Figure 3.

3D STORM imaging of actin reveals intra-axonal actin bundles. (A and A′) Widefield image of a hippocampal neuron labeled with phalloidin (F-actin) and Neurofascin-186 (NF-186; to label the axon initiation segment [AIS], brackets). Proximal axon marked with arrowheads. (A′) 3D-STORM image of the actin labeling corresponding to the boxed area in A. Color codes for Z depth from −300 nm (blue) to 300 nm (red). Note Y-shaped proximal axon, as well as distal axons from other neurons. (B and C) Zoomed XY projections (300–600 nm thick) of the boxed regions (B and C) in image A′. Note that several longitudinal actin filaments can be seen within the axon shaft (arrowheads), in addition to periodic actin rings (comb). (B′ and C′) XZ slices (800 nm thick) from axons shown in B and C (area within brackets) highlighting intra-axonal actin filaments (arrowheads, dashed ellipse localizes the axon boundary). (D) 3D STORM image of distal axons with a dendritic shaft on right. Color codes for Z depth from −300 nm (blue) to 300 nm (red). (E and E′) XY/XZ projections (300–400 nm thick) of the boxed regions E in image D. Note longitudinal actin filaments inside the distal axon (arrowheads), together with the periodic actin rings (comb). (F and F′) Same imaging parameters and scales as previous, showing an axon segment with actin rings. Bars: (A) 20 µm; (A′ and D) 5 µm; (B, C, and E–F′) 2 µm; (B′ and C′) 500 nm.