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<p>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&#xa0;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.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月25日

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  • Hong Jiang,
  • Jinnawat Jongkhumkrong,
  • Y. J. Chao,
  • Qian Wang,
  • Guiren Wang

摘要

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.

Graphical Abstract