Important factors determining the nanoscale tracking precision of dynamic microtubule ends

Summary Tracking dynamic microtubule ends in fluorescence microscopy movies provides insight into the statistical properties of microtubule dynamics and is vital for further analysis that requires knowledge of the trajectories of the microtubule ends. Here we analyse the performance of a previously developed automated microtubule end tracking routine; this has been optimized for comparatively low signal‐to‐noise image sequences that are characteristic of microscopy movies of dynamic microtubules growing in vitro. Sequences of simulated microtubule images were generated assuming a variety of different experimental conditions. The simulated movies were then tracked and the tracking errors were characterized. We found that the growth characteristics of the microtubules within realistic ranges had a negligible effect on the tracking precision. The fluorophore labelling density, the pixel size of the images, and the exposure times were found to be important parameters limiting the tracking precision which could be explained using concepts of single molecule localization microscopy. The signal‐to‐noise ratio was found to be a good single predictor of the tracking precision: typical experimental signal‐to‐noise ratios lead to tracking precisions in the range of tens of nanometres, making the tracking program described here a useful tool for dynamic microtubule end tracking with close to molecular precision. Lay Description Microtubules form part of the ‘skeleton’ of a cell and are highly dynamic, tubular polymers which grow and shrink in order to perform specific roles at different points of the cell's life cycle. A powerful method to study these microtubules is fluorescence microscopy; tracking the end position of microtubules in movies provides insight into the mechanisms that govern microtubule dynamics. It is important for further analysis of these dynamics that the position of the microtubule ends can be reliably and accurately determined throughout a microtubule's growth. Here we describe and analyse the performance of an automated microtubule end‐tracking program that has been optimised for microscopy image sequences of purified dynamic microtubules growing on a slide, outside the cellular environment. The errors associated with tracking the microtubule ends have been characterised here using simulated movies. Simulations allow a range of experimental parameters to be tested with knowledge of the true end position, to evaluate to the results of the trackin