Clearing and Labeling Techniques for Large-Scale Biological Tissues

Abstract

Clearing and labeling techniques for large-scale biological tissues enable simultaneous extraction of molecular and structural information with minimal disassembly of the sample, facilitating the integration of molecular, cellular and systems biology across different scales. Recent years have witnessed an explosive increase in the number of such methods and their applications, reflecting heightened interest in organ-wide clearing and labeling across many fields of biology and medicine. In this review, we provide an overview and comparison of existing clearing and labeling techniques and discuss challenges and opportunities in the investigations of large-scale biological systems.

Keywords: 3D volume imaging; CLARITY; SWITCH; large-scale tissue clearing; stochastic electrotransport; whole-mount labeling.

Fig. 1.

Tissue clearing techniques. (A) Light scattering in biological tissues can be reduced by removal of lipid and RI matching. (B) Simple immersion in a high-RI aqueous solution renders the tissue modestly transparent by homogenizing scattering throughout the sample. (C) Delipidation and dehydration/hyperhydration followed by refractive index matching. (Top) For solvent-based clearing, the tissue is incubated in dehydrating solvent for delipidation and dehydration, and is moved to a high-RI clearing solvent where RI matching and additional delipidation occur. (Bottom) The sample is placed in an aqueous solution that contains high concentration of non-ionic detergent and denaturant, where delipidation, hyperhydration, and RI matching take place. (D) A biological sample is first transformed into a tissue-gel hybrid by hydrogel embedding (Top) or glutaraldehyde fixation (Bottom), where the gel network increases the tissue integrity. The tissue-gel hybrid then can withstand extensive delipidation by incubation in ionic detergent (SDS) assisted by electrophoresis or heating.