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Ultrahigh accuracy imaging modality for super-localization microscopy

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Abstract

Super-localization microscopy encompasses techniques that depend on the accurate localization of individual molecules from generally low-light images. The obtainable localization accuracies, however, are ultimately limited by the image detector's pixelation and noise. We present the ultrahigh accuracy imaging modality (UAIM), which allows users to obtain accuracies approaching the accuracy that is achievable only in the absence of detector pixelation and noise, and which we found can experimentally provide a >200% accuracy improvement over conventional low-light imaging.

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Figure 1: UAIM and its applications.
Figure 2: Experimental and theoretical demonstration of UAIM.

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Acknowledgements

This work was supported in part by grant R01 GM085575 from the US National Institutes of Health and in part by the Cancer Prevention and Research Institute of Texas. We thank D. Kim and S. You for their assistance with data analysis.

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Author notes

  1. Jerry Chao and Sripad Ram: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Electrical Engineering, University of Texas at Dallas, Richardson, Texas, USA

    Jerry Chao, Sripad Ram & Raimund J Ober

  2. Department of Immunology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Jerry Chao, Sripad Ram, E Sally Ward & Raimund J Ober

Authors
  1. Jerry Chao
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  2. Sripad Ram
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  3. E Sally Ward
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  4. Raimund J Ober
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Contributions

J.C., S.R. and R.J.O. conceived the experiments, designed the experiments and analyzed the data. J.C. and S.R. performed the experiments. E.S.W. and R.J.O. provided the experimental materials and computing resources. All authors wrote the manuscript.

Corresponding author

Correspondence to Raimund J Ober.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–3 and Supplementary Notes 1–15 (PDF 1891 kb)

Single-molecule tracking of an ErbB2 cell surface receptor labeled with anti- ErbB2 Fab Atto 647N

The left panel shows the UAIM image, which was acquired at a 1,000 × magnification with an EMCCD camera. The top right panel shows the compacted version of the UAIM image, which was created by a 10 × 10 binning. The bottom right plot shows the two-dimensional trajectory of the ErbB2 receptor that is highlighted by the red box (red arrow) in the UAIM image (compacted image). The trajectory is color-coded from red to green to blue to indicate increasing time, and the video is played at the acquisition speed. For display purposes, the UAIM images were multiplied by a constant and then piecewise linearly adjusted, and the compacted images were piecewise linearly adjusted. Scale bars, 1 μm. (MOV 37032 kb)

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Chao, J., Ram, S., Ward, E. et al. Ultrahigh accuracy imaging modality for super-localization microscopy. Nat Methods 10, 335–338 (2013). https://doi.org/10.1038/nmeth.2396

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