Extended Fig. 6. Variability of imaging angles has virtually no impact on determinations of spine turnover.

We examined empirically whether variations in dendritic angle across different imaging sessions might impact determinations of spine turnover. However, the variations in dendritic angle that were actually present in our datasets were insufficient to cause illusory turnover.

(a, b) Dendritic spines can be detected when the angle (θ) between a spine and the normal vector is large (Supplementary Methods, §IV). View of a dendrite and spine in the (x, z), (a), and optical (x, y), (b), planes.

(c, d) Dendritic spines cannot be detected when the angle between a spine and the normal vector (θ) is small (Supplementary Methods, §IV). View of a dendrite and spine in the (x, z), (c), and optical (x, y), (d), planes.

(e) For every dendrite and time point, we estimated the dendrite’s angle with respect to the optical plane, using the 3D coordinates of two manually labeled points on the dendrite chosen to flank the region of dendrite containing the scored spines. Over time, individual dendrites varied about their initial angle (n = 55 dendrites tracked over 16 sessions; dataset of Fig. 3d).

(f) Distribution of the fluctuations in angle, pooled across the 55 dendrites, relative to the initial angle as seen in the first imaging session. The average magnitude of an angular fluctuation was 4.5°, and 90% of angular fluctuations were <10° in magnitude. Thus, a 5° fluctuation was typical in our dataset, whereas a 10° fluctuation was atypically large.

(g) To determine if variability in the imaging angle might impact determinations of spine turnover, we imaged 18 dendrites in fixed slices while deliberately tilting the imaging plane by 0°, 5° and 10°. We made a total of 989 spine observations. Over 95% of spines scored in the 0°-condition were also correctly scored when the specimen as tilted by 5° or 10°. Overall, the level of angular fluctuations in the in vivo imaging data has virtually no impact on turnover scores.