Intraoperative pathology remains constrained by ice crystal artifacts in frozen sections and the high cost of emerging slide-free optical methods. Here, we introduce FLASH-Path, a rapid slide-free technique enabling subcellular-resolution imaging of centimeter-scale tissues in 10 minutes. By replacing mechanical thin sectioning or optical thin-layer excitation with thin-layer (≤10 µm) fluorescent labeling using commercially available probes, FLASH-Path achieves artifact-free visualization of diverse tissues (e.g., fat, lymph nodes) incompatible with conventional frozen sections. The method integrates with retrofitted clinical fluorescence microscopes or manual observation, ensuring adaptability across resource settings. Fluorescence images are computationally transformed into H&E-like histopathology without ice crystal artifacts or hidden risks from generated images. In colorectal cancer validation, FLASH-Path outperformed frozen sections in speed and imaging area. FLASH-Path enhances clinical accessibility, image traceability, and cost-effectiveness, providing new opportunities for the clinical application and dissemination of slide-free pathology.

There are three patents pending for inventions related to slide-free sample processing, slide-free optical imaging systems, and color conversion, respectively.

National Natural Science Foundation of China (82260368), National Key Research and Development Program of China (2022YFC3400601), Innovational Fund for Scientific and Technological Personnel of Hainan Province (KJRC2023C03), Hainan Province Science and Technology Special Fund (ZDYF2022SHFZ126) and Start-up Fund from Hainan University (KYQD(ZR)-20077).

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Ethics committee of Nanchong Central Hospital gave ethical approval for this work.

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