Label-free assessment of myelin status using birefringence microscopy

Background: Label-free methods for quantifying myelination can reduce expense, time, and variability in results when examining tissue white matter pathology. New method: We sought to determine whether the optical birefringent properties of myelin could be exploited to determine myelination status of white matter in tissue sections. Sections of forebrains of mice (normal, and treated with cuprizone to cause demyelination) were examined by birefringence using a birefringence imaging system (Thorlabs LCC7201), and results compared with sections stained using Luxol Fast Blue. Results: Quantitative birefringence analysis of myelin was not only reliable in detecting demyelination, but also showed abnormalities that preceded myelin loss in cuprizone-treated mice. Comparison with existing methods: Subtle myelin pathology visible with electron microscopy but not with conventional histopathological staining was readily detected with birefringence microscopy. Conclusions: Birefringence imaging provides a rapid, label-free method of analyzing the myelin content and nanostructural status in longitudinal white matter structures, being sensitive to subtle myelin changes that precede overt pathological damage.