Confocal microscopy and passive staining with the styryl dye FM1-43: a convenient method to evaluate morphometric changes in nodes of Ranvier of single living myelinated axons
Résumé
Confocal laser scanning microscopy was used to image at high resolution single living myelinated axons isolated from the frog sciatic nerve. Before imaging, the nerve fibres were exposed to the fluorescent styryl dye FM1-43 and thereafter rinsed with dye-free solution. This procedure, which consistently stained nerve membranes for several hours, was particularly useful and especially adapted for delineating the contours of the node of Ranvier and revealing the myelin sheath layers surrounding the axon. Images of three-dimensional digital reconstructions of the structure of a single myelinated axon were obtained by “look-through” projections from series of optical sections through the axon, collected
before and during various treatments. Results were quantified, on the same myelinated axon, by measuring the internodal diameter, the nodal length (L) and the nodal diameter (D) and then by calculating the nodal volume (V) as: V = π L (D/2)2, assuming the simplest geometry in which a node of Ranvier approaches a cylinder. This method is of particular interest since it provides the possibility to detect and quantify changes in nodal volume of a given living myelinated axon under various experimental conditions. Results previously obtained with this method are shown.