Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues

Hei Ming Lai,Alan King Lun Liu,Tsz Wing Chau,Marc H Goldfinger,Wai Man Wong,John Defelice,Steve M Gentleman,Harry Ho Man Ng,Wutian Wu,Bension S Tilley

doi:10.1038/s41467-018-03359-w

Hei Ming Lai, Alan King Lun Liu + Show 8 more

Open Access

https://doi.org/10.1038/s41467-018-03359-w

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Abstract

Modern clearing techniques for the three-dimensional (3D) visualisation of neural tissue microstructure have been very effective when used on rodent brain but very few studies have utilised them on human brain material, mainly due to the inherent difficulties in processing post-mortem tissue. Here we develop a tissue clearing solution, OPTIClear, optimised for fresh and archival human brain tissue, including formalin-fixed paraffin-embedded material. In light of practical challenges with immunostaining in tissue clearing, we adapt the use of cresyl violet for visualisation of neurons in cleared tissue, with the potential for 3D quantification in regions of interest. Furthermore, we use lipophilic tracers for tracing of neuronal processes in post-mortem tissue, enabling the study of the morphology of human dendritic spines in 3D. The development of these different strategies for human tissue clearing has wide applicability and, we hope, will provide a baseline for further technique development.

Highlights

The brain is arguably the most complex organ in the human body
On the basis of our understanding of the mechanisms of tissue clearing and the properties of human neural tissues, we have deconstructed some of the current techniques and identified three major aims for this study: (1) to develop a refractive index homogenisation solution optimised for human tissue clearing research; (2) to optimise tissue clearing for prolonged formalinfixed and paraffin-embedded materials, thereby freeing up archival tissues for research; and (3) to explore the compatibility of traditional non-immunohistochemical histology staining methods with tissue clearing
We present the development of a refractive index homogenisation reagent, OPTIClear, which is optimised for tissue clearing in both fresh and archival human brain tissues, including formalin-fixed paraffin-embedded (FFPE) materials

Summary

Introduction

The brain is arguably the most complex organ in the human body. For centuries, efforts have been made to try and understand the structural and functional connections within the brain. On the basis of our understanding of the mechanisms of tissue clearing and the properties of human neural tissues, we have deconstructed some of the current techniques and identified three major aims for this study: (1) to develop a refractive index homogenisation solution optimised for human tissue clearing research; (2) to optimise tissue clearing for prolonged formalinfixed and paraffin-embedded materials, thereby freeing up archival tissues for research; and (3) to explore the compatibility of traditional non-immunohistochemical histology staining methods with tissue clearing. We present the development of a refractive index homogenisation reagent, OPTIClear (for Optical Properties-adjusting Tissue-Clearing agent), which is optimised for tissue clearing in both fresh and archival human brain tissues, including formalin-fixed paraffin-embedded (FFPE) materials This has enabled us to establish various protocols for

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