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To visualize the optically cleared tooth in 3D at the cellular level, a previously described custom-built laser-scanning confocal microscope^{53 (link)–55 (link)} was used. Three laser modules with wavelengths at 488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt), and 640 nm (MLD640, Cobolt) were utilized as excitation light sources. For laser scanning, a fast-rotating polygonal mirror with 36 facets (MC-5, aluminium coated, Lincoln Laser) and a galvanometer mirror scanner (60 H, Cambridge Technology) were used. To illuminate the optically cleared tooth with the two-dimensional raster scanning laser beam and collect fluorescence signals in an epi-detection manner, commercial objective lenses (CFI Plan Apo lambda, 10X, NA 0.45, Nikon; CFI Plan Apo lambda, 20X, NA 0.75, Nikon; and LUCPLFLN, 40X, NA 0.6, Olympus) were used. Three highly sensitive photomultiplier tubes (PMT; R9110, Hamamatsu) with bandpass filters (FF02-525/50, FF01-600/37, FF01-685/40, Semrock) were employed for detecting multicolor fluorescence signals. A three-channel frame grabber (Solios, Matrox) was used to acquire the voltage output of the photomultiplier. A custom-written software program based on the Matrox Imaging Library (MIL9, Matrox) was used for image acquisition.

Intravital imaging was performed by using a custom-built laser scanning confocal microscope (59 (link), 67 (link)). Three continuous-wave lasers with 488 nm (Cobolt, MLD), 561 nm (Cobolt, Jive) and 640 nm (Cobolt, MLD) were used as excitation lights for fluorescence imaging. Fluorescence signals were simultaneously detected by three bandpass filters (FF01-525/50, FF01-600/37, FF01-685/40; Semrock) and three photomultiplier tubes (R9110; Hamamatsu). For photoconversion of Kaede proteins, HEV in a field of view (170 × 170 μm) was irradiated by 405 nm laser (∼10 mW/mm²; Coherent; OBIS) for 5 min. Z-axis resolution of about 3 μm per section was acquired with 100 μm pinhole and 60× objective lens (LUMFLN, water immersion, NA 1.1; Olympus).