Current high-content screening (HCS) techniques involve the analysis of cellular assays using high-resolution
imaging combined with sophisticated algorithms for automated image analysis. Commercially available platforms
are invariably highly specialised and expensive. Here we present a novel assay utilising changes in fluorescence
lifetime in the vicinity of a rough Au film. A mammary carcinoma cell line was created expressing EGFP in the
membrane, and cells were plated onto multi-well slides covered with a 30 nm Au film. FLIM images show a large
reduction in lifetime for membrane-bound GFP in close proximity to the Au surface. Addition of a suitable ligand
leads to internalization of the GFP with a corresponding increase in lifetime. The degree of internalization can
be very quickly and easily checked using standard lifetime analysis techniques, with no need for image analysis.
We demonstrate the method by comparing the efficacies of two small molecule inhibitors interfering with the
internalization process.
We report on the resonant coupling between localized surface plasmon resonances (LSPRs) in nanostructured Ag
films, and an adsorbed monolayer of Rhodamine 6G dye. Hybridization of the plasmons and molecular excitons
creates new coupled polaritonic modes, which have been tuned by varying the LSPR wavelength. The resulting
polariton dispersion curve shows an anticrossing behavior which is very well fit by a simple coupled-oscillator
Hamiltonian, giving a giant Rabi-splitting energy of ~400 meV. The strength of this coupling is shown to be
proportional to the square root of the molecular density. The Raman spectra of R6G on these films show an
enhancement of many orders of magnitude due to surface enhanced scattering mechanisms; we find a maximum
signal when a polariton mode lies in the middle of the Stokes shifted emission band.
We have investigated the effects of tuning the localized surface plasmon resonance (LSPR) of a silver film on
the extinction spectrum, Raman signal, and fluorescence intensity from nearby fluorophores. We observe the
formation of hybridized modes due to strong coupling between the plasmonic and molecular excitations. The
Raman spectra of R6G on these films show an enhancement of many orders of magnitude due to surface enhanced
scattering mechanisms; we find a maximum signal when a hybridized mode lies in the middle of the Stokes shifted
emission band. The effect of fluorophore-film separation on fluorescence intensity has been investigated using an
alumina spacer layer. An enhancement in detected signal of up to 18× is observed relative to that detected from
a bare Ag film. Overall, we observe a greater than 40× increase in detected intensity from the alumina-coated
Ag film relative to fluorophores on glass; this is a result of increased collection efficiency and a greater radiative
emission rate.
Highly ordered periodic arrays of silver nanoparticles have been fabricated which exhibit surface plasmon resonances
in the visible spectrum. We demonstrate the ability of these structures to alter the fluorescence properties
of vicinal dye molecules by providing an additional radiative decay channel. Using fluorescence lifetime imaging
microscopy, we have created high resolution spatial maps of the molecular lifetime components; these show
an order of magnitude increase in decay rate from a localized volume around the nanoparticles, resulting in a
commensurate enhancement in the fluorescence emission intensity. Spatial maps of the Raman scattering signal
from molecules on the nanoparticles shows an enhancement of more than 5 orders of magnitude.
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