Dynamics of single dye molecules observed by confocal imaging and spectroscopy

The fluorescence emission of single rhodamine dye molecules (rhodamine 6G a nd rhodamine 630) at room temperature was analyzed by using scanning confoc al laser microscopy in conjunction with polarization analysis, fluorescence spectroscopy, time-resolved detection (minutes to microseconds), and excit ation saturation. Results are presented and discussed 1) for samples with d ye molecules at the glass-air interface and 2) covered with an additional t hin protective polymer film (polyvinylbutyral). Under the polymer layer, th e single-molecule fluorescence was more stable than the glass-air interface . This result may be explained by fewer spontaneous variations of the fluor escence rate, polarization changes, spectral shifts, and longer photochemic al lifetimes. Cytometry 36:217-223, 1999, (C) 1999 Wiley-Liss, Inc.