Scanning Bessel beam microscopy with a protected and corrective objective for solvent-cleared large samples

Emerging imaging strategies, such as tissue clearing and advanced light-sheet modalities, are transforming the visualization of cell differentiation, tissue reorganization, and morphogenesis in both model and non-model organisms, thereby driving future discoveries of conserved developmental programs and species-specific innovations. In this study, we present a versatile Bessel light sheet microscopy (BLX) system compatible with both immersion and capped air objectives, optimized for whole-mount three-dimensional imaging of cleared tissues. We demonstrate that autofluorescence-based light-sheet imaging enables reliable visualization of CUBIC-R-cleared mouse embryos, revealing microanatomical features such as nephrons during developmental stages. To support solvent-based clearing protocols, we designed a protective lens cap that shields objectives from solvent erosion and economically converts standard air objectives into high-performance immersion objectives. Using this capped BLX configuration, we achieved cellular-resolution imaging of whole PEGASOS-cleared mouse brains. The system's simplified design and open-source availability promote broader adoption for diverse applications in developmental biology.