In this paper, fluorescence metal nanoshells (FMNs) were synthesized for target molecule detection on tissue specimens by fluorescence imaging method. FMNs were made with 40 nm silica spherical cores and 10 nm silver shells. Ru(bpy)32+ complexes were encapsulated in the silica cores for fluorescence properties. Avidin molecules were covalently bound on FMNs and formed avidin-Ag complexes could be site-specially conjugated on bone tissue specimens. Fluorescence intensity and lifetime images were recorded on a time-resolved confocal microscope. Imaging measurements showed that the emissions by avidin-FMN complexes could be distinctly isolated as individuals from the cellular backgrounds on lifetime images even when the tissues were stained with additional organic dyes. This observation demonstrates that the metal nanoprobes can be used for single target molecule detection on tissues during fluorescence imaging measurements.
Fluorescence cell imaging can be used for disease diagnosis and cellular signal transduction. Using a metal nanoshell as molecular imaging agent, we develop a cellular model system to detect CXCR4 chemokine receptor on T-lymphatic cell surface. These metal nanoshells are observed to express enhanced emission intensity and shortened lifetimes due to the near-field interactions. They are covalently bound with anti-CXCR4 monoclonal antibodies for immunoreactions with the target sites of the CXCR4 receptors on the CEM-SS cells. The fluorescence intensity and lifetime cell images are recorded with a time-resolved confocal microscopy. As expected, the emission signals from the metal nanoshells are clearly isolated from the cellular autofluorescence due to strong intensities and distinctive lifetimes. The number of emission spots on the single cell image is estimated by direct count to the emission signals. Analyzing a pool of cell images, a maximal count number is obtained in a range of 200±50. Because there is an average of ~6000 binding sites on the cell surface, we estimate that one emission spot from the metal nanoshell may represent ~30 CXCR5 receptors. In addition, the CXCR4 receptors are estimated to distribute on ~70% area of the cell surface.
Metal nanoparticle fluorophores have been developed using metal-enhanced fluorescence (MEF) principle. Compared
with the conventional organic fluorophores, the metal fluorophores display the increasing brightness and shortening
lifetime as well as the lengthening photostability and reducing photoblinking. Conjugated the metal fluorophores on the
surfaces of cell lines, the cell images were recorded on a scanning confocal microscopy in the either emission intensity
or lifetime. The emission spots by the conjugated metal fluorophores were isolated distinctly from the cell images
because of their brighter signals and shorter lifetimes. Collected in the three-dimension, the total number of emission
signals could be counted quantitatively and the distribution could be described on the cell surfaces. It was noticed that
the emission intensity over the cell image was increased with an increase of the number of metal fluorophore on the cell
surface and simultaneously the lifetime was altered. A quantitative regression curve was achieved between the amount of
metal fluorophore on the cell surface and the emission intensity or lifetime over the entire cell image. Based on this
regression curve, the target molecules on the cell surfaces could be quantified readily through the cell intensity and/or
lifetime at the single cell level instead of the direct count to the emission spots. As novel molecule imaging agents, these
metal fluorophores are being applied in the quantification and distribution of target molecule on the cell surface for the
clinical diagnostic research.
Plasmon-controlled fluorescence (PCF) creates new opportunities to dramatically improve the fluorescence detection
efficiency. We summarize recent works on single molecule studies on metal-fluorophore interactions and suggest how
these effects will result in new classes of experimental procedures, novel probes, bioassays and devices.
Creating a versatile set of highly stable fluorophores capable of high emission
rate is crucial to studies of the individual function of biomolecules. There are continuing
efforts to increase the sensitivity of fluorescence. These efforts include modifications in
the spectral properties of the probes, increasing the detection efficiencies of the
instruments, or the use of amplification methods. Our previous results show that
plasmonic-controlled fluorescence provides a novel physical mechanism to enhance
fluorescence intensity, reduce blinking and increase photostability. The further
development of fluorophore-metal interactions for single molecule detection requires
defined structures. For example, we investigate the effects of the defined silver
nanospheres fabricated by wet chemistry methods coupling to nearby organic
fluorophores. Additionally, we are developing nanoparticles incorporated into the
Quantum Dot (QD) system. Coupling between the plasmon resonance effect and the
quantum size effect of the QD or the organic fluorophore may develop new aspects of
nano-composite material systems and also widen applications for noble imaging probes.
Metal-enhanced fluorescence (MEF) is useful in single molecule detection (SMD) by increasing the photostability,
brightness and increase in radiative decay rates of fluorophores. We have investigated MEF from an individual
fluorophore tethered to a single silver nanoparticle and also a single fluorophore between a silver dimer. The
fluorescence lifetime results revealed a near-field interaction mechanism of fluorophore with the metal particle. Finite-difference
time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal
monomer and dimer. The coupling effect of metal particles on the fluorescence enhancement was studied. We have also
investigated the photophysics of FRET near metal nanoparticles and our preliminary results suggest an enhanced FRET
efficiency in the presence of a metal nanoparticle. In total, our results demonstrate improved detectability at the single
molecule level for a variety of fluorophores and quantum dots in proximity to the silver nanoparticles due to the near-field
Fluorescence is widely used in biological research. Future advances in biology and medicine often depend on the advances in the capabilities of fluorescence measurements. In this overview paper we describe how a combination of fluorescence, and plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology. This change will be based on the use of surface plasmons which are collective oscillations of
free electrons in metallic surfaces and particles. Surface plasmon resonance is now used to measure bioaffinity reactions. However, the uses of surface plasmons in biology are not limited to their optical absorption or extinction. We have shown that fluorophores in the excited state can create plasmons which radiate into the far field; additionally fluorophores in the ground state can interact with and be excited by surface plasmons. These 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 this technology as plasmon-controlled fluorescence. We predict that plasmon-controlled fluorescence (PCF) will result in a new generation of probes and devices. PCF is likely to allow design of structures which enhance emission at specific wavelengths and the creation of new devices which control and transport the energy from excited fluorophores in the form of plasmons, and then convert the plasmons back to light.