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.

We describe a novel method for visualizing the network of axons in the unlabeled fresh wholemount retina. The intrinsic radiation of second harmonic generation (SHG) was utilized to visualize single axons of all major retinal neurons, i.e. photoreceptors, horizontal cells, bipolar cells, amacrine cells, and the retinal ganglion cells. The cell types of SHG + axons were determined using transgenic GFP/YFP mice. New findings were obtained with retinal SHG imaging: Müller cells do not maintain uniformly polarized microtubules in the processes; SHG + axons of bipolar cells terminate in the inner plexiform layer (IPL) in a subtype-specific manner; a subset of amacrine cells, presumably the axon-bearing types, emits SHG; and the axon-like neurites of amacrine cells provide a cytoskeletal scaffolding for the IPL stratification. To demonstrate the utility, retinal SHG imaging was applied for testing whether the inner retina is preserved in glaucoma, using DBA/2 mice as a model of glaucoma and DBA/2-Gpnmb + as the non-glaucomatous control. It was found that the morphology of the inner retina was largely intact in glaucoma and the pre-synaptic compartments to the retinal ganglion cells were uncompromised. It proves retinal SHG imaging as a promising technology for studying the physiological and diseased retinas in 3D.

We describe a novel microscopy for studying the network of axons in the unlabeled fresh wholemount retina. The intrinsic radiation of second harmonic generation (SHG) was utilized to visualize single axons of all major retinal neurons, i.e., photoreceptors, horizontal cells, bipolar cells, amacrine cells, and the retinal ganglion cells. The cell types of SHG+ axons were determined using transgenic GFP/YFP mice. New findings were obtained with retinal SHG imaging: Müller cells do not maintain uniformly polarized microtubules in the processes; SHG+ axons of bipolar cells terminate in the inner plexiform layer (IPL) in a subtype-specific manner; a subset of amacrine cells, presumably the axon-bearing types, emits SHG; and the axon-like neurites of amacrine cells provide a cytoskeletal scaffolding for the IPL stratification. To demonstrate the utility, retinal SHG imaging was applied for testing whether the inner retina is preserved in glaucoma, using DBA/2 mice as a model of glaucoma and DBA/2-Gpnmb+ as the non-glaucomatous control. It was found that the morphology of the inner retina was largely intact in glaucoma and the pre-synaptic compartments to the retinal ganglion cells were uncompromised. It proves retinal SHG imaging as a promising technology for studying the physiological and diseased retina in 3D.

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