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Multicolour localization microscopy by point-spread-function engineering

Abstract

Super-resolution microscopy has revolutionized cellular imaging in recent years1,2,3,4. Methods that rely on sequential localization of single point emitters enable spatial tracking at a resolution of 10–40 nm. Moreover, tracking and imaging in three dimensions is made possible by various techniques, including point-spread-function (PSF) engineering5,6,7,8,9—namely, encoding the axial (z) position of a point source in the shape that it creates in the image plane. However, efficient multicolour imaging remains a challenge for localization microscopy—a task of the utmost importance for contextualizing biological data. Normally, multicolour imaging requires sequential imaging10,11, multiple cameras12 or segmented dedicated fields of view13,14. Here, we demonstrate an alternate strategy: directly encoding the spectral information (colour), in addition to three-dimensional position, in the image. By exploiting chromatic dispersion we design a new class of optical phase masks that simultaneously yield controllably different PSFs for different wavelengths, enabling simultaneous multicolour tracking or super-resolution imaging in a single optical path.

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Figure 1: Dual-colour mask SLM implementation.
Figure 2: Dual-colour 20 μm Tetrapod mask.
Figure 3: Simultaneous 3D multicolour microsphere diffusion.
Figure 4: Multicolour super-resolution cell imaging.

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Acknowledgements

This work was supported in part by the National Institutes of Health, National Institute of General Medical Sciences Grants No. R01GM085437 and R35GM118067, and by the Stanford Nanofabrication Facility (a member of the National Nanotechnology Infrastructure Network), which is supported by the National Science Foundation Grant ECS-9731293.

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Authors and Affiliations

Authors

Contributions

Y.S. conceived the idea and performed numerical simulations, calculations, and experiments. L.E.W. made samples and performed experiments. A.S.B. conceived and implemented the calibration procedure and performed calculations. M.Y.L. fabricated dielectric phase mask. W.E.M supervised the research. All authors contributed to writing the paper.

Corresponding authors

Correspondence to Yoav Shechtman or W. E. Moerner.

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The authors declare no competing financial interests.

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Shechtman, Y., Weiss, L., Backer, A. et al. Multicolour localization microscopy by point-spread-function engineering. Nature Photon 10, 590–594 (2016). https://doi.org/10.1038/nphoton.2016.137

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