Open-top two-photon light sheet microscopy for three-dimensional tissue examination

Nondestructive three-dimensional (3D) pathology based on high-throughput 3D microscopy holds promise as a complement to traditional hematoxylin and eosin (H&E) stained slide-based two-dimensional (2D) pathology by providing rapid 3D pathological information. However, conventional techniques provided superficial information only due to shallow imaging depth. Herein, we developed open-top two-photon light sheet microscopy (OT-TPLSM) for intraoperative 3D pathology. A two-photon excitation light sheet, generated by 1D scanning of a Bessel beam illuminated the sample and planar imaging was conducted at 400 frames/s max. An imaging depth of 60-100 μm was achieved with long excitation wavelengths, and the image throughput was up to 1 cm² per 7 min. Cells and extra-cellular matrix were visualized using extrinsic fluorescence and intrinsic second harmonic generation, respectively. OTTPLSM was tested in various human cancer specimens and cancer structures were detected via 3D visualization. OT-TPLSM may have the potential for rapid and precise 3D histopathological examination.