Quantitative assessment of optical clearing methods on formalin-fixed human lymphoid tissue.

Rising interest in three-dimensional volume imaging of biological tissues for diagnostic and research purposes, calls for appropriate optical clearing methods as an indispensable requirement for high-resolution imaging on a cellular level. In recent years, many clearing protocols have emerged, though most of them focus on murine central nervous tissue. Peripheral organs or tissues of human origin have only been investigated sparsely. Therefore, we tested eight established clearing methods (BABB, Ce3D, CUBIC, ECi, ChemScale, ChemScaleQQ5, SeeDB2 and PACT) on formaldehyde-fixed human tonsils. This application-oriented taxonomy can help researchers restrict the space of their survey on clearing techniques for lymphatic tissue as it provides information on each method in regard to its efficacy, clearing speed, preservation of fluorescence labelling, toxicity, expenditure and monetary costs. We found that all of the applied clearing protocols could render the sample tissues transparent. Ce3D and PACT achieved the highest degrees of tissue transparency. Since it requires less preparing and processing time and is lower in toxicity, we recommend Ce3D for the clearing of human lymphoid tissue.