Large-scale three-dimensional fluorescence imaging is essential for comprehensive and quantitative understanding of neuronal circuitry.We describe a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days leaving many types of fluorescent dyes unquenched, including fluorescent proteins and lipophilic neuronal tracers. This method maintains a constant sample volume during the clearing procedure, an important factor to keep cellular morphology intact. After optical clearing with SeeDB, we can reach a depth of >1000 μm with confocal microscopy. When combined with two-photon microscopy, SeeDB allows us to image fixed mouse brains at millimeter-scale level.
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