BPS2026 – High-fold homogeneous expansion microscopy for ultrastructural imaging of mammalian centrioles

Mapping cellular ultrastructure demands methods that combine nanometer resolution with faithful preservation. Expansion microscopy (ExM), which enlarges specimens before imaging, provides an optics-independent route to super-resolution. While recent gel chemistries have extended expansion factors from 4× to nearly 20×, high-fold ExM has rarely been validated for ultrastructural accuracy, limiting its use for detailed organelle analysis. Here, we present high-fold homogeneous expansion microscopy (hiHomoExM), a one-step strategy achieving uniform ∼8-9× expansion while preserving centriolar architecture. hiHomoEx facilitates sample handling, supports post-expansion labeling, and ensures high labeling density. Quantitative benchmarks confirm homogeneous expansion across macro- and nanoscale levels, validating its reliability for structural biology. To push resolution further, we integrate hiHomoEx with single-molecule localization microscopy (hiHomoEx-dSTORM). This combination reaches ∼2 nm effective resolution and reveals ultrastructural features previously inaccessible, including CEP44 periodicity, CCDC77 microtubule linkages, and the canonical nine-fold symmetry of SAS6. Together, hiHomoExM and hiHomoEx-dSTORM establish a robust and accessible platform for high-fidelity ultrastructural analysis, uniting physical and optical super-resolution to map cellular architecture at molecular precision.