Interbranch axonal diameter was sampled at high spatial frequency from branch point to branch point. This representative example based on
Figure 1e, depicts a 3
rd order branch segment. (
a) Axonal diameter was measured using the average intensity projection of a
z-stack series of SIM images (red). A semi-automated, user-guided line was traced from the center of each axonal branch point to the center of the subsequent distal branch point (thin white line). Each segment was analyzed in the direction centrifugally oriented from the soma along the axon, as indicated by the white arrow. For every consecutive 40 nm of axon, a 50-pixel wide line (~2.0 µm) was drawn orthogonally to the tangent line of axonal orientation (white). Scale bar, 5 µm. (
b) Along the orthogonals, pixel fluorescence intensities were extracted (black diamonds) and a Gaussian line was fitted (grey). Fits with r
2 <0.9 were excluded from further analysis. From the Gaussian fit at each consecutive axonal position, the full-width at half-maximum (FWHM) was calculated. In this representative example, the FWHM (axonal diameter)=0.238 µm and
r2 = 0.987. (
c) Left axis: Axonal diameter measurements as a function of centrifugal distance along the axonal segment shown in (
a). Right axis: Fit (r
2) of the Gaussian function along the axonal segment. Black arrows indicate the location of the corresponding fit shown in (
b). (
d) Distribution over branch order of median Gaussian fit (r
2) prior to exclusion of points with r
2 <0.9. Individual cells in grey, average in black. (
e) Goodness of fit (r
2) is similar between myelinated and unmyelinated segments. Individual cells in grey, average in black. Abbreviations: FWHM, full-with at half-maximum.