Rapid and high‐resolution histological imaging with minimal tissue preparation has long been a challenging and yet captivating medical pursuit. Here, the authors propose a promising and transformative histological imaging method, termed computational high‐throughput autofluorescence microscopy by pattern illumination (CHAMP). With the assistance of computational microscopy, CHAMP enables high‐throughput and label‐free imaging of thick and unprocessed tissues with large surface irregularity at an acquisition speed of 10 mm2/10 s with 1.1‐µm lateral resolution. Moreover, the CHAMP image can be transformed into a virtually stained histological image (Deep‐CHAMP) through unsupervised learning within 15 s, where significant cellular features are quantitatively extracted with high accuracy. The versatility of CHAMP is experimentally demonstrated using mouse brain/kidney and human lung tissues prepared with various clinical protocols, which enables a rapid and accurate intraoperative/postoperative pathological examination without tissue processing or staining, demonstrating its great potential as an assistive imaging platform for surgeons and pathologists to provide optimal adjuvant treatment.

Histopathological examination of tissue sections is the gold standard for disease diagnosis. However, the conventional histopathology workflow requires lengthy and laborious sample preparation to obtain thin tissue slices, causing about a one-week delay to generate an accurate diagnostic report. Recently, microscopy with ultraviolet surface excitation (MUSE), a rapid and slide-free imaging technique, has been developed to image fresh and thick tissues with specific molecular contrast. Here, we propose to apply an unsupervised generative adversarial network framework to translate colorful MUSE images into Deep-MUSE images that highly resemble hematoxylin and eosin staining, allowing easy adaptation by pathologists. By eliminating the needs of all sample processing steps (except staining), a MUSE image with subcellular resolution for a typical brain biopsy (5 mm × 5 mm) can be acquired in 5 minutes, which is further translated into a Deep-MUSE image in 40 seconds, simplifying the standard histopathology workflow dramatically and providing histological images intraoperatively.

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