Time-Tagged Single Photon Counting Examination of Rotation of Receptor-Bound Quantum Dots

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Rotation of single membrane receptors can be observed by examination of polarized optical signals from receptor-bound asymmetric nanoparticles such as fluorescent quantum dots (QD) or nanogold. For example, we have examined the slow, hindered rotation of the Type I Fce receptor (FceRI) on 2H3 RBL cells using polarized fluorescence imaging of receptor-bound Qdot655. With imaging methods, only receptor rotational correlation times (RCT) slower than the camera frame time can be examined and suitable low-light cameras typically require 1ms or longer per frame. However, time-resolved phosphorescence anisotropy shows the hydrodynamic RCT of FcεRI to be about 40 μs at 25°C. To examine such rapid reorientation, an alternate approach is to illuminate individual QD on the cell surface with a focused laser beam, collect fluorescence using a confocal detector and direct signals polarized parallel and perpendicular to the laser polarization into separate APD detectors. A time-tagged single photon counter records the channel of each detected photon and its arrival time with a precision of 165 ps. The auto- and cross-correlations of the two signals are calculated directly from arrival times without binning, combined using adjustable constants such as the g-factor, and these constants optimized to obtain the maximum statistical independence between the anisotropy and intensity time-autocorrelation functions (TAC). While RCTs below 1 ns are theoretically accessible, useful rotational information exists only when RCT∗photon count rate ≥ 1. Thus, given photon count rates, our data potentially provide information on RCTs ≥ 20 μs. However, intensity changes due to QD blinking may feed through to some extent into calculated anisotropies and thus slightly distort apparent anisotropy TAC decay shapes. Efforts aimed at removing this possible complication are underway.