Applied electric fields accelerate the diffusion rate and increase the diffusion distance of DiI in fixed tissue

Lipophilic carbocyanine dyes are effective neuronal tracers in fixed tissue. However, their application has been limited by the slow diffusion, short tracing distances, and long durations of incubation in fixed tissue. We used applied dc electric fields, that exerted forces on the cationic dyes, to increase the diffusion velocity and maximal tracing distances of DiI and its analogs. Maximum diffusion distances of DiI in fixed human peripheral nerve were approximately 4 times longer then the previous reported maximum, and diffusion velocities was approximately 100 times faster in samples exposed to the electric field than in control samples. This method enabled retrograde tracing from a distal nerve branch into a proximal nerve trunk, and did not result in lateral transaxonal diffusion. Field enhanced diffusion will expand the range of uses of lipophilic dyes in fixed tissues and enable topographic mapping of peripheral nerve fascicles in post-mortem tissue.