Bimodal Cholesterol for Correlative In-Cell DNP Solid-State NMR and Confocal Microscopy of the Plasma Membrane

Cholesterol plays a pivotal role in defining the structure and signaling function of plasma membrane lipid rafts, yet direct nanoscale measurements of cholesterol-rich domains in intact cells that enable atomic scale analysis remains challenging. Here, we present AsymPol-Chol-AF647, a bimodal cholesterol-based probe that integrates a biradical (AsymPol) for Dynamic Nuclear Polarization (DNP) with a fluorescent dye (AF647), enabling correlative in-cell DNP solid-state NMR and confocal fluorescence microscopy of the plasma membrane. The cholesterol anchor ensures insertion into the plasma membrane, providing a spatially defined delivery of the polarizing agent. Confocal imaging in live cells confirms plasma membrane localization and reveals concentration-dependent perturbations to lipid raft integrity, with optimal conditions at 0.19 nmol/10⁶ cells maintaining cell viability and discrete GM1-positive microdomains (associated with lipid rafts). Under these conditions, significant global enhancements of natural abundance ¹³C of ε ≈ 14 were achieved. Analysis of signal buildup kinetics unveiled evidence for more complex hyperpolarization dynamics than the simple spherical model indicated by the microscopy. These findings lay the groundwork for future studies utilizing bimodal polarizing agents for use not just for selective cellular enhancements but also as tools for the determination of the spatial organization of cellular structures at nanometer resolution.