Aggregation and fast diffusion of dye molecules on air-glycerol interface observed by confocal fluorescence microscopy

Diffusion processes of Rhodamine 6G (Rh6G) dye molecules dissolved in a small hemisphere drop of glycerol on a cover glass were investigated by using a confocal fluorescence microscope equipped with an objective lens with a high numerical aperture (NA = 1.35). Photon burst signals from Rh6G molecules in the bulk glycerol and on the air-glycerol interface of the hemisphere drop were separately detected at a single molecule level. The analysis of the photon burst signals by a correlation function method reveals that a sizable portion of the Rh6G molecules in the drop are aggregated on the air-glycerol interface and diffuse two-dimensionally on it, while the rest diffuse molecularly in the bulk. The aggregates are found to have a diffusion constant 15 times as large as that of the Rh6G molecule in the bulk glycerol, although the aggregates have a hydrodynamical radius much larger than that of a Rh6G molecule.