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

Abstract

Introduction: 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.

Objectives: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Bioelectrical marker; Flowcytometry; Microfluidic impedance; Microglia.