Autofluorescence enhancement for label‑free imaging of myelinated fibers in mammalian brains

Year: 2021

Authors: Costantini I., Baria E., Sorelli M., Matuschke F., Giardini F., Menzel M., Mazzamuto G., Silvestri L., Cicchi R., Amunts K., Axer M., Pavone F.S.

Autors Affiliation: European Laboratory for Non‑Linear Spectroscopy, University of Florence, Florence, Italy; Department of Biology, University of Florence, Florence, Italy; National Institute of Optics, National Research Council, Rome, Italy; Department of Physics, University of Florence, Florence, Italy; Institute of Neuroscience and Medicine (INM‑1), Research Centre Jülich, Jülich, Germany
C. and O. Vogt Institute for Brain Research, University Hospital Düsseldorf, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany

Abstract: Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse’s hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.


Volume: 11 (1)      Pages from: 8038-1  to: 8038-11

More Information: We thank Markus Cremer, INM-1, Forschungszentrum Julich, Germany for brain tissue sectioning and mounting. The research leading to these results has received funding 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). This research has also been supported by the Massachusetts General Hospital (The General Hospital Corporation), Athinoula A. Martinos Center, The National Institute of Mental Health (NIMH) under award number 1U01MH117023-01, by the Italian Ministry for Education, University, and Research in the framework of the Eurobioimaging Italian Nodes (ESFRI research infrastructure)-Advanced Light Microscopy Italian Node. Finally, this research was carried out with the contribution from “Fondazione CR Firenze” (private foundation). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
KeyWords: brain imaging, myelin, glycerol, autofluorescence
DOI: 10.1038/s41598-021-86092-7

Citations: 17
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