Rapid optical tissue clearing using various anionic polymer hydrogels

Abstract

Optical tissue clearing has emerged as an indispensable technique for three-dimensional imaging. The Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tissue-hYdrogel (CLARITY) method is a useful method in optical tissue clearing. Previously, we succeeded in shortening the process time in a passive CLARITY method by replacing the commonly used poly(acrylamide (AAm)) hydrogel with poly(AAm-co-styrenesulfonate (SS)) hydrogels. In this study, different polyanionic hydrogels such as poly(AAm-co-SS), poly(AAm-co-2-acrylamido-2-methylpropane sulfonate (AMPS)), and poly(AAm-co-acrylic acid (AcA)) hydrogels were used for optical clearing of various tissues. The transparency of cleared tumor tissues was similar after the CLARITY treatment with these polyanionic hydrogels, and it was higher than that treated with the poly(AAm) hydrogel. Green fluorescent protein (GFP)-expressing brain tissues could also be cleared using these polymer hydrogels. Because the GFP fluorescence signal intensity of these tissues cleared using the poly(AAm) and poly(AAm-AcA) hydrogels was higher than that using the poly(AAm-SS) and poly(AAm-AMPS) hydrogels, the poly(AAm-AcA) hydrogel can be predicted to be the optimal candidate for the CLARITY method. Various tissues including the heart, lungs, stomach, and kidneys were also optically cleared using the poly(AAm-AcA) hydrogel without any significant swelling.