Imaging membrane curvature inside a FcϵRI-centric synapse in RBL-2H3 cells using TIRF microscopy with polarized excitation

Abstract

Total internal reflection fluorescence microscopy with polarized excitation (P-TIRF) can be used to image nanoscale curvature phenomena in live cells. We used P-TIRF to visualize rat basophilic leukemia cells (RBL-2H3 cells) primed with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) coming into contact with a supported lipid bilayer containing mobile, monovalent DNP, modeling an immunological synapse. The spatial relationship of the IgE-bound high affinity IgE receptor (FcϵRI) to the ratio image of P-polarized excitation and S-polarized excitation was analyzed. These studies help correlate the dynamics of cell surface molecules with the mechanical properties of the plasma membrane during synapse formation.