Holotomography-BasedQuantification of IntracellularViscosity Using Nanodiamonds Containing Nitrogen-Vacancy (NV) Centers

Abstract Intracellular viscosity is a fundamental mechanical property that reflects the physiological and pathological state of cells and plays a critical role in regulating molecular transport and organelle dynamics. Despite its importance, quantitative and noninvasive measurement of cytoplasmic viscosity in living cells remains challenging due to limitations in existing techniques, including phototoxicity, low throughput, and reliance on viscoelastic approximations instead of direct measurement of cytoplasmic viscosity. Here, we present a label-free approach for quantifying intracellular viscosity by integrating nanodiamond (ND)-based particle tracking with label-free refractive index-based holotomographic imaging. Polyvinylpyrrolidone (PVP)-coated nanodiamonds (PNDs) were spontaneously internalized by cells via endocytosis and visualized in real time using holotomography, enabling long-term, high-resolution tracking of individual intracellular PNDs without photobleaching or phototoxicity. Mean-squared displacement (MSD) analysis of PND trajectories was used to extract effective diffusion coefficients, from which effective intracellular viscosity was estimated using the Stokes–Einstein relationship. We validated this platform by comparing young and senescent cells, revealing a pronounced increase in intracellular viscosity in senescent cells, consistent with known aging-associated cytoplasmic alterations. Together, this work establishes a robust and noninvasive framework for quantitative effective intracellular viscosity mapping in living adherent cells, providing opportunities to study cellular aging, disease progression, and mechanobiological regulation at the subcellular level.