Multiscale, Survey-and-Target Optical Super-Resolution Histology

Super-resolution microscopy reveals fine subcellular tissue architecture, yet extending this capability to histological screening requires balancing spatial resolution with imaging area, axial access, throughput, and instrumental complexity. Here we present multiscale optical super-resolution histology (MOSH), an integrated optical-computational platform that combines tissue-scale, extended-depth-of-focus super-resolution surveys with coordinate-guided three-dimensional interrogation of selected targets. MOSH leverages stationary multifocal excitation, continuous sample translation, tunable axial refocusing, and parallelized reconstruction within a shared spatial-coordinate framework in a conventional epi-fluorescence setting. This survey-and-target strategy preserves tissue-scale context, directs volumetric acquisition to selected subcellular structures, and generates virtual H&E images that retain resolution-enhanced structural information. Across cultured cells, cytological preparations, conventional tissue sections, and intact unsectioned biopsies, MOSH achieved approximately twofold resolution enhancement, continuous lateral coverage beyond the native field of view, and tunable axial survey ranges up to 20 μm. Bridging tissue-scale histological screening and targeted, quantitative nanoscale interrogation, MOSH establishes a scalable framework for multiscale analysis of biological and clinical specimens.