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

Year: 2025

Authors: Perego L., Cheli F., Bradley S., Di Meo D., Giannoni L., Ramazzotti J., Caria F.F., Sancataldo G., Sorelli M., Mazzamuto G., Costantini I., Pavone F.S.

Autors Affiliation: Univ Florence, European Lab Nonlinear Spect LENS, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Biol, I-50019 Sesto Fiorentino, Italy; UCL, Dept Med Phys & Biomed Engn, London WC1E 6BT, England; Univ Palermo, Dept Phys & Chem, I-90128 Palermo, Italy; Univ Florence, Dept Informat Engn, I-50139 Florence, Italy; Natl Res Council Natl Inst Opt CNR INO, I-50019 Sesto Fiorentino, Italy.

Abstract: 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.

Journal/Review: LASER & PHOTONICS REVIEWS

More Information: The authors express their gratitude to the donor involved in the body donation program of the Massachusetts General Hospital Autopsy Service, USA, who made this study possible by generously donating their body to science. This project has received funding from the European Union’s Horizon Europe research and innovation programme under Grant agreement No. 101147319 (EBRAINS 2.0) and under Grant agreement No. 654148 (Laserlab-Europe). This research has also been supported by the Italian Ministry for University and Research in the framework of the Advanced Light Microscopy Italian Node of Euro-Bioimaging ERIC, from the General Hospital Corporation Centre of the National Institutes of Health under award number U01 MH117023, and BRAIN CONNECTS (award number U01 NS132181). The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health-USA. The work was also supported by Fondazione Cassa di Risparmio di Firenze (Human Brain Optical Mapping project). Additional funding was provided by the University of Florence (D.R. no. 464 del 02/04/2024) for the project Smart hy drogels with enhanced toughness to enable human brain tissue clearing (SMART-brain), CUP: B97G24000240005 and by RICTD2025_2026-CUP: B97G24000240005, by LENS and CNR for the technical and scientific support to the Italian National Node FOE 2022-CUP B53C24004790001. Open access publishing facilitated by Universita degli Studi di Firenze, as part of the Wiley-CRUI-CARE agreement.
KeyWords: connectomics; high-speed volumetric imaging; image processing; light-sheet microscopy; multiplexing; neuroanatomy; neuropathology
DOI: 10.1002/lpor.202501725