High-throughput phenotyping of microglial membrane Capacitance: Linking label-free dielectric signatures to polarization continuum and phospholipid remodeling

• A high-throughput single-cell dielectric phenotyping (54 cells/s) method positions C sm as a label-free metric for microglial polarization states. • Quantifies C sm as a continuous polarization metric, overcoming M1/M2 dichotomy. • PPARγ/SIRT1 activators reversibly modulate C sm , demonstrating dynamic polarization control. • Elucidates membrane lipid remodeling as the biophysical basis for dielectric biomarker functionality. The dynamic monitoring of microglial polarization remains constrained by the static M1/M2 dichotomy and a lack of robust biomarkers, limiting therapeutic development for neurological disorders. Recent advances in bioelectrical characterization, however, have revealed that cellular processes correlate with distinct dielectric properties, suggesting a potential new approach for label-free cellular analysis. This study aimed to establish specific membrane capacitance (C sm ) as a label-free, continuous metric for microglial polarization states and to elucidate the underlying biophysical mechanisms. We employed high-throughput single-cell dielectric phenotyping via a microfluidic impedance cytometry platform (54 cells/s) to characterize the ability of C sm to capture the continuous spectrum between M1/M2 phenotypes. Complementary lipidomic and proteomic analyses, alongside pharmacological interventions, were used to investigate the molecular basis of dielectric changes. We demonstrate that M1-polarized microglia exhibit a significantly elevated C sm compared to M2 or resting (M0) states. This shift of C sm was continuous and dose-dependent to polarizing stimuli and was mechanistically linked to membrane lipid remodeling, specifically an increased lysophosphatidylcholine/phosphatidylcholine ratio (LPC/PC) regulated by Pla2g4a/Lpcat1. Furthermore, PPARγ/SIRT1 activators reversibly modulated these continuous changes of dielectric signatures. Pharmacological validation confirmed C sm ’s sensitivity to membrane reorganization. Our results establish C sm as a real-time, single-cell functional metric for the continuum of microglial polarization, directly linking biophysical measurements to subcellular biochemistry. This work identifies C sm as a promising screening tool for neuroimmunomodulators and a predictive biomarker for therapeutic response, providing a scalable platform for neuroinflammation research.