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High-speed super-resolution imaging of rotationally symmetric structures using SPEED microscopy and 2D-to-3D transformation

Nature Protocols volume 16, pages 532–560 (2021)Cite this article

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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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Fig. 1: A schematic of excitation optical paths for both vertical and inclined illumination.
Fig. 2: 3D information derived from 2D single-molecule data using a 2D-to-3D transformation.
Fig. 3: Representative data from SPEED microscopy experiments.
Fig. 4: Representatives of 3D probability density maps in three rotationally symmetric systems.
Fig. 5: Schematic workflow for SPEED microscopy.
Fig. 6: Optical paths in the SPEED microscope and other microscopes.
Fig. 7: Validation of the reproducibility of SPEED microscopy and its 2D-to-3D histogram.
Fig. 8: A schematic showing data normalization and 2D-to-3D transformation.

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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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  1. These authors contributed equally: Yichen Li, Mark Tingey.

Authors and Affiliations

  1. Department of Biology, Temple University, Philadelphia, PA, USA

    Yichen Li, Mark Tingey & Weidong Yang

  2. Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA

    Andrew Ruba

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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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Key references using this protocol

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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