docked with a packaging terminase-ATPase. Fluorescence resonance energy transfer and fluorescence correlation spectroscopic (FRET-FCS) studies of a branched (Y-junction) DNA substrate with a procapsid-anchoring leader segment and a single dye molecule situated at the junction point reveal that the "Y-DNA" stalls in proximity to the procapsid portal fused to GFP. Comparable structure Y-DNA substrates containing energy transfer dye pairs in the Y-stem separated by 10 or 14 base pairs reveal that B-form DNA is locally compressed 22-24% by the linear force of the packaging motor. Torsional compression of duplex DNA is thus implicated in the mechanism of DNA translocation.

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and lambda phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that approximately 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and 50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the 50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.

Fluorescence technology is fully entrenched in all aspects of biological research. To a significant extent, future advances in biology and medicine depend on the advances in the capabilities of fluorescence measurements. As examples, the sensitivity of many clinical assays is limited by sample autofluorescence, single-molecule detection is limited by the brightness and photostability of the fluorophores, and the spatial resolution of cellular imaging is limited to about one-half of the wavelength of the incident light. We believe a combination of fluorescence, plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology. Surface plasmons are collective oscillations of free electrons in metallic surfaces and particles. Surface plasmons, without fluorescence, are already in use to a limited extent in biological research. These applications include the use of surface plasmon resonance to measure bioaffinity reactions and the use of metal colloids as light-scattering probes. However, the uses of surface plasmons in biology are not limited to their optical absorption or extinction. We now know that fluorophores in the excited state can create plasmons that radiate into the far field and that fluorophores in the ground state can interact with and be excited by surface plasmons. These reciprocal interactions suggest that the novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location, and direction of fluorophore emission. We refer to these phenomena as plasmon-controlled fluorescence (PCF). We predict that PCF will result in a new generation of probes and devices. These likely possibilities include ultrabright single-particle probes that do not photobleach, probes for selective multiphoton excitation with decreased light intensities, and distance measurements in biomolecular assemblies in the range from 10 to 200 nm. Additionally, PCF is likely to allow design of structures that enhance emission at specific wavelengths and the creation of new devices that control and transport the energy from excited fluorophores in the form of plasmons, and then convert the plasmons back to light. Finally, it appears possible that the use of PCF will allow construction of wide-field optical microscopy with subwavelength spatial resolution down to 25 nm.

Water-soluble gold nanoparticles with an average diameter of 5 nm were prepared with carboxylic acid terminated thiol ligands. These ligands contain zero to eight methylene moieties. CdTe nanocrystals with an average diameter of 5 nm were synthesized with aminoethanethiol capping. These nanocrystals displayed characteristic absorption and emission spectra of quantum dots. The amine terminated CdTe nanocrystals and carboxylic-acid-terminated gold nanoparticles were conjugated in aqueous solution at pH 5.0 by electrostatic interaction, and the conjugation was monitored with fluorescence spectroscopy. The CdTe nanocrystals were significantly quenched upon binding with gold nanoparticles. The quenching efficiency was affected by both the concentration of gold nanoparticles in the complex and the length of spacer between the CdTe nanocrystal and Au nanoparticle. The observed quenching was explained using Förster resonance energy transfer (FRET) mechanism, and the Förster distance was estimated to be 3.8 nm between the donor-acceptor pair.

We studied the effect of metal particles on Förster resonance energy transfer (FRET) between nearby donor-acceptor pairs. The studies included the effect of donor-acceptor distance, silver particle size, and distance from the metal surface. The metal particles were synthesized with average diameters of 15, 40, and 80 nm, respectively. A Cy5-labeled oligonucleotide was chemically bound to a single silver particle with a distance of 2 or 10 nm from the surface of metal core. A Cy5.5-labeled complementary oligonucleotide was bound to the particle-conjugated oligonucleotide by hybridization. The spacer length between donor-acceptor was adjusted by the number of base pairs. FRET between the donor-acceptor pair was investigated by dual-channel single-molecule fluorescence detection. Both the emission intensities and lifetimes indicated that FRET was enhanced efficiently by the metal particles. The results showed an increase of apparent energy transfer distance with the size of silver particle and distance from the metal core. Simulations by finite-difference time-domain (FDTD) calculations were used to compare with the experimental results. The local fields at the location of the donor-acceptor pair appeared to correlate with the FRET efficiency. These results will aid in the design of metal particles for using FRET to determine biomolecule proximity at distances beyond the usual Förster distance.

Surface plasmon-coupled emission (SPCE) is the directional radiation of light into a glass substrate due to excited fluorophores above a thin metal film. The sharp angular distribution of SPCE is a striking phenomenon and is in stark contrast with the isotropic fluorescence emission. In this paper, we show that SPCE can occur with thin platinum films at green and red wavelengths and was found to be mostly p-polarized. This SPCE emission is the result of near-field interactions of the excited fluorophores with the thin platinum film, and is not simply a reflective or transmissive phenomenon. Our preliminary observation suggests that platinum nanostructures can be part of several novel bio-analysis surfaces.

Labeled silica beads with an average diameter of 100 nm were synthesized by incorporating with 20-600 muM Ru(bpy)(3)(2+) complexes. Silver shells were deposited on the beads layer-by-layer with the shell thickness of 5-50 nm. The emission band became narrower and the intensity was enhanced depending on the shell thickness. Self-quenching of the probe was observed at high concentration. Poisson statistics were employed to analyze self-quenching of the fluorophores. The estimated quenching distance was extended from 6 to 16 nm with shell growth from 0 to 50 nm. Moreover, the silver shells were also labeled with Rhodamine 6G. Fluorescence enhancement and reduced lifetime were also observed for silver-silica shell containing R6G. We found that by adjustment of probe concentration and silver shell thickness, a Ru(bpy)(3)(2+)-labeled particle could be 600 times brighter than an isolated Ru(bpy)(3)(2+) molecule. We expect labeled metal core-shell structures can become useful probes for high sensitivity and/or single particle assay.

We use the finite-difference time-domain method to predict how fluorescence is modified if the fluorophore is located between two silver nanoparticles of a dimer system. The fluorophore is modeled as a radiating point dipole with orientation defined by its polarization. When a fluorophore is oriented perpendicular to the metal surface, there is a large increase in total power radiated through a closed surface containing the dimer system, in comparison to the isolated fluorophore and the case of a fluorophore near a single nanoparticle. The increase in radiated power indicates increases in the relative radiative decay rates of the emission near the nanoparticles. The angle-resolved far-field distributions of the emission in a single plane are also computed. This is informative as many experimental conditions involve collection optics and detectors that collect the emission along a single plane. For fluorophores oriented perpendicular to the metal surfaces, the dimer systems lead to significant enhancements in the fluorescence emission intensity in the plane. In contrast, significant emission quenching occurs if the fluorophores are oriented parallel to the metal surfaces. We also examine the effect of the fluorophore on the near-field around the nanoparticles and correlate our results with surface plasmon excitations.

We report that self-assembled monolayers of colloidal silver nanoparticles can increase the intensity of the surface plasmon-coupled emission (SPCE) signal from sulforhodamine 101 (S101). The S101 was spin coated on a glass slide coated with a layer of continuous silver, and a silica layer upon which the nanoparticle layer was self-assembled. Of the various colloid sizes studied, the 40 nm colloids showed both the highest enhancements in the SPCE signal and the largest extent of plasmon coupling, defined as the ratio of SPCE to Free Space signal. Our findings reveal a new technique that can be potentially employed to increase the sensitivity of SPCE applications.

We study the nature of fluorescence scattering by a radiating fluorophore placed near a metal nanoparticle with the finite-difference time-domain method. Angle-resolved light-scattering distributions are contrasted with those that result when ordinary plane waves are scattered by the nanoparticle. For certain sized nanoparticles and fluorophore dipoles oriented parallel to the metal surface, we find that the highest scattered fluorescence emission is directed back toward the fluorophore, which is very different from plane-wave scattering. The largest enhancements of far-field radiation are found when the dipole is oriented normal to the surface. We also examined the effect of the fluorophore on the near field around the particle. The fields can be enhanced or quenched compared to the isolated fluorophore and exhibit strong dependence on fluorophore orientation, as well as interesting spatial variations around the nanoparticle.