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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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.
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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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). https://doi.org/10.1038/nmeth.2835
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