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 a 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.