Rapid Single-cell Measurement of Transient Transmembrane Water Flow under Osmotic Gradient

Aquaporins (AQPs) are critical for transmembrane water transport in response to osmotic gradients, but their gating and regulatory mechanisms remain poorly understood. A central challenge is the lack of methods to measure water flow across AQPs from individual cells with the spatiotemporal resolution and sensitivity equivalent to patch-clamp recordings of ion fluxes-a limitation stemming from the electrically silent nature of water flow. Here, we present a novel optical technique-Flow-Induced Fluorescence Increase Velocimetry (FIFIV) based on Laser-Induced Fluorescence Photobleaching Anemometry (LIFPA)-that enables direct, real-time monitoring of cytoplasmic flow induced by transmembrane water transport under osmotic pressure gradients. Using small molecular fluorescent dyes to label cytoplasm in single adherent MDA-MB-231 breast cancer cells, we show detection of instantaneous extremely low transmembrane water flow signals on the order of 1 μm/s following localized hypotonic stimulation. This approach circumvents the limitations of traditional volume-based osmotic permeability assays, achieving single-cell sensitivity and temporal resolution comparable to electrophysiological measurements of ion flux. FIFIV potentially provides a new optical approach for probing transmembrane water-flow-induced intracellular dynamics, and a foundation for future investigations of AQP regulation and gating mechanisms in physiopathological studies and drug discovery.