A qualitative comparison of ten tissue clearing techniques.

The understanding of spatially complex biological systems is greatly aided by the availability of high resolution information on their tissue architecture, as is provided by optical sectioning microscopy like confocal and light sheet microscopy. In addition, genetically encoded fluorescent reporter proteins reveal tissue architecture without the need for staining procedures. Owing to opacity caused by scattering and absorption, light microscopy in tissue is limited to thin tissue layers of a few micrometers traditionally provided by histological sections. Aiming to allow deeper imaging, during the last decade massive efforts to develop tissue clearing protocols produced a flurry of novel clearing techniques for whole organ visualization, now available to microscopists. In particular, new tissue clearing methods were developed that avoid the use of organic solvents, aiming to retain the integrity of genetically encoded fluorescent proteins. So far, these methods have not been directly compared and selection of the right technique can be a non-trivial task. Here, we have aimed to compare different tissue clearing approaches side by side in a standardized manner. We provide qualitative data on their clearing capability of mouse brain, lung, heart, kidney and muscle, as well as embryos and fetuses at the developmental stages E10.5, E12.5 and E15.5 and discuss possible applications.