Mechanical de-skewing enables high-resolution imaging of thin tissue slices with a mesoSPIM light-sheet microscope

Optical clearing combined with light-sheet microscopy enables high-resolution imaging of extended tissue at scale. However, standard mesoSPIM systems are optimised for intact organs and are not suited to thin tissue slices. We present an oblique compensation scanning method using obliquely mounted samples held between refractive-index-matched slides in a 3D-printed frame. This enables mechanical de-skewing during acquisition, minimising post-processing requirements. We demonstrate feasibility in fluorescent bead phantoms and rabbit heart tissue, achieving a 4.8 × reduction in processing time and a 1.5 × improvement in axial resolution ((13.15±1.36) μm to (8.72±1.80) μm) compared to conventional z scan. The oblique compensation acquisition method extends mesoSPIM’s utility to fragile, laterally extended tissue sections.