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Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms

Nature Methods volume 10, pages 653–658 (2013)Cite this article

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Abstract

Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.

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Figure 1: sCMOS camera–specific algorithms enable unbiased SMSN at the theoretical limit.
Figure 2: Unbiased, fast SMSN of fixed microtubules, demonstrating high-throughput capabilities.
Figure 3: Live-cell SMSN at 0.5- to 2-s temporal resolution.
Figure 4: Video-rate live-cell nanoscopy of transferrin receptor clusters in live EA.hy926 cells.

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Acknowledgements

We thank J. Munro, P. Pellett, L. Schroeder, F. Bottanelli and M. Gudheti for helpful discussions about the buffer and sample preparation, J. Spatz for support, and P. de Camilli, O. Idevall-Hagren, T. Gould, E. Allgeyer and E. Kromann for helpful comments on the manuscript. We thank P. Xu (Chinese Academy of Sciences) for providing the mEos3.2 plasmid for initial experiments and G. Patterson (US National Institutes of Health) for the human clathrin light chain plasmid. This work was supported by grants from the Wellcome Trust (095927/A/11/Z), US National Institutes of Health (R01 CA098727 to W.M.) and Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences.

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Author notes

  1. Tobias M P Hartwich and Felix E Rivera-Molina: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA

    Fang Huang, Tobias M P Hartwich, Felix E Rivera-Molina, Whitney C Duim, Jordan R Myers, Derek Toomre & Joerg Bewersdorf

  2. Department of Biophysical Chemistry, University of Heidelberg, Heidelberg, Germany

    Tobias M P Hartwich

  3. Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, Stuttgart, Germany

    Tobias M P Hartwich

  4. Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA

    Yu Lin & Joerg Bewersdorf

  5. Integrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, Connecticut, USA

    Yu Lin & Joerg Bewersdorf

  6. Yale College, Yale University, New Haven, Connecticut, USA

    Jane J Long

  7. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA

    Pradeep D Uchil & Walther Mothes

  8. National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida, USA

    Michelle A Baird & Michael W Davidson

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  1. Fang Huang
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Contributions

F.H. and J.B. conceived the project. F.H., T.M.P.H., Y.L. and J.B. built the setup and designed the bead experiments. All authors designed the biological imaging experiments. F.H., T.M.P.H., Y.L., J.J.L., P.D.U. and J.R.M. performed the fixed-cell experiments. F.H., F.E.R.-M., W.C.D. and J.J.L. performed the live-cell experiments. M.A.B. and M.W.D. generated the mEos3.2 and tdEos plasmids. F.H. wrote the software and performed the simulations and analysis. All authors wrote the manuscript.

Corresponding author

Correspondence to Joerg Bewersdorf.

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Competing interests

F.H. and J.B. are co-inventors on a patent application related in part to the material presented here. J.B. is consultant, equity holder and member of the scientific advisory board of Vutara, Inc., which makes super-resolution microscopes.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Table 1 and Supplementary Note (PDF 4240 kb)

Supplementary Data

ZIP archive of obtained localization estimates and uncompressed super-resolution images for Figure 2a. (i) Uncompressed super-resolution image stretched for visualization purpose. (ii) Uncompressed 2D histogram image. (iii) List of localization estimates containing x, y position estimates and their averaged localization uncertainty (square root of their mean variance from CRLBsCMOS). Units are in pixels (103 nm). (ZIP 53695 kb)

Supplementary Software

Example of the developed algorithms implemented in Matlab and CUDA. (ZIP 50737 kb)

Super-resolution video of mEOS3.2-labeled CCPs in a live HeLa cell

Raw data were recorded as described in the Online Methods. Acquired images were analyzed using single-emitter fitting (Online Methods). 1,200 frames were combined to reconstruct each super-resolution image corresponding to a 2-s time window. Localization estimates in each image were binned into 20-nm pixels for display. To generate the video, we combined all 40 super-resolution images into a three-dimensional (3D) data stack and smoothed with a 3D Gaussian kernel with σx,y = 20 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar, 5 μm. (AVI 105080 kb)

Super-resolution video of mEOS3.2-labeled CCPs in a second live HeLa cell

This video corresponds to the data set shown in Figure 3a–c. Raw data were recorded as described in the Online Methods. Acquired images were analyzed using single-emitter fitting (Online Methods). 1,200 frames were combined to reconstruct each super-resolution image corresponding to a 2-s time window. Localization estimates in each image were binned into 20-nm pixels for display. To generate the video, we combined all 22 super-resolution images into a 3D data stack and smoothed with a 3D Gaussian kernel with σx,y = 20 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar, 5 μm. (AVI 57801 kb)

Super-resolution video for a small cutout of a larger data set of mEOS3.2-labeled CCPs in a live HeLa cell (1)

Raw data were recorded as described in the Online Methods. Acquired images were analyzed using single-emitter fitting (Online Methods). 1,200 frames were combined to reconstruct each super-resolution image corresponding to a 2-s time window. Localization estimates in each image were binned into 10-nm pixels for display. To generate the video, we combined all 28 super-resolution images into a 3D data stack and smoothed with a 3D Gaussian kernel with σx,y = 10 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar, 500 nm. (AVI 55160 kb)

Super-resolution video for a small cutout of a larger data set of mEOS3.2-labeled CCPs in a live HeLa cell (2)

This video corresponds to the data set shown in Figure 3a–c. Raw data were recorded as described in the Online Methods. Acquired images were analyzed using single-emitter fitting (Online Methods). 1,200 frames were combined to reconstruct each super-resolution image corresponding to a 2-s time window. Localization estimates in each image were binned into 10-nm pixels for display. To generate the video, we combined all 19 super-resolution images into a 3D data stack and smoothed with a 3D Gaussian kernel with σx,y = 10 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar, 500 nm. (AVI 37431 kb)

Super-resolution video for a small cutout of a larger data set of mEOS3.2-labeled CCPs in a live HeLa cell (3)

This video corresponds to the data set shown in Figure 3a–c. Raw data were recorded as described in the Online Methods. Acquired images were analyzed using single-emitter fitting (Online Methods). 1,200 frames were combined to reconstruct each super-resolution image corresponding to a 2-s time window. Localization estimates in each image were binned into 10-nm pixels for display. To generate the video, we combined all 29 super-resolution images into a 3D data stack and smoothed with a 3D Gaussian kernel with σx,y = 10 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar, 500 nm. (AVI 57130 kb)

Super-resolution video of tdEos-labeled human PDHA1 in COS-7 cells

Raw data were recorded as described in the Online Methods. Acquired images were analyzed using our multi-emitter fitting algorithm. 200 frames were combined to reconstruct each super-resolution image corresponding to a 0.5-s time window. Localization estimates in each image were binned into 20-nm pixels for display. To generate the video, we combined all 144 super-resolution images into a 3D data stack and smoothed with a 3D Gaussian kernel with σx,y = 20 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 25 frames per second. Scale bar , 5 μm. (AVI 225904 kb)

Super-resolution video of mEOS3.2-labeled EB3 in live HeLa cells

Raw data were recorded as described in the Online Methods. This video corresponds to one of the data sets shown in Supplementary Figure 11. Acquired images were analyzed using our multi-emitter fitting algorithm. 600 frames were combined to reconstruct each super-resolution image corresponding to a 1-s time window. Localization estimates in each image were binned into 20-nm pixels for display. To generate the video, we combined all 21 super-resolution images into a 3D data stack and smoothed with a 3D Gaussian kernel with σx,y = 20 nm and σt = 2 s to aid visualization. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar , 5 μm. (AVI 41371 kb)

Super-resolution video of tdEos-labeled peroxisome membrane protein in live COS-7 cells

This video corresponds to the dat aset shown in Figure 3d–f. Raw data were recorded as described in Online Methods. Acquired images were analyzed using our single-emitter fitting algorithm. 300 frames were combined to reconstruct each super-resolution image corresponding to a 0.5-s time window. Localization estimates in each image were binned into 20-nm pixels for display. To generate the video, we smoothed all 165 super-resolution images with a 2D Gaussian kernel with σx,y = 20 nm to aid visualization and then combined them into a 3D data stack. The resulting stack was converted into a video playing back at 15 frames per second. Scale bar, 5 μm. (AVI 433405 kb)

Super-resolution video of Alexa Fluor 647–labeled transferrin receptor cluster dynamics as shown in Figure 4a

Each frame corresponds to a 31-ms reconstructed super-resolution image and is played back at four frames per second. (AVI 1538 kb)

Super-resolution video of Alexa Fluor 647–labeled transferrin receptor cluster dynamics as shown in Figure 4b

Each frame corresponds to a 31-ms reconstructed super-resolution image and is played back at four frames per second. (AVI 1922 kb)

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Huang, F., Hartwich, T., Rivera-Molina, F. et al. Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms. Nat Methods 10, 653–658 (2013). https://doi.org/10.1038/nmeth.2488

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