Abstract Multiple sclerosis (MS) is a demyelinating disease of the central nervous system affecting more than 2.3 million people worldwide. The pathogenic mechanism is not well understood and the diagnosis does not occur early enough to prevent further neurodegeneration. Evidence suggests that demyelination in the cerebrum is an important lesion of MS correlating well with cognitive defects in the disease. However, the clinical significance has not been fully established because cortical myelin cannot be observed in vivo over a long period. Here we propose to develop optical methods for studying cortical myelin in living animals. A new imaging modality will be employed, namely third-harmonic generation microscopy (THGM), which we recently demonstrated for label-free visualization of myelin in intact nerves of the peripheral nervous system. Powerful imaging capabilities will be developed suitable for advancing our knowledge of cortical myelination. First, THGM will be validated for probing cortical demyelination and remyelination in living MS animal models. Second, wavefront control will be implemented to facilitate large-scale THGM imaging of cortical myelin in deep mouse brain. Third, super-resolution THGM morphometry will be achieved by image restoration and probabilistic modeling. Upon successful completion, this project will open a new field of investigations in basic science as well as translational medicine to elucidate the axon-glial cell interaction underlying cortical demyelination and remyelination.