Slide-Free Histopathology via Fluorescent Layer-Adaptive Subcellular Imaging for Less Artifacts and Faster Intraoperative Diagnosis

Intraoperative pathology is limited by freezing artifacts, complex workflows, and cost in conventional frozen-section procedures. This study introduces Fluorescent Layer-adaptive Artifact-free Subcellular Histopathology (FLASH-Path), a rapid, slide-free histopathological technique that enables subcellular-resolution imaging of centimeter-scale specimens within 10 min. The core technologies mainly include (1) controlling the slow-release of dual-targeted dye molecules in hyaluronic acid solution to achieve thin-layer staining; (2) real-time depth-of-field expansion imaging and large-area image scanning; and (3) conversion of fluorescent images into a hematoxylin and eosin staining appearance without loss of image details. The imaging results of colorectal samples demonstrated that the images obtained by FLASH-Path exhibited no freezing artifacts, and the image quality was equivalent to that of traditional paraffin sections, which makes FLASH-Path more conducive to pathological analysis, such as nucleus-to-cytoplasm ratio calculation. The FLASH-Path technology offers superior clinical accessibility, diagnostic traceability, and cost-effectiveness, making it highly promising for emergency applications, particularly in low-resource environments.