Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (FcεRI) is extensively cross-linked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids (the lipid analog diI-C18 or glycosphingolipids)) and lipid-anchored proteins coredistribute with cross-linked IgE-FcεRI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C18 and Alexa 488-labeled IgE-FcεRI in the domains where these molecules colocalize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.
Bibliographical noteFunding Information:
This work was supported, in part, by The Pennsylvania State University (E.D.S. and A.A.H.), the Penn State Materials Research Institute (E.D.S. and A.A.H.), the Penn State Materials Research Science and Engineering Center (under National Science Foundation grant DMR 0213623) (E.D.S. and A.A.H.), and the Lehigh-Penn State Center for Optical Technologies (E.D.S. and A.A.H.), which is supported by the Commonwealth of Pennsylvania. Acknowledgment is also made to the Donors of the American Chemical Society Petroleum Research Fund (E.D.S.) and to the Johnson & Johnson/Penn State Innovative Technology Research Seed Grant (A.A.H.) for partial support of this research. A.A.H. is also grateful for the generosity of Coherent Lasers for the loan of a pulse picker (Mira 9200, Coherent).