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

## TL;DR

A new optical method called FIFIV allows real-time measurement of water flow through aquaporins in single cells, offering insights into their function and regulation.

## Contribution

The paper introduces FIFIV, a novel optical technique for measuring transmembrane water flow in single cells with high spatiotemporal resolution.

## Key findings

- FIFIV detects transmembrane water flow in single MDA-MB-231 breast cancer cells at speeds of 1 μm/s.
- The method distinguishes between osmotic gradients using fluorescence signal peaks.
- FIFIV achieves single-cell sensitivity and temporal resolution comparable to electrophysiological methods.

## Abstract

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.

A 1X PBS buffer droplet (∆c = 0) and DI water droplet (∆c≠0) is injected extracellularly on the left side of an adherent cell respectively. For ∆c = 0, fluorescence intensity decays exponentially. For ∆c≠0, however, there is a peak in the signal.

A 1X PBS buffer droplet (∆c = 0) and DI water droplet (∆c≠0) is injected extracellularly on the left side of an adherent cell respectively. For ∆c = 0, fluorescence intensity decays exponentially. For ∆c≠0, however, there is a peak in the signal.