Optical clearing, in combination with recently developed optical imaging techniques, enables visualization and acquisition of high-resolution, three-dimensional images of biological structures deep within the tissue. Many different approaches can be used to reduce light absorption and scattering within the tissue, but there is a paucity of research on the quantification of clearing efficacy. With the use of a custom-made spectroscopy system, we developed a way to quantify the quality of clearing in biological tissue and applied it to the mouse brain. Three clearing techniques were compared: BABB (1:2 mixture of benzyl alcohol and benzyl benzoate, also known as Murray’s clear), pBABB (peroxide BABB, a modification of BABB which includes the use of hydrogen peroxide), and passive CLARITY. We found that BABB and pBABB produced the highest degree of optical clearing. Furthermore, the approach allows regional measurement of light attenuation to be performed, and our results show that light is most attenuated in regions with high lipid content. We provide a way to choose between the multiple clearing protocols available, and it could prove useful for evaluating images that are acquired with cleared tissues.
Optical clearing allows the reduction of light scattering in biological tissue, enabling 3D morphological information to be
obtained deep within tissue using techniques such as optical projection tomography and light sheet microscopy. However, the
extent of the clearing is dependent on the technique that is used. There is therefore a need for methods to quantify the quality
of the clearing process and thereby to compare clearing techniques. In this study, we developed such a method using a
custom spectroscopy system and applied it to compare three techniques that were applied to mouse brain: BABB (Murray’s
clear), pBABB (a modification of BABB which includes the use of hydrogen peroxide), and passive CLARITY.