Third Harmonic Generation: A Method for Visualizing Myelin in the Murine Cerebral Cortex
Here we present the use of Third Harmonic Generation (THG) for the label-free imaging of myelinated axons in the murine cerebral cortex. Myelin plays an important role in the processes of learning and disease. However, much of the myelin biology research thus far has focused on white matter tracts where myelin is more visible. Much is still unknown, particularly with regard to myelin in gray matter. First, we engage in THG microscopy using an optical parametric oscillator pumped by a titanium-sapphire laser to demonstrate the utility of the technique for imaging myelin in vivo. Second, we investigate the use of a custom built low-repetition rate laser to substantially increase THG signal. We characterize the improvements and limitations of this light source with regards to THG microscopy. Lastly, we demonstrate a method for the estimation of the g-ratio from THG images by the use of a Bayesian model. The g-ratio is an important physical property relating to the thickness of the myelin sheath; modulation in the g-ratio could give clues to its underlying function. THG microscopy is uniquely adept at providing the data necessary for a g-ratio estimation.
Biophysics, Molecular and Cellular Neuroscience, Optics, Nonlinear optical microscopy, Label-free imaging, Myelin, Gaussian Mixture Model
City University of New York - Graduate Center
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The Graduate School and University Center of The City University of New York