Label-free tracking of subcortical white matter degradation <i>in vivo</i> using third harmonic generation microscopy in a mouse model of multiple sclerosis

Characterization of myelin degradation in the white matter (WM) is important for understanding neurodegeneration. We demonstrate label-free <i>in vivo</i> imaging of myelin structure in the WM of mice, through intact cortex, using third harmonic generation (THG) microscopy at 1320-nm excitation. Longitudinal THG imaging of the same axons in the cuprizone mouse model of multiple sclerosis revealed dynamics of myelin blistering. Further, we measured intranodal distance at nodes of Ranvier <i>in vivo</i> and developed a novel metric of myelin structural change based on spatial concentration of the brightest THG signal. We also demonstrated compatibility with three-photon excited fluorescence microscopy by imaging GFP-labeled microglia in the WM in parallel with THG microscopy, thereby enabling detailed tracking of subcortical myelin and other cellular dynamics in neurodegenerative disease models.