Imaging Membrane Curvature inside a FcεRI-Centric Synapse in RBL-2H3 Cells Using TIRF Microscopy with Polarized Excitation

Rosa Machado; Justin Bendesky; Madison Brown; Kathrin Spendier; Guy M Hagen

doi:10.3390/jimaging5070063

Imaging Membrane Curvature inside a FcεRI-Centric Synapse in RBL-2H3 Cells Using TIRF Microscopy with Polarized Excitation

J Imaging. 2019 Jul;5(7):63. doi: 10.3390/jimaging5070063. Epub 2019 Jul 4.

Authors

Rosa Machado¹, Justin Bendesky¹, Madison Brown^{1

2}, Kathrin Spendier^{1

2}, Guy M Hagen¹

Affiliations

¹ UCCS Center for the Biofrontiers Institute, University of Colorado at Colorado Springs, Colorado Springs, CO 80918, USA.
² Department of Physics and Energy Science, University of Colorado at Colorado Springs, Colorado Springs, CO 80918, USA.

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.

Keywords: FcεRI; IgE receptor; P-TIRF; RBL-2H3; plasma membrane; rat basophilic leukemia cells; supported lipid bilayer; total internal reflection fluorescence microscopy.

Grants and funding

R15 GM128166/GM/NIGMS NIH HHS/United States