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Rapid Sequential in Situ Multiplexing With DNA-Exchange-Imaging

Yu Wang, Johannes B. Woehrstein, Noah Donoghue, Mingjie Dai, Maier S. Avendaño, Ron C.J. Schackmann, Jason J. Zoeller, Shan Shan H. Wang, Paul W. Tillberg, Demian Park, Sylvain W. Lapan, Edward S. Boyden, Joan S. Brugge, Pascal S. Kaeser, George M. Church, Sarit S. Agasti, Ralf Jungmann, Peng Yin
doi: https://doi.org/10.1101/112227
Yu Wang
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA.Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Johannes B. Woehrstein
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.Present address: Department of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539 Munich, Germany, Max Planck Institute of Biochemistry, 82152 Martinsried near Munich, Germany.
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Noah Donoghue
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA.Warren Alpert Medical School, Brown University, Providence, Rhode Island, 02903, USA.
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Mingjie Dai
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.Program in Biophysics, Harvard University, Boston, Massachusetts, 02138, USA.
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Maier S. Avendaño
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Ron C.J. Schackmann
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Jason J. Zoeller
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Shan Shan H. Wang
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, 02115, USA.Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Paul W. Tillberg
Media Lab, MIT, Cambridge, Massachusetts, 02139, USA.Department of Electrical Engineering and Computer Science, MIT, Cambridge, Massachusetts 02139, USA.
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Demian Park
Media Lab, MIT, Cambridge, Massachusetts, 02139, USA.
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Sylvain W. Lapan
Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Edward S. Boyden
Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, 02139, USA.Media Lab, MIT, Cambridge, Massachusetts, 02139, USA.
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Joan S. Brugge
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Pascal S. Kaeser
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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George M. Church
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Sarit S. Agasti
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.Present address: New Chemistry Unit and Chemistry & Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bangalore, India.
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Ralf Jungmann
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.Present address: Department of Physics and Center for Nanoscience, Ludwig Maximilian University, 80539 Munich, Germany, Max Planck Institute of Biochemistry, 82152 Martinsried near Munich, Germany.
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Peng Yin
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, 02115, USA.Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA.
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Abstract

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 °C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Footnotes

  • ↵* Emails: P.Y. (py{at}hms.harvard.edu), R.J. (jungmann{at}biochem.mpg.de), and S.S.A. (sagasti{at}jncasr.ac.in)

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Rapid Sequential in Situ Multiplexing With DNA-Exchange-Imaging
Yu Wang, Johannes B. Woehrstein, Noah Donoghue, Mingjie Dai, Maier S. Avendaño, Ron C.J. Schackmann, Jason J. Zoeller, Shan Shan H. Wang, Paul W. Tillberg, Demian Park, Sylvain W. Lapan, Edward S. Boyden, Joan S. Brugge, Pascal S. Kaeser, George M. Church, Sarit S. Agasti, Ralf Jungmann, Peng Yin
bioRxiv 112227; doi: https://doi.org/10.1101/112227
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Rapid Sequential in Situ Multiplexing With DNA-Exchange-Imaging
Yu Wang, Johannes B. Woehrstein, Noah Donoghue, Mingjie Dai, Maier S. Avendaño, Ron C.J. Schackmann, Jason J. Zoeller, Shan Shan H. Wang, Paul W. Tillberg, Demian Park, Sylvain W. Lapan, Edward S. Boyden, Joan S. Brugge, Pascal S. Kaeser, George M. Church, Sarit S. Agasti, Ralf Jungmann, Peng Yin
bioRxiv 112227; doi: https://doi.org/10.1101/112227

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