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Photometry unlocks 3D information from 2D localization microscopy data

Abstract

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.

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Figure 1: Photometry-based 3D super-resolution imaging.
Figure 2: Virtual 3D imaging obtained by TRABI from 2D dSTORM data on different structures.
Figure 3: 3D super-resolution imaging of microtubules in U2OS cells, stained with AF647-labeled antibodies.

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Acknowledgements

We thank J. Enderlein (University of Göttingen) for providing us with software for the refractive index mismatch correction and D. Birch for critically reading the manuscript. We are very grateful to T. Klein, as well as to K. Schücker, R. Benavente (University of Würzburg) and L. Lavis (Janelia Research Campus) for the provision of raw data and to L. Pließ for support with cell culture.

Author information

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Sebastian van de Linde.

Ethics declarations

Competing interests

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

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–19 and Supplementary Notes 1 and 2. (PDF 5981 kb)

Source data to Supplementary Figure 1

Source data to Supplementary Figure 2

Source data to Supplementary Figure 3

Source data to Supplementary Figure 4

Supplementary Software

TRABI 1.0 Software. (ZIP 161 kb)

Source data

Source data tables for Supplementary Figures 1, 3, 4 and 10 (ZIP 32 kb)

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. (MOV 1350 kb)

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. (MOV 906 kb)

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. (MOV 4415 kb)

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. (MOV 729 kb)

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. (MOV 479 kb)

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Franke, C., Sauer, M. & van de Linde, S. Photometry unlocks 3D information from 2D localization microscopy data. Nat Methods 14, 41–44 (2017). https://doi.org/10.1038/nmeth.4073

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