A high-throughput nanoscale imaging platform for direct visualization and rapid measurement of telomeres at single-molecule resolution

Telomere length is a biomarker of aging, cancer and telomeropathies, with some of the strongest health-related associations occurring at the extremes of the telomere-length spectrum. Critically short telomeres can drive chromosome instability, cellular stress and senescence, yet conventional assays largely provide population-average measurements that obscure these biologically important molecules, and the underlying heterogeneity of individual chromosome ends. In addition, amplification bias associated with highly repetitive telomeric sequences and inter-laboratory variability can limit measurement reproducibility. Although emerging technologies provide higher-resolution analyses, they often require high DNA input, specialized sample preparation, or complex instrumentation and bioinformatic tools, restricting broader adoption. Here, we present a high-throughput single-molecule platform that overcomes these barriers through an automated, user-friendly nanoscale imaging workflow compatible with standard genomic DNA preparations. The method combines a unique dual CRISPR-Cas9 telomere boundary labeling strategy with amplification-free magnetic-bead-based enrichment, preserving the native telomere-length distribution while minimizing representation bias. Using only 500 ng of input DNA and 2–4 h of hands-on processing, the assay accurately resolves intact telomeres spanning ~50 bp to >50 kb within the same sample, enabling simultaneous characterization of the full telomere-length spectrum. It also demonstrates high reproducibility across technical replicates and independent operators (ICC up to 0.90). Validation in telomere-length models, replicative aging systems, and clinical biospecimens, together with subtelomeric barcoding for chromosome-arm assignment, establishes a scalable platform for chromosome-resolved telomere profiling, biomarker development, and future clinical implementation in aging, cancer, and telomere biology.