Imaging Subresolution Membrane Curvature in Living Cells by Back Focal Plane Positioning Polarized Total Internal Reflection Microscopy (TIRFM)

Abstract

Induction of membrane curvature is an essential step in the formation of endocytic vesicles and viral particles. Axelrod and co-workers have shown that polarized total internal reflection fluorescence microscopy (pol-TIRFM) can be used to visualize membrane curvature in cells labeled with the lipophilic fluorophore DiI. We describe an approach for creating s-pol and p-pol TIRF fields using a commercial microscope system equipped with a 2-D scan head to position polarized laser beams at distinct azimuthal positions in the back focal plane (BFP) of a high numerical aperture TIRF lens. This configuration allows for reduction of interference fringing of the coherent light by alternating illumination at azimuthal positions separated by 180 degrees. Additional reduction of fringing could be obtained by sweeping the laser through small arcs in the BFP of about 8 degrees with minimal impact on the polarization of the TIRF fields. We investigated the performance of this pol-TIRFM approach for visualization of endocytosis and viral budding in cells labeled with DiI and either the endocytic marker clathrin-YFP or viral budding marker HIV Gag-YFP.