Quad-SPIM: A High-Speed, Multi-Color Light-Sheet Microscope for 3D Imaging of Large Cleared Human Brain Tissues

Light-sheet fluorescence microscopy (LSFM) is a powerful tool for high-resolution volumetric imaging of biological samples, offering fast acquisition speeds and reduced photodamage. However, its application to large, optically cleared human tissues—such as postmortem brain specimens—remains technically challenging due to constraints in speed, resolution, spectral flexibility, and sample handling. In this work, a custom-built, high-speed, multi-color LSFM system specifically designed for large-scale imaging of cleared human brain tissue with subcellular resolution is presented. The system enables simultaneous four-channel acquisition and achieves imaging speeds exceeding 3 cm3 h−1, thanks to a custom optical design and an optimized software for data handling. The platform features a large and stable imaging chamber with precise sample positioning, and an optimized detection path that provides four-channel imaging, avoiding cross-talk and preserving image quality across extended fields of view. The capabilities and performance of the microscope by imaging postmortem, optically cleared human brain samples, revealing cytoarchitectonic and vascular features across centimeter-scale volumes are demonstrated. This LSFM platform provides a robust and scalable solution for high-throughput imaging of large human tissues, with potential applications in neuroanatomy, connectomics, and neuropathology.