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J Microsc. 2016 Jan;261(1):67-78. doi: 10.1111/jmi.12316. Epub 2015 Oct 7.

Important factors determining the nanoscale tracking precision of dynamic microtubule ends.

Author information

1
The Francis Crick Institute, Lincoln's Inn Fields Laboratory, London, U.K.
2
Present address: Gatsby Computational Neuroscience Unit, University College London, London, U.K.
3
Centre for Mathematics and Physics in Life Sciences and Experimental Biology (CoMPLEX), University College London, London, U.K.
4
Present address: MRC Laboratory for Molecular Cell Biology, University College London, London, U.K.
5
Present address: Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003, Barcelona, Spain.

Abstract

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.

KEYWORDS:

Image processing; TIRF microscopy; image simulation; microtubule; tracking

PMID:
26444439
PMCID:
PMC4832305
DOI:
10.1111/jmi.12316
[Indexed for MEDLINE]
Free PMC Article

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