Precise Alternation Between Image-Forming Sample Planes Enables Quantitative Monitoring of Receptor-Arrestin...

Investigations of G protein-coupled receptors (GPCRs) interactions with non-visual arrestins in living cells are essential to understanding the complex molecular mechanisms of GPCR-based signaling. 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 alternatively 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 arrestin-2 toward the plasma membrane in both cellular cross-sections and at the basolateral membrane. This method enables direct comparison of receptor and arrestin dynamics across regions of individual cells with very high precision, eliminating the need for averaging over numerous cells in order to denoise biologically relevant signals, and thereby capturing physiological cell-to-cell variability.