A new phosphorus-rhodamine voltage-sensing dye for optical membrane potential imaging

Accurate measurement of membrane potential dynamics is essential for understanding cellular excitability and signaling. While electrophysiological methods provide high temporal resolution, their invasiveness and low throughput limit their applicability in complex biological systems. Voltage-sensing dyes (VSDs) offer a powerful optical alternative, yet achieving near-infrared (NIR) emission, high sensitivity, and rapid response remains challenging. Here, we report the design, synthesis, and characterization of a novel phosphorus-rhodamine-based VSD (VSD 1) incorporating a phenyl substituent on the phosphorus atom. This modification enhances σ*-π* conjugation and electron-withdrawing effects, leading to a pronounced bathochromic shift with absorption and emission maxima at 715 and 744 nm, respectively-the most red-shifted spectral profile among reported rhodamine VSDs. VSD 1 shows strong fluorescence quenching in the resting state, suggesting efficient nonradiative deactivation, although the exact mechanism was not experimentally determined. Voltage-clamp fluorometry in Xenopus laevis oocytes demonstrates robust voltage sensitivity, with a linear fluorescence-voltage relationship and a ΔF/F of 4.7±1.4% per 100 mV in the physiologically relevant range from - 100 to 50 mV. Notably, the dye responds to membrane potential changes on the microsecond timescale, enabling faithful tracking of action potentials in cardiac and neuronal models. These properties establish VSD 1 as a promising next-generation optical probe for high-speed, minimally invasive imaging of membrane potential in excitable cells and complex tissues.