Analysis to identify minimum and maximal intervals for the dCCD method. A.
A simple model to illustrate the limits of image intervals used for the dCCD method. Colored EB3-GFP labels are represented by linear arrays of colored squares equivalent to the pixels in digital images. The minimum interval (left) is defined as the time needed to detect the movement of the array from the nth frame (shown in green) to the n+1th frame (in red), leading to a new array with at least one red square followed by yellow and green squares in the merged dCCD images. The maximal interval (right) is defined as time allowed for the movement of one array length (4 pixels shown here) in the n+1th frame. B. Relationship of accuracy and time interval based on the numerical simulation using the criteria for minimum (left) and maximum (right) intervals defined in (A). For the minimum interval, accuracy is calculated as the number of ends that have moved at least one pixel length over 1000 total ends used in the simulation, whereas for the maximum interval, accuracy is calculated as the number of ends moved for less than the label length over 1000 simulated ends. Note the opposite relationship between the accuracy and the minimum or maximum interval. For fast growing microtubules (>14 µm/min), intervals >1 sec provide 98% accuracy for the minimum interval and <1 sec intervals give the same accuracy for the maximal interval. For slow growing ends found in the COS cell (4 µm/min), intervals between 3 sec and 7 sec can provide 95% accuracy.