R9110

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.

A picosecond mode-locked Ti:sapphire laser (Tsunami, Spectra-Physics; repetition rate $=80$ MHz) and a picosecond acoustic-optic tunable filter (AOTF) mode-locked Ti:sapphire laser (Megaopt; repetition rate $=80$ MHz) were used as excitation light sources, as shown in Fig. 4, serving as ${\omega }_p$ and ${\omega }_s$ laser beams, respectively. The synchronization system of the two lasers was previously reported^{40 (link),41 (link)}. The wavelengths of these lasers were tuned to 709 nm and 888 nm to excite the ${\mathrm{CH}}_2$ symmetric stretching vibration of lipids at 2845 cm ${}^{-1}$ . Two laser beams were superimposed at a long-wavelength-pass filter and were focused with an objective lens (CFI Apo LWD, $\times 25$ , $\mathrm{NA}=1.1$ , WI, Nikon). Backward-propagating CARS was collected with the same objective lens and was detected with a photomultiplier tube (PMT; R9110, Hamamatsu). The powers of the two excitation beams on the sample plane were 31 mW for 709 nm and 11 mW for 888 nm. We observed 100 nerve images (field of view of $273\mathrm{\mu }\mathrm{m}$ , $500\times 500$ pixels, imaging rate: 66.7 images/min, exposure time: 0.9 s) at one position with CARS microscopy, and prepared a total of 9100 nerve images at 91 positions. An average image of 100 images at one position was used as a ground truth image.

To visualize liver tissues obtained from rabbit VX2 liver tumors, a custom-built video-rate confocal laser-scanning microscope was used 20 (link)-22 (link). Three continuous-wave laser modules with output wavelengths of 488 nm (MLD488, Cobolt), 561 nm (Jive, Cobolt), and 640 nm (MLD640, Cobolt) were used as excitation light sources. Two-dimensional laser scans were achieved using a rapidly rotating polygonal mirror with 36 facets (MC-5, Lincoln Laser) for fast-axis scanning, and a galvanometer scanning mirror (6230H, Cambridge Technology) for y-axis scanning. The multi-color fluorescence signals were captured by a high NA commercial objective lens (CFI Plan Apo λ, 10X, NA0.45, Nikon; UPLSAP0, 4X, NA 0.16, Olympus), and detected by three photomultiplier tubes (PMT; R9110, Hamamatsu). The output signals of each PMT were digitally acquired by frame grabber (Solios, Matrox), and the images were recorded and displayed with custom-written imaging software based on the Matrox Imaging Library (MIL9, Matrox) and Visual C++ 23 (link).