Fluorescence Photoactivation Imaging of Neurofilament Transport in Myelinated Mouse Axons

Begin with a perfusion assembly placed on the stage of an inverted microscope.

The perfusion assembly contains a dissected peripheral nerve secured between a coverslip and a micro aqueduct slide with continuous saline perfusion.

The nerve’s myelinated axons express the green fluorophore-tagged neurofilaments.

Using brightfield illumination, focus on the myelinated axons.

Switch to confocal mode, photobleach with high laser power, then capture a low-autofluorescence reference image using low laser power.

Draw parallel lines along the axon to define the photoactivation window length. Use this as a guide to create a rectangular activation region.

Illuminate this region with a specific wavelength to activate the green fluorophore-tagged neurofilaments, causing them to fluoresce. Acquire the image.

After activation, capture time-lapse images.

Over time, the fluorescent neurofilaments move in both directions and spread into adjacent flanking regions.

The appearance of fluorescence in the flanking regions adjacent to the activation region confirms neurofilament transport.