Abstract
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.
Highlights
The opacity of tissues arises as a result of light scattering at the boundaries of the heterogeneous intermix of hydrophilic and hydrophobic components with different refractive indices within the tissue. [1,2] A technique called CLARITY [1] has been developed by delipidating tissues with the detergent sodium dodecyl sulphate (SDS), which removes the major hydrophobic components to achieve tissue clarity for deep microscopic imaging
In this study we examined the theoretical aspects behind the practically complex hydrogel-embedding step, and demonstrated that a formaldehyde-crosslinked tissue serves as an effective protective physical framework for detailed three-dimensional histological evaluation, leading us to propose the omission of acrylamide embedding in CLARITY
We observed that the time taken to adequately delipidate a tissue block depended largely on the conditions of formaldehyde fixation instead of the concentration of acrylamide used for embedding, and the usage of Electrophoretic tissue clearing (ETC) did not alter the time course of tissue clearing
Summary
The opacity of tissues arises as a result of light scattering at the boundaries of the heterogeneous intermix of hydrophilic and hydrophobic components with different refractive indices within the tissue. [1,2] A technique called CLARITY [1] has been developed by delipidating tissues with the detergent sodium dodecyl sulphate (SDS), which removes the major hydrophobic components to achieve tissue clarity for deep microscopic imaging. The original CLARITY technique included the hydrogel-embedding step based on two hypothetical reasons [1]: firstly, delipidation removes lipid bilayers essential for cellular integrity; secondly, acrylamide might be used to crosslink formaldehyde-modified amines (called formaldimines) on proteins via a nucleophilic addition reaction. The attached acrylamide will be polymerized, forming a physical framework to prevent excessive protein loss from tissues during SDS-mediated delipidation, whereas lipids and other biomolecules lacking the amine groups can be washed off. This is an attractive hypothesis but there is little experimental evidence to support it and there is no available comparisons on tissue morphologies between acrylamide-embedded and non-embedded samples. We aimed to investigate the role of an acrylamide-based hydrogel in tissue clearing by SDS-mediated delipidation (hereafter referred to as clearing), and subsequently we propose the replacement of the complex hydrogel-embedding step by standard passive formaldehyde-fixation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
