Tissue clearing of both hard and soft tissue organs with the pegasos method

Tissue clearing technique enables visualization of opaque organs and tissues in 3-dimensions (3-D) by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissues that can be cleared with each individual protocol, which inevitably led to the presence of blind-spots within whole body or body parts imaging. Hard tissues including bones and teeth are still the most difficult organs to be cleared. In addition, loss of endogenous fluorescence remains a major concern for solvent-based clearing methods. Here, we developed a polyethylene glycol (PEG)-associated solvent system (PEGASOS), which rendered nearly all types of tissues transparent and preserved endogenous fluorescence. Bones and teeth could be turned nearly invisible after clearing. The PEGASOS method turned the whole adult mouse body transparent and we were able to image an adult mouse head composed of bones, teeth, brain, muscles, and other tissues with no blind areas. Hard tissue transparency enabled us to reconstruct intact mandible, teeth, femur, or knee joint in 3-D. In addition, we managed to image intact mouse brain at sub-cellular resolution and to trace individual neurons and axons over a long distance. We also visualized dorsal root ganglions directly through vertebrae. Finally, we revealed the distribution pattern of neural network in 3-D within the marrow space of long bone. These results suggest that the PEGASOS method is a useful tool for general biomedical research.