Optical Clearing and Index Matching of Tissue Samples for High-resolution Fluorescence Imaging Using SeeDB2

Tissue clearing techniques are useful for large-scale three-dimensional fluorescence imaging of thick tissues. However, high-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we present a water-based optical clearing and mounting media, SeeDB2, which is designed for high numerical aperture (NA) objective lenses with oil or glycerol immersion. Using quick and simple soaking procedures, the refractive indices of samples can be matched either to that of immersion oil (1.52) or glycerol (1.46), thus minimizing light scattering and spherical aberrations. Fine morphology and various fluorescent proteins are highly preserved during the clearing and imaging process. Our method is useful for the three-dimensional fluorescence imaging of neuronal circuitry at synaptic resolution using confocal and super-resolution microscopy. SeeDB2 is also useful as a mounting media for the super-resolution imaging of fluorescent proteins.