Full-Spectrum Spectroscopic Characterization of Extracellular pH Effects on FRET-Based Genetic Voltage Indicators Expressed in Xenopus laevis Oocytes

Genetically encoded voltage indicators convert voltage-dependent conformational transitions of membrane proteins into optical signals. pH sensitivity is a critical yet often under-characterized property and becomes particularly important when extending imaging to organellar membranes. This chapter presents a step-by-step protocol for full-spectrum spectroscopic analysis of extracellular pH effects on FRET-based voltage indicators expressed in Xenopus laevis oocytes, beginning with assembly and alignment of the measurement rig. Oocytes enable real-time, full-spectrum spectroscopic measurements within a single cell under stable electrophysiological control, providing well-defined mechanistic characterization under conditions difficult to achieve in other systems.