Fig. 5

Appearance of skeletonized kymograms—considerations for data quality control and transect size selection. a Representative skeletonized kymograms obtained from SDCM recordings from cotyledon epidermis of wt seedlings expressing Lifeact-mRFP using a 20 μm transect. With good quality video recordings, most kymograms should contain one or a few distinct long traces (left), with only a fraction possibly showing a meshwork or fragmented pattern (right). Too many meshwork kymograms result in most lifetime and lateral motility values close or equal to the maximum, preventing meaningful interpretation, while fragmented kymograms are indicative of noisy and thus possibly unreliable primary data. b Skeletonized kymograms obtained from the same place within a representative SDCM recording from fh1 cotyledon epidermis expressing Lifeact-mRFP (top) or from a VAEM recording of wt cotyledon epidermis expressing GFP-MAP4 (bottom) using the indicated transect sizes. Green frames indicates a recommended range of transect sizes for capturing both rare and frequent events (dotted line means sub-optimal, but possibly still usable, settings). While these examples illustrate lifetime measurements, analogous considerations also apply for quantification of lateral motility. Longest traces in the time dimension are shown in yellow, asterisks denote specific problems or features (see key in the figure)