Precise Alternation between Image-Forming Sample Planes Enables Quantitative Monitoring of Receptor-Arrestin Interaction Dynamics at the Plasma Membrane of Live Cells

Quantitative analysis of these interactions remains challenging in live cells, particularly when attempting to repeatedly image distinct cellular regions with high precision. Here, we describe the implementation of an optical imaging stabilization approach that integrates the recently developed Focal Readjustment for Enhanced Vertical Resolution (FREVR) technology into a multiphoton microscope, enabling high-precision alternation between image-forming sample planes with <20 nanometer repeatability and stability over time.

Using this setup, we monitored the dynamic recruitment of arrestin-2 (Arr2) to the plasma membrane of HEK-293 cells expressing muscarinic acetylcholine M₂ receptors (M₂R) by alternately imaging distinct planes of interest, the basolateral membrane and a membrane cross-section. Following stimulation of M₂R by agonist ligand, we observed a pronounced redistribution of cytoplasmic Arr2 toward the plasma membrane in both cellular cross sections and at the basolateral membrane.