BABB clearing

BABB (stands for benzyl alcohol-benzyl benzoate, also known as Murry's clear) is a tissue clearing method used to turn biological tissues transparent. It was developed by Andrew Murray & Marc Kirschner in the 1980s. BABB clearing was used to clear the largest sized tissue to date, and hence has the highest clearing efficacy of all methods. These include a whole mouse or even a whole human brain.

Process

The tissue, usually flueroscently labelled with dyes or antibodies, is first dehydrated in graded methanol, followed by immersion in a mixture of 1:2 v/v benzyl alcohol:benzyl benzoate, which has a high refractive index of 1.55 - 1.56.

Principle

Tissue clearing works by reducing the inhomogeneities in refractive indices within the tissue. Since BABB is an organic solvent and is immiscible with water, the tissue - usually in a hydrated state - must first be dehydrated with increasing concentrations of methanol by osmotic dehydration before being immersed into BABB.

Due to the high refractive index of BABB and its even permeation into the tissue, the refractive index of the tissue is now adjusted to 1.55-1.56 by the infiltrated solvent mixture, achieving the required optical transparency for microscopy and three-dimensional (3D) histological studies.

Limitations

Bones scatter light due to the presence of hydroxyapatite crystals. Bone tissues must be decalcified to remove the crystals before leaving a protein matrix, which is then amenable to BABB clearing.

Safety

Although widely perceived as toxic, both components have very low toxicity to humans and are non-volatile. The confusion probably arises from its nature as an organic solvent and hence capable of dissolving glues, paints and plastics, especially when in contact with microscopy components such as microscope objectives, and hence wrongly perceived as corrosive.