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 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.