Fig. 1 |. Major tissue-clearing methods and their key features.

a | Hydrophobic methods rely on a complete dehydration of the tissue, followed by lipid extraction and refractive index (RI) matching using organic solvents. Flydrophobic methods are generally fast and can clear tissues fully. However, some hydrophobic methods can bleach the signal of fluorescent proteins rapidly. b | Hydrophilic methods are based on water-soluble solutions and are usually associated with higher biosafety and compatibility than hydrophobic methods. A potent hydrophilic method such as CUBIC starts with decolourization, which is followed by delipidation, RI matching and (optionally) expansion; some hydrophilic methods need longer incubation times for intact organs. c | Hydrogel embedding forms the third major category of tissue clearing. Hydrogel-based methods use either monomer and initiator molecules to make a synthetic gel or polyepoxide to make a reinforced tissue gel. Hydrogel-based methods can allow retainment of enough RNAs for assays such as fluorescence in situ hybridization and can be used to expand tissues several fold owing to the hydrogel mesh that glues the tissues. Some hydrogel-based methods need longer incubation times for intact organs.