Open-top Bessel-Gauss Beam Two-photon Light Sheet Microscopy for Three-dimensional Pathology

Abstract

Nondestructive 3D pathology based on 3D optical microscopy holds promise to complement hematoxylin and eosin (H&E) based 2D pathology by providing 3D pathological information in a high-throughput manner. However, shallow imaging depth in opaque tissue is still limitations. Herein, we developed open-top two-photon light sheet microscopy (OT-TPLSM) for deep 3D pathology. Two-photon excitation light sheet was generated by scanning a Bessel-Gauss beam, and the selective planar imaging was conducted at 400 frames/s max. High imaging depth was achieved with long excitation wavelengths and long wavelength fluorophores. OT-TPLSM was tested in various human cancer specimens and unsupervised deep learning was applied for generating virtual H&E. OT-TPLSM has the potential for rapid and precise 3D histology.