Rationalisation and Validation of an Acrylamide-Free Procedure in Three-Dimensional Histological Imaging

Three-dimensional visualization of intact tissues is now being achieved by turning tissues transparent. CLARITY is a unique tissue clearing technique, which features the use of detergents to remove lipids from fixed tissues to achieve optical transparency. To preserve tissue integrity, an acrylamide-based hydrogel has been proposed to embed the tissue. In this study, we examined the rationale behind the use of acrylamide in CLARITY, and presented evidence to suggest that the omission of acrylamide-hydrogel embedding in CLARITY does not alter the preservation of tissue morphology and molecular information in fixed tissues. We therefore propose a novel and simplified workflow for formaldehyde-fixed tissue clearing, which will facilitate the laboratory implementation of this technique. Furthermore, we have investigated the basic tissue clearing process in detail and have highlighted some areas for targeted improvement of technologies essential for the emerging subject of three-dimensional histology.

完整组织的三维可视化现已可通过组织透明化手段实现。CLARITY是一种独特的组织透明化技术，其核心特征为利用去污剂从固定组织中去除脂质以获得光学透明性。为维持组织完整性，已有研究提出采用丙烯酰胺基水凝胶（acrylamide-based hydrogel）对组织进行包埋。本研究探究了CLARITY技术中使用丙烯酰胺（acrylamide）的原理，并通过实验证据证实，在CLARITY流程中省去丙烯酰胺-水凝胶包埋步骤，并不会改变固定组织的形态学与分子信息保存效果。据此，我们提出一种针对甲醛固定组织的新型简化透明化工作流程，可助力该技术在实验室中的落地应用。此外，我们还对组织透明化的基础流程进行了详尽研究，并针对三维组织学这一新兴学科所需的关键技术，指明了若干可定向优化的方向。