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.
Yang et al. (Sun,) studied this question.