The main aim of this work was to develop necessary tools and methods in order to improve the quality of study of microtubules in vivo using digital fluorescence microscopy. Through combination of microtubule modeling, model convolution, creating programs for automated image analysis, and imaging itself, such issues as using fixed samples with long exposure times can be overcome. We used the MATLAB® numerical computing environment to create computer scripts that are able to analyze images obtained via digital fluorescence microscope and get important insights about the dynamic nature of microtubules and their tip structures in vivo. The foundation of this project was our previously published MATLAB® microtubule contour tracking code . After necessary modifications to the script had been made, the testing the MATLAB® code on the simulated images of microtubules revealed the typical accuracy of tip tracking in living LLC-PK1 cells with our microscope system was around 36 nm, but it could be as good as ~15 nm if observed microtubule tips are blunt. By using the same algorithm, we also have established that most microtubules within living cells are not blunt, but exhibit highly variable tapered structures (p-value<10-69). Using our conversion tables we were able to determine the mean taper length of microtubules within living LLC-PK1 cells to be 418 nm (with the standard deviation of 420 nm) and in fixed cells of the same type it was 387 nm (with 420 nm as the standard deviation).
University of Minnesota M.S. thesis. June 2010. Major: Biomedical Engineering. Advisor: David J. Odde. 1 computer file (PDF); v, 69 pages. Ill. (some col.)
Demchouk, Alexei Olegovich.
Microtubule tip tracking and tip structures at the nanometer scale using digital fluorescence microscopy..
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