We have implemented a new laser microscopic method, polarized fluorescence depletion (PFD), for measuring the rotational dynamics of functional membrane proteins on individual, microscopically selected cells under physiological conditions. This method combines the long lifetimes of triplet-state probes with the sensitivity of fluorescence detection to measure macromolecular rotational correlation times from 10μsec to > 1 ms. As examples, the rotational correlation time of Fc{sub}ε receptors (Fc{sub}εR) on the surface of 2H3 rat basophilic leukemia cells is 79.9±4.4μsec at 4°C when labeled with eosin conjugates of IgE. This value is consistent with the known 100 kDa receptor size. When labeled with intact F4 anti-Fc{sub}εR monoclonal antibody, the rotational correlation time for Fc{sub}εR is increased about 2-fold to 170.8±6.5μsec, consistent with receptor dimer formation on the plasma membrane and with the ability of this antibody to form Fc{sub}εR dimers on 2H3 cell surfaces. We have also examined the rotational diffusion of the luteinizing hormone receptor on plasma membranes of small ovine luteal cells. Luteinizing hormone receptors (LHR), when occupied by ovine luteinizing hormone (oLH), have a rotational correlation time of 20.5±0.1μsec at 4°C. When occupied by human chorionic gonadotropin (hCG), LHR have a rotational correlation time of 46.2±0.4μsec suggesting that binding of hCG triggers additional LHR interactions with plasma membrane proteins. Together these studies suggest the utility of PFD measurements in assessing molecular size and molecular association of membrane proteins on individual cells. Relative advantages of time- and frequency-domain implementations of PFD are also discussed.
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