Quantitative Analysisof Cytoplasmic Viscosity inColorectal Cancer Cells by Differential Dynamic Microscopy of GeneticallyEncoded Nanoparticles

Abstract The viscosity of the cytoplasm plays a key role in regulating molecular diffusion and cellular mechanics, yet quantifying it in living cells remains technically challenging. Genetically encoded multimeric nanoparticles (GEMs) have emerged as useful probes for intracellular microrheology; however, current analyses rely on single-particle tracking, which is limited by probe density, imaging noise, and expression variability. Here, we combine GEMs with differential dynamic microscopy (DDM) to enable quantitative, noninvasive, and rapid measurement of intracellular viscosity using standard wide-field fluorescence imaging. DDM extracts particle dynamics from ensemble spatiotemporal intensity fluctuations, yielding diffusion coefficients and viscosity estimates, even in crowded or heterogeneous environments where tracking fails. Validation with fluorescent nanoparticles diffusing in water confirmed that DDM accurately reproduced theoretical viscosities across a wide range of particle sizes and concentrations. Comparison with single-particle tracking (SPT) demonstrated equivalent precision under dilute conditions and improved the performance under crowding. To showcase the potential of this approach, we applied GEM-DDM to colorectal cancer cell lines with different metastatic potentials. Cytoplasmic viscosity correlated with aggressiveness, increasing from 1.9 to 2.3 cP in poorly metastatic lines to 3.6–3.7 cP in highly metastatic lines, consistent with greater macromolecular crowding and cytoplasmic reorganization reported in aggressive cells. Together, these results establish GEM-DDM as a fast, reproducible, and accessible platform for intracellular microrheology to link the physical state of the cytoplasm to cell function and disease progression.