Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy

Abstract

A new technique that allows high-resolution in vivo imaging of myelinated fibers without the use of a fluorescent marker is described by Aaron Schain and colleagues. This label-free approach, which does not require histological or immunocytochemical staining, uses spectral confocal reflectance microscopy, can be performed on a conventional confocal microscope and can be used for deep-tissue transcranial imaging up to 400 μm deep, longitudinally tracking fine changes in axonal myelination. It has potential for the in vivo analysis of normal myelin development, as well as demyelinating diseases of the CNS and peripheral nervous system. We report a newly developed technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser-scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we carried out chronic brain imaging up to 400 μm deep, capturing de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans.