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Microtubule Segmentation and 3 D Reconstruction in Total Internal Reflection Fluorescence Microscopy ( TIRFM )

The interaction of the microtubules and their assembly with the cell membrane plays numerous critical roles in a cell. For example, it directs neurotransmitter vesicles to synapses and modulates membrane adhesions pivotal for cell movement and mitosis. Abnormal function of the assembly is involved in neurodegenerative diseases and cancer. A technique that enables the direct observation of microtubule function near the cell membrane is TIRF microscopy. To date most image and data analysis of the microtubule assembly has been done by visual inspection and manual tracing. In this work we have developed a method to automatically extract microtubules from TIRF microscopy images so as to enable a more extensive and higher throughput quantitative study of the microtubule assembly. The microtubules are first segmented in 2D in terms of consecutive segments. The segments are streamlines of HamiltonJocobi cost maps. Subsequently, the fluorescence is used to implement a limited reconstruction of the microtubules in 3D space. Last, we show examples of our method applied to phantom data as well as real TIRF microscopy images from fluorescently tagged living cells.