Comparison of Different Tissue Clearing Methods for Three-Dimensional Reconstruction of Human Brain Cellular Anatomy Using Advanced Imaging Techniques

Year: 2021

Authors: Scardigli M., Pesce L., Brady N., Mazzamuto G., Gavryusev V., Silvestri L., Hof PR., Destrieux C., Costantini I., Pavone FS.

Autors Affiliation: European Laboratory for Non-linear Spectroscopy, University of Florence, Florence, Italy
Department of Physics and Astronomy, University of Florence, Florence, Italy
National Institute of Optics, National Research Council, Rome, Italy
Nash Family Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, United States
UMR 1253, iBrain, INSERM, Université de Tours, Tours, France
Department of Biology, University of Florence, Florence, Italy

Abstract: The combination of tissue clearing techniques with advanced optical microscopy facilitates the achievement of three-dimensional (3D) reconstruction of macroscopic specimens at high resolution. Whole mouse organs or even bodies have been analyzed, while the reconstruction of the human nervous system remains a challenge. Although several tissue protocols have been proposed, the high autofluorescence and variable post-mortem conditions of human specimens negatively affect the quality of the images in terms of achievable transparency and staining contrast. Moreover, homogeneous staining of high-density epitopes, such as neuronal nuclear antigen (NeuN), creates an additional challenge. Here, we evaluated different tissue transformation approaches to find the best solution to uniformly clear and label all neurons in the human cerebral cortex using anti-NeuN antibodies in combination with confocal and light-sheet fluorescence microscopy (LSFM). Finally, we performed mesoscopic high-resolution 3D reconstruction of the successfully clarified and stained samples with LSFM.

Journal/Review: FRONTIERS IN NEUROANATOMY

Volume: 15      Pages from: 752234-1  to: 752234-13

More Information: This project received funding from the European Union’s Horizon 2020 Research and Innovation Framework Program under grant agreement No. 654148 (Laserlab-Europe), European Union’s Horizon 2020 Framework Program for Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2) and No. 945539 (Human Brain Project SGA3) and under the Marie Sklodowska-Curie grant agreement No. 793849 (MesoBrainMicr), General Hospital Corporation Center of the National Institutes of Health under award number U01 MH117023, and Italian Ministry for Education in the framework of Euro-Bioimaging Italian Node (ESFRI Research Infrastructure). Finally, this research was carried out with the contribution from 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.
KeyWords: clearing techniques; optical microscopy; immunofluorescence; light-sheet fluorescence microscopy; expansion microscopy
DOI: 10.3389/fnana.2021.752234

Citations: 10
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