Fluorescence lifetime, anisotropy and intensity dependent single molecule fluorescence correlation spectroscopy (I-FCS) are used to investigate the mechanism of fluorescence saturation in a free and nucleotide bound fluorophore (NR6104) in an antioxidising ascorbate buffer. Nucleotide attachment does not appreciably affect the fluorescence lifetime of the probe and there is a decrease in the rate of intersystem crossing relative to that of triplet state deactivation. The triplet state fraction is seen to plateau at 72% (G-attached) and 80% (free fluorophore) in agreement with these observations. Measurements of translational diffusion times show no intensity dependence for excitation intensities between 1 and 10⁵kW cm^-2 and photobleaching is therefore negligible. The dominant mechanism of fluorescence saturation is thus triplet state formation. I-FCS measurements for Rhodamine 6G in water were compared with those in the ascorbate buffer. In water the triplet fraction was saturated at considerably higher powers (45% at ca. 1.5 × 10³kW cm^-2) than in the ascorbate buffer (55%ca. 1 1kW cm^-2)

Date Published: 1 September 2009
PDF: 12 pages
Proc. SPIE 7393, Nanophotonic Materials VI, 73930L (1 September 2009); doi: 10.1117/12.826043

Published in SPIE Proceedings Vol. 7393:
Nanophotonic Materials VI
Stefano Cabrini; Taleb Mokari, Editor(s)