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

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.

Tissue libraries enable rapid determination of conditions that preserve antibody labeling in cleared mouse and human tissue

“…Starting from a comprehensive collection of published IHC protocols ( Richardson et al, 2021 ; Kim et al, 2016 ; Scardigli et al, 2021 ; Richardson and Lichtman, 2015 ), we selected a subset of commonly varied conditions to create 12 test protocols for proof of principle. Each protocol is nearly identical, only varying by a single condition from at least one other protocol to allow for a complete understanding of how each change influences staining.…”

“…This is rarely the case and complete, specific and homogenous staining is now the most common bottleneck preventing the widespread use of tissue clearing in samples that cannot be modified to express fluorescent proteins such as human tissue. Therefore, most tissue clearing methods have been optimized for studies in rodents, and only a few recent studies apply clearing techniques to human tissue ( Scardigli et al, 2021 ; Lai et al, 2018 ; Susaki et al, 2020 ; Matos et al, 2010 ; Zhao et al, 2020 ). Here, we present a simple optimization strategy and automated analysis routine that allows researchers to tune their immunohistochemistry (IHC) protocols for labeling protein targets in cleared and uncleared brain tissue.…”

Direct optical nanoscopy unveils signatures of cytokine-induced β-cell structural and functional stress

“…In this sub-millimetric organ, the presence of multiple cells tightly packed in a three-dimensional architecture makes it even more complicated to achieve single-cell identification and sub-cellular analysis, mainly because of the refractive-index mismatch caused by the variety of biomolecules with different optical properties contained in the islet (e.g., lipids, fibers, and proteins), and to light scattering and/or absorption by the same molecules. To tackle this issue, ExM protocol variations 61,62 that guarantee multiplexed localization, molecular specificity, and tissue clearing to increase light penetration 63 and signal-to-noise ratio, potentially allowing rapid reconstruction of whole-stained islets, and hence perform a more accurate molecular interrogation in diabetic and non-diabetic tissues.…”

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy

“…The principle of tissue clearing relies on the homogenization of the refractive index (RI) inside and outside the sample, thus allowing good penetration of the dyes and optical sectioning of intact samples. Each clearing method has advantages and limitations according to the tissue to be analyzed (e.g., size, fixation, conservation) and the goal to be achieved (e.g., resolution, acquisition speed) [ 8 , 9 , 10 ].…”