A new label-free method is presented for measuring myeloarchitecture of the murine cerebral cortex in vivo and ex vivo . Growing evidence suggests that cortical myelination plays significant roles in neuronal plasticity and pathologies, such as multiple sclerosis (MS), but illuminating the mechanism requires longitudinal imaging of the same brains. Here we demonstrate imaging unlabeled myelinated fibers in a live mouse brain by third-harmonic generation (THG). Contrary to other label-free microscopies based on reflectance, fibers of all orientations could be visualized, i.e., radial and tangential to the pia, which is suitable for revealing the three-dimensional connectivity. The depth of THG imaging in an intact brain was approximately 200 μm, so the network of myelinated fibers could be captured into layers 2/3 in vivo . THG provides a novel base for reconstruction of morphology. Semi-automatic tracing of THG-positive axons unraveled the depth-dependent distribution of the myelin lattice. Finally, a unique light property of THG was exploited for the estimation of the g-ratio. The demonstrated THG morphometry of the length density, orientation, and sheath thickness of cortical myelin could be useful for elucidating its function and how it is modulated during learning and disease.

Here we demonstrate high-pulse-energy multiphoton microscopy (MPM) for intravital imaging of neurons and oligodendrocytes in the murine brain. Pulses with an order of magnitude higher energy (~ 10 nJ) were employed from a ytterbium doped fiber laser source at a 1-MHz repetition rate, as compared to the standard 80-MHz Ti:Sapphire laser. Intravital imaging was performed on mice expressing common fluorescent proteins, including green (GFP) and yellow fluorescent proteins (YFP), and TagRFPt. One fifth of the average power could be used for superior depths of MPM imaging, as compared to the Ti:Sapphire laser: A depth of ~ 860 µm was obtained by imaging the Thy1-YFP brain in vivo with 6.5 mW, and cortical myelin as deep as 400 µm ex vivo by intrinsic third-harmonic generation using 50 mW. The substantially higher pulse energy enables novel regimes of photophysics to be exploited for microscopic imaging. The limitation from higher order phototoxicity is also discussed.