Ultrahigh accuracy imaging modality for super-localization microscopy

Journal name:
Nature Methods
Volume:
10,
Pages:
335–338
Year published:
DOI:
doi:10.1038/nmeth.2396
Received
Accepted
Published online

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.

At a glance

Figures

  1. UAIM and its applications.
    Figure 1: UAIM and its applications.

    (a) Visual comparison of a UAIM image and a conventional EMCCD image. Images are of an Atto 647N molecule from which ~72 (UAIM) and ~84 (conventional) photons per image were detected on average; images were acquired with effective pixel sizes of 16 and 253.97 nm using 1,000× and 63× magnifications, respectively. The mean photon count in the brightest pixel is 0.22 for the UAIM image and 21.85 for the conventional image. The mesh representation, which displays intensity as height, contrasts more conspicuously the spiky appearance of the UAIM image with the relatively smooth appearance of the conventional image. Scale bars, 0.5 μm. (b) Super-resolution imaging of a LAMP1+ cellular structure using UAIM. Top, image of the structure, formed by summing the 5,063 UAIM (1,000×) images from which single Alexa 647 molecules were localized to produce a super-resolution image. Bottom, super-resolution image constructed from the location estimates of individual Alexa 647 molecules. The average number of photons detected per molecule is 128.94. Scale bars, 1 μm. (c) Single-molecule tracking using UAIM. Top, trajectory of an ErbB2 receptor (Supplementary Video 1) determined by localization of its Atto 647N label from 594 UAIM (1,000×) images. On average, 102.85 photons per image were detected from the Atto 647N dye. Bottom left, one of the 594 UAIM images. The red box encloses the tracked Atto 647N molecule. Bottom right, compacted (10 × 10–binned) version of the same UAIM image that facilitates visualization of the tracked Atto 647N molecule (red arrow). Scale bars, 1 μm.

  2. Experimental and theoretical demonstration of UAIM.
    Figure 2: Experimental and theoretical demonstration of UAIM.

    (a) Comparison of the s.d. of the maximum-likelihood estimates of the x0 coordinate of fluorescent beads imaged using UAIM (blue stars) and conventional EMCCD imaging (blue circles). Each s.d. corresponds to a different bead that is identified by its per-image mean photon count. For each s.d., the corresponding limit of accuracy (magenta) is shown. Likewise, the corresponding ultimate limit of accuracy (black), which assumes an ideal detector that introduces neither noise nor pixelation, is shown. The UAIM and conventional images were acquired with effective pixel sizes of 16 and 253.97 nm using 1,000× and 63× magnifications, respectively. (b) Theoretical analysis of point-source localization. Decreasing the effective pixel size by increasing the magnification for EMCCD imaging at a high level of signal amplification (g = 1,000) yields a limit of accuracy (stars) that approaches the ultimate limit (blue line). The red markers at effective pixel sizes of 373.31, 224.00 and 160.00 nm (magnifications of M = 42.86, 71.43 and 100) correspond approximately to standard magnifications of 40× and 63× and exactly to the standard magnification of 100×. For the same range of effective pixel sizes, the limits of accuracy corresponding to the common excess noise–based supposition (dots) and to CCD imaging with a readout noise s.d. of two electrons per pixel (circles) are shown. (See Supplementary Note 9 for more details.)

Videos

  1. Single-molecule tracking of an ErbB2 cell surface receptor labeled with anti- ErbB2 Fab Atto 647N
    Video 1: 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.

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Author information

  1. These authors contributed equally to this work.

    • Jerry Chao &
    • Sripad Ram

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

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Author details

Supplementary information

Video

  1. Video 1: Single-molecule tracking of an ErbB2 cell surface receptor labeled with anti- ErbB2 Fab Atto 647N (37,033 KB, Download)
    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.

PDF files

  1. Supplementary Text and Figures (2 MB)

    Supplementary Figures 1–3, Supplementary Tables 1–3 and Supplementary Notes 1–15

Additional data