Protocol for microfluidic-based high-precision general polarization fluorescence microscopy of lipid packing in membrane vesicles

Lipid packing plays a critical role in membrane organization and function, but its precise quantification remains challenging in fluidic environments. Here, we present a protocol to integrate confocal fluorescence generalized polarization (GP) imaging with a polydimethylsiloxane (PDMS)-based microfluidic platform. We describe steps for lipid preparation, giant unilamellar vesicle (GUV) fabrication, microfluidic trapping, and GP analysis using polarity-sensitive dyes. This protocol enables robust measurement of membrane order and is readily adaptable to diverse lipid compositions and aqueous environments. For complete details on the use and execution of this protocol, please refer to Zhao et al. 1 , 2 • Steps for microfluidic device fabrication and assembly • Procedures for giant unilamellar vesicle (GUV) production and entrapment • Instructions for confocal general polarization (GP) imaging and lipid packing assessment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Lipid packing plays a critical role in membrane organization and function, but its precise quantification remains challenging in fluidic environments. Here, we present a protocol to integrate confocal fluorescence generalized polarization (GP) imaging with a polydimethylsiloxane (PDMS)-based microfluidic platform. We describe steps for lipid preparation, giant unilamellar vesicle (GUV) fabrication, microfluidic trapping, and GP analysis using polarity-sensitive dyes. This protocol enables robust measurement of membrane order and is readily adaptable to diverse lipid compositions and aqueous environments.