Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Year: 2024

Authors: Di Meo D., Ramazzotti J., Scardigli M., Cheli F., Pesce L., Brady N., Mazzamuto G., Costantini I., Pavone FS.

Autors Affiliation: Univ Florence, European Lab Nonlinear Spect LENS, Florence, Italy; Univ Florence, Dept Expt & Clin Med, Div Physiol, Florence, Italy; Univ Pisa, Dept Phys, Pisa, Italy; CNR, Natl Inst Opt CNR INO, Pisa, Italy; Univ Florence, Dept Phys & Astron, Florence, Italy; Univ Florence, Dept Biol, Florence, Italy.

Abstract: Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH – H2O2 – Antigen Retrieval – 2,2’-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with lightsheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi -labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high -throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Journal/Review: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS

Volume: (203)      Pages from: e65960-1  to: e65960-13

More Information: We thank Bruce Fischl, Massachusetts General Hospital, A.A. Martinos Center for Biomedical Imaging, Department of Radiology, for providing the human brain specimens analyzed in this study. This project received funding from the European Union’s Horizon 2020 Research and Innovation Framework Programme under grant agreement No. 654148 (Laserlab-Europe), from the European Union’s Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2) and No. 945539 (Human Brain Project SGA3), from the General Hospital Corporation Center of the National Institutes of Health under award number U01 MH117023, and from the Italian Ministry for Education in the framework of Euro-Bioimaging Italian Node (ESFRI research infrastructure). Finally, this research was carried out with the contribution of Fondazione CR Firenze. 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. Figure 1 was created with BioRender.com.
KeyWords: Whole-body; Tissue
DOI: 10.3791/65960

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