Surface-enhanced resonance Raman scattering (SERRS) spectra of various rhodamine dyes, of pyronine G and thiopyronine adsorbed on isolated silver clusters were recorded at the ensemble level and at the single-molecule level with a high-resolution confocal laser microscope equipped with a spectrograph and a CCD-detector. Comparing single-molecule spectra with ensemble spectra, various inhomogeneous spectral features, such as line splitting, spectral wandering, spectral diffusion and abrupt spectral jumps between different metastable spectral states, are revealed positions and the relative intensities of the vibronic bands. Resonance enhancement is investigated with respect to single-molecule surface-enhanced Raman scattering (SERS) spectroscopy and is found to be responsible for approximately three orders of magnitude in sensitivity. A significant influence of the substituents on the single-molecule SERRS sensitivity is found, showing that various chemical effects are responsible for surface enhancement in addition to the electromagnetic enhancement effect.
We studied the emission of mutants of the red fluorescent protein DsRed by room temperature single molecule fluorescence spectroscopy. Bulk samples of the DsRed variant E8 show mixed green and red fluorescence of equivalent intensities individually spectrally similar to arrested green and mature red fluorescent forms of DsRed. Investigations at the single molecule level indicate that, like DsRed, E8 is not monomeric at single molecule concentrations. The entities visualized are composed of green and red emitting proteins without a fixed ratio of green to red fluorescing units. We find indications for only weak, if any, fluorescence resonance energy transfer (FRET) between red and green chromophores within one E8 entity.
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We report on the imaging of single dye molecules in thin polymer films under ambient conditions by means of scanning near-field optical microscopy. The long-term mechanical stability and high detection efficiency of our instrument allow the imaging of single dye molecules over several hours with a high signal-to-noise ratio. The local excitation by the near-field tip, the low excitation power and the fact that the dye molecules are embedded in the three dimensional polymer structure drastically reduce photodestruction as compared with conventional microscopy. The observation of many molecules in the same film allows to probe them under identical experimental conditions and to statistically analyze their global behavior. We find that the molecules diffuse and rotate within the polymer matrix. The single dye molecules act as nanometer-sized probes and reflect the local structure of the polymer.
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