Real-Time Investigation of Plasma Membrane Deformation and Fusion Pore Expansion Using Polarized Total Internal Reflection Fluorescence Microscopy

Abstract

Polarized Total Internal Reflection Fluorescence Microscopy (pTIRFM) allows for real-time observation of plasma membrane deformations. The technique provides insights into the dynamics of biological processes requiring rapid and localized changes in membrane shape. Such processes include exocytosis, endocytosis, cytokinesis, and cell motility. In this chapter, we describe how to implement a polarization-based TIRF imaging system to monitor exocytosis in adrenal chromaffin cells.

A primer on resolving the nanoscale structure of the plasma membrane with light and electron microscopy

“…Thus, the orientation of those fluorophores can be mapped by tracking the resulting emission generated by two oppositely polarized fields (Zenisek et al, 2002;Taraska and Almers, 2004). Recently, this method has gained traction to image the behavior of exocytic and endocytic vesicles at the plasma membrane (Anantharam et al, 2010(Anantharam et al, , 2011Passmore et al, 2014;Scott et al, 2018). For example, the realtime shape of exocytic vesicles has been monitored when dense core vesicles fuse with the membrane in chromaffin cells and when clathrin-coated pits curve (Anantharam et al, 2010(Anantharam et al, , 2011Scott et al, 2018).…”