Optimizing tissue clearing and imaging methods for human brain tissue

Abstract

Objectives To identify optimum sample conditions for human brains, we compared the clearing efficiency, antibody staining efficiency, and artifacts between fresh and cadaver samples. Methods Fresh and cadaver samples were cleared using X-CLARITY™. Clearing efficiency and artifact levels were calculated using ImageJ, and antibody staining efficiency was evaluated after confocal microscopy imaging. Three staining methods were compared: 4-day staining (4DS), 11-day staining (11DS), and 4-day staining with a commercial kit (4DS-C). The optimum staining method was then selected by evaluating staining time, depth, method complexity, contamination, and cost. Results Fresh samples outperformed cadaver samples in terms of the time and quality of clearing, artifacts, and 4′,6-diamidino-2-phenylindole (DAPI) staining efficiency, but had a glial fibrillary acidic protein (GFAP) staining efficiency that was similar to that of cadaver samples. The penetration depth and DAPI staining improved in fresh samples as the incubation period lengthened. 4DS-C was the best method, with the deepest penetration. Human brain images containing blood vessels, cell nuclei, and astrocytes were visualized three-dimensionally. The chemical dye staining depth reached 800 µm and immunostaining depth exceeded 200 µm in 4 days. Conclusions We present optimized sample preparation and staining protocols for the visualization of three-dimensional macrostructure in the human brain.

Seeing the Forest and Its Trees Together: Implementing 3D Light Microscopy Pipelines for Cell Type Mapping in the Mouse Brain

“…Remarkable advances in tissue clearing and 3D immunolabeling make it possible to label target cells with specific antibodies in intact biological samples. This enables scientists to bypass complex breeding schemes and care for excessively large colonies accelerating new discoveries and extending brain mapping technology to new species including humans (Lai et al, 2018;Kim et al, 2021). Here, we provide a brief summary of a few widely used clearing and chemical labeling methods to examine cell type distribution in the whole brain.…”

Quantitative optical coherence microscopy of neuron morphology in human entorhinal cortex

“…One unsolved challenge in traditional histology is that substantial distortions of the thin slices cause irreducible errors in volumetric reconstruction that are often greater than the structure of interest. The development of various tissue clearing methods provides a tempting solution to promote volumetric optical imaging in fluorescence labeled tissue samples, which supports molecular probing and cell typing in the brain (Murray et al, 2015;Swaney et al, 2019;Yun et al, 2019;Ueda et al, 2020;Kim et al, 2021). Despite the success of whole brain imaging in small animals, the application of clearing in thick sections of postmortem human tissues remains challenging due to the high myelin density.…”