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To understand brain functions, it is important to observe directly how multiple neural circuits are performing in living brains. However, due to tissue opaqueness, observable depth and spatiotemporal resolution are severely degraded in vivo. Here, we propose an optical brain clearing method for in vivo fluorescence microscopy, termed MAGICAL (magical additive glycerol improves clear alive luminance). MAGICAL enabled two-photon microscopy to capture vivid images with fast speed, at cortical layer V and hippocampal CA1 in vivo. Moreover, MA