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

Abstract

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.

3‐dimensional visualization of implant‐tissue interface with the polyethylene glycol associated solvent system tissue clearing method

“…30 Our laboratory is working to improve PEGASOS method to overcome these limitations. First of all, PEGASOS treatment leads to differential shrinkage among soft and hard tissues, which may cause anisotropic distortion for organs composed of multiple tissue types.…”

“…In addition, the treatment procedure preserves endogenous fluorescence better than other solventbased clearing methods, including 3DISCO, uDISCO and FluoClear 30. In our other study, we were able to acquire images up to 3 mm depth on a cleared dog tibia bone sample 30. Inside structures and implant surface can be directly visualized with a regular confocal or two-photon microscope without sectioning.…”

“…to nearly invisible after treatment. The favourable transparency achieved with PEGASOS can be attributed to the combination of decalcification, decolourization, delipidation and the high RI (1.543) of BB-PEG clearing medium, which is close to the average RI (1.53) of decalcified bone 30,42,43. The fluorescence preservation is critical for acquiring multi-channel 3-D images after tissue clearing and enables applications of multiple transgenic mouse models to investigate implant-tissue interface.Combining with fluorescent reporter mouse lines, multi-colour 3-D investigation of implant-bone interface is becoming possible.…”

“…30 In the current study, we introduced the application of the PEGASOS method on studying the implant-bone interface. 30 In the current study, we introduced the application of the PEGASOS method on studying the implant-bone interface.…”

“…In addition, the treatment procedure preserves endogenous fluorescence better than other solventbased clearing methods, including 3DISCO, uDISCO and FluoClear 30. In addition, the treatment procedure preserves endogenous fluorescence better than other solventbased clearing methods, including 3DISCO, uDISCO and FluoClear 30.…”