Photometry unlocks 3D information from 2D localization microscopy data

Journal name:
Nature Methods
Volume:
14,
Pages:
41–44
Year published:
DOI:
doi:10.1038/nmeth.4073
Received
Accepted
Published online

We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

At a glance

Figures

  1. Photometry-based 3D super-resolution imaging.
    Figure 1: Photometry-based 3D super-resolution imaging.

    (a) The z-dependent photometric parameter P is created by determining the intensities in two apertures of the molecule's emission pattern according to P = I2 I1−1. (b) Principle of TRABI, which uses a circular aperture with radius r1 (solid circle) and an exclusion zone defined by 2r1 (dashed circle); I1 is determined by subtracting background (IBG) from raw intensity (Iraw,1); Iraw,1 and IBG are not estimated in frames in which localizations from neighboring spots interfere. (c) Left, dSTORM imaging of surface-immobilized Cy5-labeled DNA. Right, number of photons obtained by fitting using rapidSTORM (free fit; black), classical AP (r1 = 865 nm, r2 = 1,131 nm; gray) and TRABI (r1 = 865 nm, nBG = 7; magenta). (d) Histograms of P for different axial positions of the single-molecule surface adjusted with a piezo-scanner. Dashed lines indicate the median of the distribution. (e) P as a function of the axial position. Dots represent the median of the distribution, solid lines are used to guide the eye; I1 was determined by TRABI (r1 = 865 nm), and I2 was determined either by TRABI (r2 = 333 nm, black curve) or through free (green) or fixed fitting (magenta, FWHM = 300 nm) in rapidSTORM. (f) Reconstructed dSTORM image of a single-molecule surface tilted by an angle of 0.56°, color-coded by P. Arrowheads indicate x positions of magnified single-molecule localization patterns (insets). Scale bars represent 1 μm (c), 5 μm (f, top) and 100 nm (f, insets).

  2. Virtual 3D imaging obtained by TRABI from 2D dSTORM data on different structures.
    Figure 2: Virtual 3D imaging obtained by TRABI from 2D dSTORM data on different structures.

    (a) Left, conventional 2D dSTORM image of the SC by staining SYCP3 with AF647-labeled antibodies15. Right, virtual 3D image, that is, xyP(z), obtained by TRABI by analyzing the conventional 2D data. (b) Magnified views of insets (i, ii) shown in a; arrowheads indicate twists of the lateral element of the SC (i). Magnified views of ii are shown as xy and P(z)y projections (Supplementary Video 1). (c) 3D analysis of active zones in the neuromuscular junction of Drosophila larva, stained with Cy5-labeled antibodies, showing the presynaptic protein Bruchpilot in rab3 mutants16 (Supplementary Video 2). Inset, P(z) projection of one active zone. Top left, corner shows the conventional dSTORM image. (d) 3D analysis of conventional 2D data on F-actin in COS-7 cells stained with Si-rhodamine-labeled phalloidin17 (Supplementary Video 3). Top left, conventional dSTORM image. Scale bars represent 5 μm (a), 1 μm (b), 200 nm (b, bottom), 500 nm (c) and 2 μm (d).

  3. 3D super-resolution imaging of microtubules in U2OS cells, stained with AF647-labeled antibodies.
    Figure 3: 3D super-resolution imaging of microtubules in U2OS cells, stained with AF647-labeled antibodies.

    (a) 3D dSTORM image using BP-TRABI, color-coded in z. (b) Axial localization precision (s.d.) of BP-TRABI and BP-FWHM were determined to 17 nm and 23 nm, respectively. (ce) Magnified views of insets shown in a. (c) xz projection of the entire xy view. (d) xz projection of the boxed region of the xy view. (e) zy projection of the boxed region in xy; cross-section profile of adjacent filaments as seen with BP-TRABI and BP-FWHM; solid lines indicate double Gaussian fits. Using BP-TRABI, indicated filaments were resolved with highest resolution and axially separated by 94 nm (averaged in y as indicated by dashed boxes). Regions shown in c and d are available as Supplementary Videos 4 and 5. Scale bars represent 5 μm (a), 200 nm (c,d) and 100 nm (e).

Videos

  1. Virtual 3D imaging of synaptonemal complex
    Video 1: Virtual 3D imaging of synaptonemal complex
    Rendered virtual 3D structure of synaptonemal complex of the inset shown in Fig. 2b (ii). Left: x-y projection color-coded in P(z); right: 360° rotational view, scale bar 500 nm.
  2. Virtual 3D imaging of active zone protein Bruchpilot in Drosophila
    Video 2: Virtual 3D imaging of active zone protein Bruchpilot in Drosophila
    Rendered virtual 3D structure of the presynaptic protein Bruchpilot in Drosophila neuromuscular junction of a detail shown in Fig. 2c. Left: x-y projection color-coded in P(z); right: 360° rotational view, scale bar 200 nm.
  3. Virtual 3D imaging of F-Actin in COS-7 cells
    Video 3: Virtual 3D imaging of F-Actin in COS-7 cells
    Virtual 3D image stack of the data shown in Fig. 2d and Supplementary Fig. 15. P(z) interval in 5% steps.
  4. 3D imaging of microtubules in U2OS cells
    Video 4: 3D imaging of microtubules in U2OS cells
    Rendered 3D microtubule structure of the inset shown in Fig. 3c. Left: x-y projection, right: 3D TRABI 360° rotational view, x-y-z scale 20 nm/px, Scale bar 250 nm.
  5. 3D imaging of microtubules in U2OS cells
    Video 5: 3D imaging of microtubules in U2OS cells
    Rendered 3D microtubule structure of the inset shown in Fig. 3d. Left: x-y projection, right: 3D TRABI 360° rotational view, x-y-z scale 20 nm/px, Scale bar 250 nm.

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Author information

  1. Present address: Department of Physics, University of Strathclyde, Glasgow, UK.

    • Sebastian van de Linde

Affiliations

  1. Department of Biotechnology and Biophysics, University of Würzburg, Würzburg, Germany.

    • Christian Franke,
    • Markus Sauer &
    • Sebastian van de Linde

Contributions

C.F. and S.v.d.L. designed the TRABI algorithm, developed software, performed experiments and evaluated the data. C.F., M.S. and S.v.d.L. discussed results and commented on the manuscript. S.v.d.L. conceived the project and wrote the manuscript.

Competing financial interests

C.F., M.S. and S.v.d.L. are in the process of filing a patent application.

Corresponding author

Correspondence to:

Author details

Supplementary information

Video

  1. Video 1: Virtual 3D imaging of synaptonemal complex (1.31 MB, Download)
    Rendered virtual 3D structure of synaptonemal complex of the inset shown in Fig. 2b (ii). Left: x-y projection color-coded in P(z); right: 360° rotational view, scale bar 500 nm.
  2. Video 2: Virtual 3D imaging of active zone protein Bruchpilot in Drosophila (906 KB, Download)
    Rendered virtual 3D structure of the presynaptic protein Bruchpilot in Drosophila neuromuscular junction of a detail shown in Fig. 2c. Left: x-y projection color-coded in P(z); right: 360° rotational view, scale bar 200 nm.
  3. Video 3: Virtual 3D imaging of F-Actin in COS-7 cells (4.31 MB, Download)
    Virtual 3D image stack of the data shown in Fig. 2d and Supplementary Fig. 15. P(z) interval in 5% steps.
  4. Video 4: 3D imaging of microtubules in U2OS cells (730 KB, Download)
    Rendered 3D microtubule structure of the inset shown in Fig. 3c. Left: x-y projection, right: 3D TRABI 360° rotational view, x-y-z scale 20 nm/px, Scale bar 250 nm.
  5. Video 5: 3D imaging of microtubules in U2OS cells (479 KB, Download)
    Rendered 3D microtubule structure of the inset shown in Fig. 3d. Left: x-y projection, right: 3D TRABI 360° rotational view, x-y-z scale 20 nm/px, Scale bar 250 nm.

PDF files

  1. Supplementary Text and Figures (6,125 KB)

    Supplementary Figures 1–19 and Supplementary Notes 1 and 2.

Zip files

  1. Supplementary Software (165 KB)

    TRABI 1.0 Software.

  2. Source data (32 KB)

    Source data tables for Supplementary Figures 1, 3, 4 and 10

Additional data