Spectral confocal reflectance microscopy helps visualize myelinated axons in vivo without any labeling.
Myelin wraps tightly around axons to insulate them and to increase conductance speed. Defects in myelination can impair signal propagation along the axon and lead to nerve damage. For studying myelin in vivo, genetically encoded reporters or complicated microscopy techniques have so far been necessary. Jaime Grutzendler and his colleagues at Yale University recently developed a simple, label-free imaging technique based on the reflective properties of myelin. “This technique opens a way to study cortical myelin in vivo, to understand its development, maintenance in adulthood and different pathologies,” explains Grutzendler.
The new technique, called spectral confocal reflectance microscopy (SCoRe), is simple yet powerful. Under a confocal microscope, myelinated axons reflect light when illuminated with laser beams of different wavelengths. These reflections are discontinuous but cover the whole axon when merged into a single image, and they arise at the interphase between axons and the surrounding myelin sheaths, probably because of the difference in refractive index between axons and lipid-rich myelin. Owing to the high levels of reflection even at low laser powers, the researchers could visualize myelinated axons up to 400 micrometers deep in the cortex in an anesthetized mouse.
In the sciatic nerve and spinal cord of mice, SCoRe generates colorful images of myelinated axons. For each axon, the color pattern is patchy but one particular hue dominates. Grutzendler thinks that “the hue ... has something to do with the overall diameter of the axons and the number of layers of myelin that are wrapping around the axon processes. The intrinsic patchiness ... is probably some local heterogeneity of the myelin.” These characteristics distinguish an axon from its neighbors and can be used to identify axons at different time points, as the color pattern stays almost constant over time. Grutzendler's team found that, in contrast to the heterogeneity of myelinated axons in the peripheral nervous system, axons in the cortex are more homogeneous in diameter and color pattern.
When imaging the sciatic nerve in vivo, the researchers observed structures with a vertical reflection pattern within the myelinated axons. These structures were revealed to be Schmidt-Lanterman incisures, which are channels that are important for myelin maintenance and are rich in gap junctions. Grutzendler believes that SCoRe will be a useful technique to study myelin pathologies caused by mutations in gap-junction proteins.
Grutzendler and his team used SCoRe microscopy for several applications. In so-called shiverer mice, myelin is not compacted owing to a mutation in the gene encoding myelin basic protein; consequently, the researchers observed very little reflectance present in the cortex of these mice. In addition, they found that SCoRe could detect changes in myelin after nerve injury in the peripheral nervous system. The researchers also observed reflection from myelinated axons in fixed human brain tissue.
In the future, Grutzendler wants to use the technique to study cortical myelin, which is less well studied than subcortical myelin. He is interested in pathologies in the nervous system but also plans to address simple questions such as what the turnover of myelin is and what role neural activity has in myelin formation. SCoRe might be useful not only for myelin research but also in other areas: for example, for tracing neurons. Finally, it could be interesting to follow traumatic brain injuries in humans via SCoRe. Grutzendler concedes that “this is fairly futuristic at this point, but not completely impossible.”
Schain, A.J. et al. Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy. Nat. Med. 20, 443–449 (2014).