Abstract
Various super-resolution imaging techniques have been developed to break the diffraction-limited resolution of light microscopy. However, it still remains challenging to obtain three-dimensional (3D) super-resolution information of structures and dynamic processes in live cells at high speed. We recently developed high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy and its two-dimensional (2D)-to-3D transformation algorithm to provide an effective approach to achieving 3D sub-diffraction-limit information in subcellular structures and organelles that have rotational symmetry. In contrast to most other 3D super-resolution microscopy or 3D particle-tracking microscopy approaches, SPEED microscopy does not depend on complex optical components and can be implemented onto a standard inverted epifluorescence microscope. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside sub-micrometer biological channels or cavities at high spatiotemporal resolution. After data collection, post-localization 2D-to-3D transformation is applied to obtain 3D super-resolution structural and dynamic information. The complete protocol, including cell culture and sample preparation (6–7 d), SPEED imaging (4–5 h), data analysis and validation through simulation (5–13 h), takes ~9 d to complete.
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Data availability
The data used to generate example data figures can be found on our lab’s GitHub repository: https://github.com/YangLab-Temple/Data.
Code availability
The code used in this work is available at https://github.com/YangLab-Temple/Master under the GNU General Public License v3.0. Specifically, the code for the reproducibility rate can be found at https://github.com/YangLab-Temple/Master/tree/master/reproducibility%20rate.
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Acknowledgements
The authors acknowledge S. J. Junod for his assistance during the preparation of this manuscript. We also thank the many collaborators who have greatly helped us complete the research included in this manuscript: E. C. Schirmer (University of Edinburgh), K. J. Verhey (University of Michigan), N. G. Walter (University of Michigan) and R. Y. H. Lim (University of Basel). The project was supported by grants from the National Institutes of Health (GM094041, GM097037, GM116204 and GM22552 to W.Y.).
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Y.L., M.T. and A.R. produced the figures and tables and wrote and edited the manuscript. W.Y. edited the manuscript and provided appropriate guidance.
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Ma, J. & Yang, W. Proc. Natl Acad. Sci. USA 107, 7305–7310 (2010): https://doi.org/10.1073/pnas.0908269107
Ma, J. et al. Nat. Struct. Mol. Biol. 23, 239–247 (2016): https://doi.org/10.1038/nsmb.3174
Luo, W. et al. Sci. Rep. 7, 15793 (2017): https://doi.org/10.1038/s41598-017-16103-z
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Li, Y., Tingey, M., Ruba, A. et al. High-speed super-resolution imaging of rotationally symmetric structures using SPEED microscopy and 2D-to-3D transformation. Nat Protoc 16, 532–560 (2021). https://doi.org/10.1038/s41596-020-00440-x
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DOI: https://doi.org/10.1038/s41596-020-00440-x
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