3D Imaging of Cleared Tissues

Tissue clearing technique enables visualization of opaque organ and tissue in 3‐dimension by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissue 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, endogenous fluorescence loss remains to be 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 tissue transparent and preserved endogenous fluorescence. Bones and teeth could be turned to 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. We were able to image intact mouse brain at sub‐cellular resolution and to trace individual neurons and axons over a long distance. We were able to visualize dorsal root ganglions directly through vertebrae. Finally, we revealed the neural network distribution pattern in 3‐dimension within the long bone marrow space. These results suggest that the PEGASOS method is a useful tool for general biomedical research. Support or Funding Information This work was supported by startup funding from Texas A&M University, NIH/NIDCR grant K08DE025090, NIH/NIDCR grant R21 DE027928 and NIH/NIDCR grant R01 DE 028291 to Hu Zhao.Thy1‐EGFP spinal cord imaged with a tiling light sheet microscope