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Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing

View ORCID ProfileNiclas Gimber, View ORCID ProfileSebastian Strauss, View ORCID ProfileRalf Jungmann, View ORCID ProfileJan Schmoranzer
doi: https://doi.org/10.1101/2021.11.19.469218
Niclas Gimber
1Advanced Medical Bioimaging Core Facility, Charité-Universitätsmedizin, Berlin, Germany
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  • For correspondence: jan.schmoranzer@charite.de niclas.gimber@charite.de
Sebastian Strauss
2Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany
3Max Planck Institute of Biochemistry, Martinsried, Germany
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Ralf Jungmann
2Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany
3Max Planck Institute of Biochemistry, Martinsried, Germany
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Jan Schmoranzer
1Advanced Medical Bioimaging Core Facility, Charité-Universitätsmedizin, Berlin, Germany
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  • For correspondence: jan.schmoranzer@charite.de niclas.gimber@charite.de
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ABSTRACT

Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach ‘DNA-PAINT’ robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by ‘Exchange-PAINT’ that requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3 – 6 nm) and multicolor registration of dual and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://github.com/gtadeus/sdmixer2

  • https://github.com/ngimber/Converter_ThunderSTORM_SDmixer

  • https://github.com/ngimber/SD-DNA-PAINT

  • https://github.com/ngimber/SMLM_VoronoiTesselation

  • https://github.com/ngimber/SMLM_NearestNeighbor

  • ABBREVIATIONS

    SMLM
    Single-molecule Localization Microscopy
    STORM
    Stochastic Optical Reconstruction Microscopy
    SD
    Spectral Demixing
    PAINT
    Points Accumulation in Nanoscale Topography
    fm-DNA-PAINT
    Frequency Multiplexing DNA-PAINT
    NeNA
    Nearest Neighbor Based Analysis
    NN
    Nearest Neighbor
    AB
    Antibody
    NB
    Nanobody
    F(ab’)1
    Antigen Binding Fragment
  • Copyright 
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    Posted November 19, 2021.
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    Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing
    Niclas Gimber, Sebastian Strauss, Ralf Jungmann, Jan Schmoranzer
    bioRxiv 2021.11.19.469218; doi: https://doi.org/10.1101/2021.11.19.469218
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    Simultaneous multicolor DNA-PAINT without sequential fluid exchange using spectral demixing
    Niclas Gimber, Sebastian Strauss, Ralf Jungmann, Jan Schmoranzer
    bioRxiv 2021.11.19.469218; doi: https://doi.org/10.1101/2021.11.19.469218

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