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.
Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.
Rapid and high-resolution histological imaging with minimal tissue preparation has long been a challenging and yet captivating medical pursue. Here, we 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 seconds with 1.1-um lateral resolution. Moreover, the CHAMP image can be transformed into a virtually stained histological image (Deep-CHAMP) through unsupervised learning within 15 seconds, where significant cellular features are quantitatively extracted with high accuracy. The versatility of CHAMP is experimentally demonstrated using mouse brain/kidney 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.
<p>The datasets for transforming autofluorescence images to the histochemically stained images were acquired from a human breast biopsy tissue and a human liver cancer tissue. Tissues of breast cancer and liver cancer were extracted surgically or through tissue biopsy. The tissues were formalin-fixed and paraffin-embedded (FFPE). Thin tissue slices, with a thickness of 4 µm, were sectioned and placed on a quartz slide. The tissue slices were deparaffined prior to imaging. The autofluorescence images were acquired from a wide-field inverted microscope equipped with a 10X/0.3 numerical aperture (NA) objective lens (Plan Fluorite, Olympus Corp.), an infinity-corrected tube lens (TTL-180-A, Thorlabs Inc.), and a monochrome scientific complementary metal-oxide-semiconductor camera (pco.panda 4.2, PCO. Inc.). A deep ultraviolet light-emitting diode of 265 nm (M265L4, Thorlabs Inc.) was used as an excitation light source because of its high absorption in cell nuclei [28], consequently providing high nuclear contrast without labels [29]. After acquiring the autofluorescence image, the same slide was stained with H&E and its bright-field images were captured using a whole-slide scanner equipped with a 20X/0.75 NA objective lens (NanoZoomer-SQ, Hamamatsu Photonics K.K). All human experiments were carried out in conformity with a clinical research ethics review approved by the Institutional Review Board of the Chinese University of Hong Kong/ New Territories East Cluster (reference number: 2021.597).</p>
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