Axially swept open-top light-sheet microscopy for densely labeled clinical specimens

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.