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.
Directional fluorescence emission of a sulforhodamine 101 in polyvinyl alcohol film has been observed from samples deposited on semi-transparent silver mirror. The fully p-polarized fluorescence emerges through the glass prism in form of hollow cone. The angle of this cone of emission depends on the thickness of the sample, and does not depend on the mode of excitation. The angular dependence of surface plasmon-coupled emission (SPCE) on the sample thickness has been discussed as well as its relevance to the surface plasmon resonance (SPR) analysis.
Measurements of time-dependent photon migration appear to provide more information for biomedical optical image reconstruction than continuous wave measurements. Yet the ultimate success of photon migration imaging (PMI) for biomedical optical tomography depends upon developing a method which can rapidly measure `time-of-flight' information, and, in near-real time, extract important information required for image reconstruction. Image reconstruction requires information to (1) detect, (2) locate the position and volume, and (3) characterize the optical properties of an optical heterogeneity that would otherwise be obscured by tissue-like scattering. In this presentation, we report PMI `images' of an obscured absorber obtained from two-dimensional time-dependent photon migration measurements which arise from single point source illumination of a scattering medium with modulated light. These PMI `images' along with a theoretical basis for PMI, suggest the potential to rapidly detect and locate the three- dimensional position of an absorber from two-dimensional frequency-domain measurements of phase, (Theta) ((rho) ,f), and modulation, M((rho) ,f). Independent single-pixel measurements and Monte Carlo simulations of (Theta) ((rho) ,f) and M((rho) ,f) confirm the PMI `images' and the hypothesis for PMI.
Fluorescence lifetime imaging (FLIM) is a new methodology in which the image contrast is derived from the fluorescence lifetime, not the local concentration and/or intensity of the fluorophore, at each point in a two-dimensional image. In our apparatus, the lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier. The phase-sensitive images obtained with various phase shifts of the gain- modulation signal are used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. Pixel-to-pixel scanning is not required to obtain the images. As an example of biochemical imaging we created lifetime images of the calcium concentration based on Ca<SUP>2+</SUP>-induced lifetime changes of calcium green (CaG), which is shown to be highly sensitive to [Ca<SUP>2+</SUP>]. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which allows Ca<SUP>2+</SUP> imaging using Ca<SUP>2+</SUP> probes which do not display spectral shifts. The concept of fluorescence lifetime imaging has numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence, the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.