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Many histological techniques are used to identify and characterize myelin in the mammalian nervous system. Due to the high content of lipids in myelin sheaths, coherent anti-stokes Raman scattering (CARS) microscopy is a label-free method that allows identifying myelin within tissues. CARS excites the CH 2  vibrational mode at 2845 cm −1  and CH 2  bonds are found in lipids. In this study, we have used CARS for a new biological application in the field of spinal cord analysis. We have indeed compared several parameters of spinal cord myelin sheath in three different species,  i.e ., mouse, lemur, and human using a label-free method. In all species, we analyzed the dorsal and the lateral  funiculi  of the adult thoracic spinal cord. We identified g-ratio differences between species. Indeed, in both  funiculi,  g-ratio was higher in mice than in the two primate species, and the myelin g-ratio in lemurs was higher than in humans. We also detected a difference in g-ratio between the dorsal and the lateral  funiculi  only in humans. Furthermore, species differences between axon and fiber diameters as well as myelin thickness were observed. These data may reflect species specificities of conduction velocity of myelin fibers. A comparison of data obtained by CARS imaging and fluoromyelin staining, a method that, similar to CARS, does not require resin embedding and dehydration, displays similar results. CARS is, therefore, a label-free alternative to other microscopy techniques to characterize myelin in healthy and neurological disorders affecting the spinal cord.

Coherent Anti-Stokes Raman Scattering Microscopy: A Label-Free Method to Compare Spinal Cord Myelin in Different Species