Label-Free Tracking of Subcortical White Matter Degradation In Vivo Using Third Harmonic Generation Microscopy in a Mouse Model of Multiple Sclerosis

Early detection and characterization of myelin degradation in the white matter (WM) is important for understanding neurodegenerative diseases such as multiple sclerosis (MS) and dementia. Here, we demonstrate a label-free in vivo imaging technique using third harmonic generation (THG) microscopy at 1320-nm excitation, which enables visualization of subcellular myelin structural changes deep in the mouse brain. By applying longitudinal THG imaging of the same axons in the cuprizone mouse model of MS, we captured the formation, evolution, and regression of myelin blisters in the corpus callosum through intact cortex - a depth inaccessible to other optical methods. We also confirmed the utility of conducting THG imaging in parallel with three-photon excited fluorescence imaging, which allowed us to determine that myelin blistering events were not correlated with the location of microglia cell bodies. Further, we used post-mortem immunohistochemistry to establish the ability to identify and measure the intranodal distance at nodes of Ranvier using in vivo THG imaging, and we found increased intranodal distances after cuprizone administration. We also developed a novel quantitative metric based on spatial concentration of the brightest THG signal that occurs before more overt changes like myelin blistering. Overall, THG microscopy offers a powerful method for detailed tracking of subcortical myelin dynamics, providing new opportunities to investigate disease mechanisms and potential therapeutic interventions in MS and other neurodegenerative diseases.