Dodecagon light-sheet fluorescence microscopy for large-volume imaging without striping artifacts

Light-sheet fluorescence microscopy (LSFM) enables high spatial and temporal resolution with minimal photodamage. However, conventional LSFM techniques often suffer from striping artifacts in the resulting images due to light scattering and absorption within samples, leading to uneven illumination that negatively impacts the accuracy of subsequent image analyses. To address this limitation, we introduce dodecagon light-sheet fluorescence microscopy (dodecaLSFM), a novel approach that maximizes angular diversity to achieve homogeneous illumination and suppress striping artifacts. dodecaLSFM employs diffraction optics and cylindrical lenses to generate twelve light sheets, providing 360° omnidirectional illumination that significantly enhances illumination uniformity compared to traditional mSPIM, mDSLM, and ultramicroscopy systems, which use only one or two illumination planes. We quantitatively characterize the relationship between illumination angular diversity and striping artifact suppression and demonstrate the effectiveness of dodecaLSFM by achieving high-resolution imaging of the whole mouse brain vasculature following tissue clearing, allowing precise morphometric analysis of vascular networks without striping artifacts. Furthermore, we show that combining dodecaLSFM with expansion microscopy (ExM) enables whole-organ 3D imaging at cellular resolution. This approach provides a scalable solution for large-volume imaging that supports quantitative structural studies, such as whole-organ vascular network analysis, across diverse biological applications.