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Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT


Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

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Figure 1: DNA-PAINT.
Figure 2: Spectrally multiplexed DNA-PAINT super-resolution imaging of microtubules and mitochondria inside fixed cells.
Figure 3: Exchange-PAINT.
Figure 4: Multiplexed 2D and 3D Exchange-PAINT super-resolution imaging in fixed cells.

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We thank J. Nicoludis and M.T. Strauss for help with DNA origami design, T. Schlichthaerle for transmission electron microscopy imaging support and M. Zhang for help with DLD1 cells. We thank C. Steinhauer for help with DNA-PAINT software development and fruitful discussions. We thank R.D. Barish for critical reading and commenting on the manuscript. This work is supported by a US National Institutes of Health (NIH) Director's New Innovator Award (1DP2OD007292), an NIH Transformative Research Award (1R01EB018659), an NIH grant (5R21HD072481), an Office of Naval Research (ONR) Young Investigator Program Award (N000141110914), ONR grants (N000141010827 and N000141310593), a US National Science Foundation (NSF) Faculty Early Career Development Award (CCF1054898), an NSF grant (CCF1162459) and a Wyss Institute for Biologically Engineering Faculty Startup Fund to P.Y., and an NIH Director's New Innovator Award (1DP2OD004641) and a Wyss Institute for Biologically Inspired Engineering Faculty Award to W.M.S. R.J. acknowledges support from the Alexander von Humboldt-Foundation through a Feodor-Lynen Fellowship. M.S.A. and M.D. acknowledge support from Howard Hughes Medical Institute International Student Research Fellowships.

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R.J., M.S.A. and J.B.W. contributed equally to this work. R.J. and M.S.A. conceived of the study, designed and performed the experiments, analyzed the data and wrote the manuscript. J.B.W. designed and performed the experiments, analyzed the data and wrote the manuscript. M.D. performed the experiments, analyzed the data and developed the drift correction software. W.M.S. supervised the project, discussed the results and critiqued the paper. P.Y. conceived of, designed and supervised the study, interpreted the data and wrote the manuscript. All authors reviewed and approved the manuscript.

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Correspondence to Peng Yin.

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R.J., M.D., M.S.A., J.B.W. and P.Y. have filed a provisional US patent application regarding the current work.

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Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Tables 1–8 and Supplementary Protocol (PDF 29243 kb)

Supplementary Software

Parallelized spot finding and 2D Gaussian fitting software implemented in LabVIEW (ZIP 1769 kb)

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Jungmann, R., Avendaño, M., Woehrstein, J. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods 11, 313–318 (2014).

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