Quantitative Analysis of Cytoplasmic Viscosity in Colorectal Cancer Cells by Differential Dynamic Microscopy of Genetically Encoded Nanoparticles

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 powerful 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, non-invasive, and rapid measurement of intracellular viscosity using standard wide-field fluorescence imaging. DDM extracts particle dynamics from ensemble spatiotemporal intensity fluctuations, yielding reliable diffusion coefficients and viscosity values 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 superior robustness 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 1.9-2.3 cP in poorly metastatic 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, providing new opportunities to link the physical state of the cytoplasm to cell function and disease progression. Statement of significancePhysical properties such as cytoplasmic viscosity influence how molecules move and interact within cells, affecting metabolism, signalling, and disease progression. Measuring viscosity in living cells has been technically challenging and often invasive. Here, we introduce GEM-DDM as a quantitative, non-invasive image-based analysis method combining genetically encoded multimeric nanoparticles (GEMs) with differential dynamic microscopy (DDM) to measure intracellular viscosity using standard wide-field microscopy. We validate its accuracy against established single particle tracking (SPT) methods and demonstrate its biological relevance by showing that cytoplasmic viscosity increases with metastatic potential in cancer cells. This approach provides an accessible platform for studying how the physical state of cells influences their function and pathology.