Orientation imaging and reorientation dynamics of single dye molecules

We introduce new techniques for obtaining single-molecule orientation images and analyzing the reorientation dynamics of single fluorescent molecules. Polarization modulation is used with confocal microscopy to measure the absorption dipole orientation and reorientation dynamics of individual dye molecules physisorbed to glass and embedded in thin, spin-cast polymer films under ambient conditions. Discrete jumps in absorption dipole orientation are observed for a significant fraction of dye molecules in all samples tested, and in all cases the distribution of first-jump times can be fitted by a stretched exponential function. A sub-population of dye molecules that is stationary on the time scale of these experiments (32 s) is observed and persists even at high excitation power. Data analysis techniques are developed for quantifying the reorientation dynamics of single molecules, and a novel method for quickly identifying rotationally mobile molecules in a scan field is introduced. A dependence of the reorientation dynamics on film thickness is identified: DiIC(18) molecules reorient with higher frequency and a broader distribution of jump rates in progressively thinner polymer films.