5 January 2017 3D reconstruction of cortical microtubules using multi-angle total internal reflection fluorescence microscopy
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Proceedings Volume 10245, International Conference on Innovative Optical Health Science; 1024506 (2017) https://doi.org/10.1117/12.2265976
Event: International Conference on Innovative Optical Health Science, 2016, Shanghai Everbright International Hotel, China
Abstract
Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different illumination angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve ∼40 nm axial resolution in experimental conditions where SNR is as low as 2, with ∼35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.
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Luhong Jin, Peng Xiu, Xiaoxu Zhou, Jiannan Fan, Cuifang Kuang, Xu Liu, Yingke Xu, "3D reconstruction of cortical microtubules using multi-angle total internal reflection fluorescence microscopy", Proc. SPIE 10245, International Conference on Innovative Optical Health Science, 1024506 (5 January 2017); doi: 10.1117/12.2265976; https://doi.org/10.1117/12.2265976
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