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Single terrylenediimide molecules diluted in a 20-nm-thick polyvinylbutyral polymer film were localized and observed by scanning confocal fluorescence microscopy. A modular and compact confocal microscope and the high optical stability of the molecules allowed a repeated imaging and observation over >5 h at room temperature. Most of the molecules showed several “on-off-on” transitions (blinking) on a time scale from seconds to hours, before permanent bleaching occurred. We determined that >1.5 × 10^7 fluorescence photons are emitted from the most-stable molecules before the final bleaching step occurs. Despite the “on-off-on” transitions, however, the overall change in fluorescence intensity, either integrated over each image of a time series or summed for several individual molecules, resembled an exponential-like decay, familiar from measurements of many-molecule ensembles. We also observed the polarization of the fluorescence from single molecules during excitation with circular polarized light. From these measurements, possible rotations of the molecular dipoles were studied. Over a span of 5 h, the polarization angle in most cases did not change by >15-20°. This may explain the slow and small intensity changes but excludes molecular rotation as a reason for the blinking behavior

Fluorescence Blinking and Photobleaching of Single Terrylenediimide Molecules Studied with a Confocal Microscope