(A) Time course and individual steps of tissue clearing with or without immunostaining. (B) Three-dimensional imaging of the organ of Corti within the temporal bone. Top view (left), lateral view (middle), and the schematic presentation of the organ of Corti with its axis parallel to the modiolus. The size of the organ of Corti is indicated in the X, Y, and Z coordinates. Scale bar, 500 μm. (C) Computational processes of linearization, cell detection, and modeling. (D) Side-by-side comparison of 3DISCO, iDISCO, CLARITY, and CUBIC. Transmitted light images of samples before and after clearing, together with MYO7A staining. Scale bar, 500 μm. (E) Manual dissection of the iDISCO-processed sample confirmed MYO7A staining in the sensory epithelium. Scale bars, 500 μm (upper image) and 100 μm (lower image). (F) Lateral and horizontal views of the reconstructed three-dimensional images of the organ of Corti stained with anti-MYO7A. CUBIC with decalcification and original ScaleS failed to detect the deepest part of the organ of Corti (green dotted lines). With modified ScaleS, the entire structure of the organ of Corti could be visualized. Scale bar, 500 μm. (G) Modified ScaleS sample of the organ of Corti stained with anti-MYO7A antibody, together with transmitted light images before (upper left) and after (upper right) treatment. Scale bar, 500 μm. (H) Three steps of the modified ScaleS protocol. The initial decalcification step is followed by a clearing step, which mainly removes lipids from the extracellular matrix. Finally, the RI of the sample is matched with mounting solution. (I) Preservation of GFP fluorescence after modified ScaleS treatment. Scale bar, 10 μm. (J) Preservation of rhodamine-phalloidin signal after modified ScaleS treatment, which includes sorbitol to stabilize cytoskeletal polymers. Scale bar, 10 μm. IHC, inner hair cell; OHC, outer hair cell; RI, refractive index.