Imaging intracellular diffusion in a crowded cytoplasm

    Imaging intracellular diffusion in a crowded cytoplasm

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    Understanding how molecules move inside cells is a central question in cell biology, as diffusion underpins essential processes such as signaling, metabolism and intracellular transport. Yet, the cytoplasm is far from a simple fluid: it is a densely packed and highly structured environment filled with organelles, membranes and macromolecular assemblies.

    In this study(1), researchers from Institut Jacques Monod and CentraleSupélec combine advanced microscopy techniques with biophysical modelling to investigate how this complex architecture impacts molecular mobility.

    By linking quantitative imaging with theoretical approaches, they reveal that intracellular diffusion is strongly shaped by cytoplasmic organization.

    Seeing and quantifying cytoplasmic organization

    Using confocal microscopy, the researchers examined the spatial organization of the cytoplasm in living cells and identified regions with different levels of accessibility. By using freely diffusing GFP as a probe, they estimated the fraction of accessible volume and showed that the cytoplasm is heterogeneous at the subcellular scale.

    These measurements revealed variations in crowding associated with the local distribution of intracellular structures, providing a quantitative description of the physical environment in which diffusion occurs.

    Figure 1. Characterization of MDCK intracellular architecture using optical microscopy. The cytoplasm features heterogeneous regions with different obstacle density, akin to porous media. (C) Confocal image of free-GFP fluorescence, which is highly heterogeneous in the cell. (H) Zoom on the white rectangle present in C. Large dark spheroids are present (left). Their volume is estimated by image segmentation (right). (1)

    Measuring diffusion across scales with FRAP and FCS

    Diffusion was quantified by combining Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), which probe complementary spatial and temporal scales.

    The measurements show that GFP diffusion in the cytoplasm is significantly reduced compared to aqueous conditions and varies depending on local crowding. In particular, regions with higher obstacle density exhibit lower diffusion coefficients.

    The data further indicate that nanoscale structures are the main contributors to these variations, while larger structures have a more limited and stable effect under the conditions tested.

    From imaging to predictive models of the cytoplasm

    By integrating experimental measurements with theoretical modelling, the study shows that the cytoplasm can be described as a porous medium. In this framework, nanoscale obstacles occupy a large fraction of the available space and largely determine the effective diffusion of macromolecules.

    Figure 4. Presentation of the multiscale model for the cytoplasmic diffusion of free-GFP. Nano-obstacles result in tortuous and porous hydrodynamic hindrances that can strongly reduce the diffusivity of particles with GFP size. (A) Schematic of the cytoplasm decomposition into three scales.(1)

    The model reproduces the experimental observations and provides a way to relate structural parameters to diffusion coefficients. It also enables predictions for other conditions, suggesting potential applications for studying intracellular transport of biomolecules, including small therapeutic compounds.

    All microscopy experiments were conducted at the ImagoSeine facility (France-BioImaging Paris-Centre node).

    (1) Destrian O, Moisan N, Mège RM, Ladoux B, Goyeau B, Chabanon M. Cytoplasmic crowding acts as a porous medium reducing macromolecule diffusion. Proc Natl Acad Sci U S A. 2026 Jan 27;123(4):e2519599123. doi: 10.1073/pnas.2519599123. Epub 2026 Jan 23. PMID: 41576074

    • April 3, 2026

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