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

Irene Costantini,Francesco Giardini,Markus Axer,Ludovico Silvestri,Katrin Amunts,Francesco Saverio Pavone,Michele Sorelli,Giacomo Mazzamuto,Riccardo Cicchi,Miriam Menzel,Felix Matuschke,Enrico Baria

doi:10.1038/s41598-021-86092-7

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.

Highlights

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics
To demonstrate that the fluorescence signal is emitted by myelinated fibers, we labeled on a mouse brain section treated with MAGIC with a myelin-specific exogenous dye: the FluoroMyelin red[19]
We found that the primary glyph orientation of the control mouse is mostly constituted by in-plane contributions in all the selected region of interest (ROI), while six out of eight areas of the Reeler sample were distributed in the out-of-plane direction (Fig. 4i and Table 1)

Summary

Introduction

Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Cutting-edge microscopy techniques such as micro-optical sectioning tomography (MOST)[5,6], serial two-photon tomography (STP)[7], block-face serial microscopy (FAST)[8], and light sheet microscopy (LSM)[9,10], permit the reconstruction of large volumes of tissue and even entire organs with micrometer resolution They need a source of contrast to detect the structure of interest. The necessity of prelabeled conditions prevents the possibility of studying archived samples To overcome these limitations, we developed MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a simple label-free method that opens the possibility of performing sub-micron resolution fluorescence imaging of myelinated fibers in 3D at the mesoscale level of different mammalian brains. We demonstrated the widespread applicability of the methodology by investigating neuronal filament organization in 3D in different mammalian brains such as mouse, rat, vervet monkey, and humans

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