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.

Grasp the proximal end of the nerve and slowly lay it down onto a cover slip. Then, straighten it under gentle tension. Place the micro aqueduct slide over the nerve with the grooved slide down and position the direction of flow parallel to the nerve. Flip the cover slip and micro aqueduct assembly over and place it in the perfusion chamber housing with the slide opposed to the outer gasket.

To secure the perfusion chamber, place it in the metal housing and rotate the locking ring. Slowly depress the saline syringe plunger and fill the perfusion chamber. Keep the inlet and outlet tubing, outlet flask, and syringe elevated above the chamber itself at all times during the setup and imaging. Transfer the perfusion assembly to an inverted microscope stage and mount the saline syringe into the syringe pump. Then, start the motor and adjust the speed for a flow rate of 0.25 millilitres per minute.

Connect the inline solution heater and set it to 37 degrees Celsius. Connect the objective heater and set it to 37 degrees Celsius. Apply oil to the objective and insert the perfusion chamber into the stage mount. Apply oil to the chamber heater pad and attach it to the perfusion chamber. Then, connect it and set it to 37 degrees Celsius.

Lock the perfusion chamber into the stage adapter and bring the objective oil into contact with the cover slip on the underside of the chamber. Use brightfield illumination to focus on the layer of axons on the bottom surface of the nerve closest to the cover slip surface. Myelinated axons can be identified by the presence of a myelin sheath, which is visible under brightfield-transmitted light illumination without contrast enhancement.

Acquire a brightfield reference image, recording the directionality of the nerve. Then acquire a confocal image using a 488 nanometer laser and an emission filter appropriate for photoactivatable GFP. Set the laser power to approximately five times normal imaging power and acquire an image with an exposure time of three to four minutes. Then acquire another confocal image to record the pre-activation autofluorescence after the bleaching step.

Draw a line parallel to the axons with a length equal to the desired activation window size of the brightfield image. Using this line as a guide, draw a rectangular region of interest across the field of view perpendicular to the axons. Activate the GFP fluorescence in this region by pattern excitation with the 405 nanometer light, making sure that an image is acquired just prior to and immediately following activation. As the activation finishes, start a one-minute timer. When it goes off, start acquisition of a time-lapse series.