Fig. 1. Schematic diagrams of tissue optical clearing^[22]. (a) Organic solvent-based clearing; (b) hydrophilic reagent-based clearing

Fig. 2. General principles for bone tissues clearing^[30]. (a) Similar treatment steps have been adapted by most tissue-clearing methods; (b) adult mouse mandible was cleared by the PEGASOS method and images were taken after each treatment step

Fig. 3. Bone CLARITY makes the complete bone transparent^[14]

Fig. 4. Clearing and imaging of mouse tibia performed by PACT-deCAL^[34]. (a) Tibia without clearing; (b) tibia after clearing; (c) confocal microscopic image of the tibia corresponding to the upper-right box region in Fig. 4 (b); (d) confocal microscopic image of the tibia corresponding to the bottom-left box region in Fig. 4 (b)

Fig. 5. Clearing mouse femur performed by uDISCO^[37]. (a) Images of tibia and femur in mice before clearing; (b) images of tibia and femur in mice after clearing

Fig. 6. Whole marmoset infant body by PI staining (propidium iodide), clearing and LSFM(light-sheet fluorescence microscope) imaging ^[41]. (a) Cleared and PI-stained infant marmoset body; (b) reconstituted whole-body 3D image acquired with a custom-built LSFM after staining and clearing

Fig. 7. FDISCO achieves individual organs clearing in mouse and rat^[42]. (a) Comparison of multiple organs of mouse before and after clearing; (b) comparison of multiple organs of rat before and after clearing

Fig. 8. Clearing performance of both hard and soft organs cleared by MACS^[43]

Fig. 9. Deep imaging of HSCs in digitally reconstructed bone marrow^[32]. (a)(b) Flow cytometry analysis of bone marrow stem cell types; (c)(d) tibia before and after clearing; (e)~(l) deep imaging of HSCs in bone marrow

Fig. 10. Bone CLARITY makes the whole bone transparent and makes endogenous fluorescence to be retained^[14]. (a) Maximum intensity projection fluorescence image of tibia; (b) maximum intensity projection fluorescence image of vertebral body

Fig. 11. A vDISCO panorama shows the meningeal vessels under the complete skull^[16]. (a) Meningeal vessels of middle cerebral artery occlusion (MCAO) model of stroke (at the arrow) invaded by immune cells; (b) meningeal vessels of normal mouse uninvaded by immune cells

Fig. 12. VDISCO panoramic imaging of SMCs under a complete skull^[16]. (a)(b) A 6-month-old VEGFR3-YFP mouse head in sagittal views; (c)(d) a 6-month-old VEGFR3-YFP mouse head in axial views; (e)(f) details of SMCs after marking with different methods

Fig. 13. PEGASOS passive immersion procedure clears hard tissue organs^[15]. (a) Brief description of the PEGASOS passive immersion procedure for clearing hard tissue organs; (b) femurs, short vertebrae segment (<3 cm length) and mandible were harvested from adult mice (60 days of age) and imaged before and after clearing; (c) (c') short vertebrae from Tie2-Cre; Ai14 mice was imaged through the bone to reveal the inside vasculatures and enlarged view of the rectangular area in Fig. 13(c); (d) (d') tooth from Tie2-Cre; Ai14 was imaged after clearing to reveal the vascular network within the dental pulp and enlarged view of the rectangular area in Fig. 13(d)

Fig. 14. PEGASOS provides a new method for three-dimensional imaging of hard tissues and organs^[15]. (a) Skull was imaged with a stereomicroscope before clearing; (a') skull was imaged with a stereomicroscope after clearing; (b) two-photon microscope image of the skull after clearing; (c)(c') two-photon microscope image of mandible after clearing and enlarged view of the rectangular area in Fig. 14(c); (d) alveolar bone of mandible corresponding to the rectangular area in Fig. 14(c'); (e) (e') two-photon microscope image of the femur after clearing and enlarged view of the rectangular area in Fig. 14(e); (f) alveolar bone of femur corresponding to the rectangular area in Fig. 14(e'); (g) (g') tooth within the mandible was imaged with a two-photon microscope and optical section was obtained to show the pulp chamber; (h)(h') intact knee joint was cleared and optical sections acquired at different depths are displayed

Fig. 15. Comparison of skulls before and after SOCS^[17-20]. (a)--(c) White-light images of intact skull, the transparent skull images after SOCS treatment for 25 min and after removing rectangle area A; (d)--(f) corresponding magnified white-light images of the rectangle area A shown in Fig. 15(a)--(c); (g)--(i) speckle contrast images of the rectangle area A shown in Fig. 15(a)--(c)

Fig. 16. Schematic diagrams of SOCW technique for cortical imaging. (a) Main experimental steps; (b) brain fixation device of mouse; (c) brain structure map of mouse; (d) SOCW diagram; (e)--(g) different clearing methods followed by mouse of different ages

Table 1. Several bone tissue optical clearing techniques and their advantages and disadvantages