Figure 5. Sample tilting is necessary to achieve full coverage of microtubules in orthogonal sections.

(A) Example electron micrographs taken from a −45° to +45° tilt series of a K-fiber. Observable microtubules are marked with dots (bottom row). Black dots represent microtubules that are unique to that tilt frame. White dots represent the accumulating microtubules identified in previous tilt frames. The total number of microtubule annotations were pooled together onto one frame (far right) giving a fair overview of the whole K-fiber. Scale bar 100 nm. (B & C) A dual tilt series of one K-fiber was carried out. (B) Representative electron micrographs taken from the central region of both X and Y tilts (A-top). All microtubules observed in each tilt series were annotated (bottom; X axis in white, Y axis in black). The sum of microtubules from both tilts were pooled together on to a single blank image, any microtubules that were common to both tilts were marked grey. Scale bar, 100 nm. (C) A pie chart showing the percentage of total microtubules that were unique to each tilt and also the common ones.