Universal Super-Resolution Multiplexing by DNA Exchange.

Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20× higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.

[1]  B. McConaughy,et al.  Nucleic acid reassociation in formamide. , 1969, Biochemistry.

[2]  M. Heilemann,et al.  Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. , 2008, Angewandte Chemie.

[3]  Michael R. Diehl,et al.  Multiplexed and Reiterative Fluorescence Labeling via DNA Circuitry , 2010, Bioconjugate chemistry.

[4]  F. Simmel,et al.  Isothermal assembly of DNA origami structures using denaturing agents. , 2008, Journal of the American Chemical Society.

[5]  N. Daigle,et al.  Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging , 2013, Science.

[6]  Johnny Tam,et al.  Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore , 2014, PloS one.

[7]  Qing Li,et al.  Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue , 2013, Proceedings of the National Academy of Sciences.

[8]  F. Simmel,et al.  Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. , 2010, Nano letters.

[9]  Shawn M. Douglas,et al.  DNA-nanotube-induced alignment of membrane proteins for NMR structure determination , 2007, Proceedings of the National Academy of Sciences.

[10]  Peng Yin,et al.  Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. , 2012, Nature chemistry.

[11]  Keith A. Lidke,et al.  Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore , 2015, PloS one.

[12]  R. Blake,et al.  Thermodynamic effects of formamide on DNA stability. , 1996, Nucleic acids research.

[13]  Amina A. Qutub,et al.  Multiplexed in situ immunofluorescence using dynamic DNA complexes. , 2012, Angewandte Chemie.

[14]  X. Zhuang,et al.  Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons , 2013, Science.

[15]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[16]  Alberto Diaspro,et al.  The 2015 super-resolution microscopy roadmap , 2015, Journal of Physics D: Applied Physics.

[17]  Leonid A. Mirny,et al.  Super-resolution imaging reveals distinct chromatin folding for different epigenetic states , 2015, Nature.

[18]  P. Rothemund Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[19]  P. Tinnefeld,et al.  Simple and aberration-free 4color-STED--multiplexing by transient binding. , 2015, Optics express.

[20]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[21]  Philip Tinnefeld,et al.  Fluoreszenzmikroskopie unterhalb der optischen Auflösungsgrenze mit konventionellen Fluoreszenzsonden , 2008 .

[22]  Johannes B. Woehrstein,et al.  Multiplexed 3D Cellular Super-Resolution Imaging with DNA-PAINT and Exchange-PAINT , 2014, Nature Methods.

[23]  R. Hochstrasser,et al.  Wide-field subdiffraction imaging by accumulated binding of diffusing probes , 2006, Proceedings of the National Academy of Sciences.