Multiplexed 3D Cellular Super-resolution Imaging with DNA-PAINT and Exchange-PAINT

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2014 Feb 4

PMID 24487583

Citations 429

Authors

Ralf Jungmann

Maier S Avendano

Johannes B Woehrstein

Mingjie Dai

William M Shih

Peng Yin

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Relay-projection microscopic telescopy.

Yi W, Zhu S, Fu M, Gu N, Qi J, Liu S Light Sci Appl. 2025; 14(1):117.

PMID: 40050280 PMC: 11885427. DOI: 10.1038/s41377-025-01800-6.

Nucleic Acid Framework-Enabled Spatial Organization for Biological Applications.

Zhang R, Zuo X, Yin F Chem Bio Eng. 2025; 2(2):71-86.

PMID: 40041004 PMC: 11873853. DOI: 10.1021/cbe.4c00164.

Light sheet illumination in single-molecule localization microscopy for imaging of cellular architectures and molecular dynamics.

Cheng S, Nakatani Y, Gagliano G, Saliba N, Gustavsson A Npj Imaging. 2025; 2(1):49.

PMID: 40018679 PMC: 11860233. DOI: 10.1038/s44303-024-00057-9.

Polarity-JaM: an image analysis toolbox for cell polarity, junction and morphology quantification.

Giese W, Albrecht J, Oppenheim O, Akmeric E, Kraxner J, Schmidt D Nat Commun. 2025; 16(1):1474.

PMID: 39922822 PMC: 11807127. DOI: 10.1038/s41467-025-56643-x.

Separating nanobubble nucleation for transfer-resistance-free electrocatalysis.

Guo S, Yu M, Lee J, Qiu M, Yuan D, Hu Z Nat Commun. 2025; 16(1):919.

PMID: 39843478 PMC: 11754753. DOI: 10.1038/s41467-024-55750-5.

References

Aitken C, Marshall R, Puglisi J . An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J. 2007; 94(5):1826-35. PMC: 2242739. DOI: 10.1529/biophysj.107.117689. View

Schweller R, Zimak J, Duose D, Qutub A, Hittelman W, Diehl M . Multiplexed in situ immunofluorescence using dynamic DNA complexes. Angew Chem Int Ed Engl. 2012; 51(37):9292-6. PMC: 3517005. DOI: 10.1002/anie.201204304. View

Bates M, Dempsey G, Chen K, Zhuang X . Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection. Chemphyschem. 2012; 13(1):99-107. PMC: 3353744. DOI: 10.1002/cphc.201100735. View

Tokunaga M, Imamoto N, Sakata-Sogawa K . Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods. 2008; 5(2):159-61. DOI: 10.1038/nmeth1171. View

Vogelsang J, Kasper R, Steinhauer C, Person B, Heilemann M, Sauer M . A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew Chem Int Ed Engl. 2008; 47(29):5465-9. DOI: 10.1002/anie.200801518. View

Wu N, Czajkowsky D, Zhang J, Qu J, Ye M, Zeng D . Molecular threading and tunable molecular recognition on DNA origami nanostructures. J Am Chem Soc. 2013; 135(33):12172-5. DOI: 10.1021/ja403863a. View

Hell S, Wichmann J . Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 2009; 19(11):780-2. DOI: 10.1364/ol.19.000780. View

Schoen I, Ries J, Klotzsch E, Ewers H, Vogel V . Binding-activated localization microscopy of DNA structures. Nano Lett. 2011; 11(9):4008-11. DOI: 10.1021/nl2025954. View

Xu K, Babcock H, Zhuang X . Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat Methods. 2012; 9(2):185-8. PMC: 3304438. DOI: 10.1038/nmeth.1841. View

10.

Vaughan J, Jia S, Zhuang X . Ultrabright photoactivatable fluorophores created by reductive caging. Nat Methods. 2012; 9(12):1181-4. PMC: 3561463. DOI: 10.1038/nmeth.2214. View

11.

Rasnik I, McKinney S, Ha T . Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods. 2006; 3(11):891-3. DOI: 10.1038/nmeth934. View

12.

Opazo F, Levy M, Byrom M, Schafer C, Geisler C, Groemer T . Aptamers as potential tools for super-resolution microscopy. Nat Methods. 2012; 9(10):938-9. DOI: 10.1038/nmeth.2179. View

13.

Betzig E, Patterson G, Sougrat R, Lindwasser O, Olenych S, Bonifacino J . Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006; 313(5793):1642-5. DOI: 10.1126/science.1127344. View

14.

Douglas S, Dietz H, Liedl T, Hogberg B, Graf F, Shih W . Self-assembly of DNA into nanoscale three-dimensional shapes. Nature. 2009; 459(7245):414-8. PMC: 2688462. DOI: 10.1038/nature08016. View

15.

Lin C, Jungmann R, Leifer A, Li C, Levner D, Church G . Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. Nat Chem. 2012; 4(10):832-9. PMC: 3523708. DOI: 10.1038/nchem.1451. View

16.

Giannone G, Hosy E, Levet F, Constals A, Schulze K, Sobolevsky A . Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys J. 2010; 99(4):1303-10. PMC: 2920718. DOI: 10.1016/j.bpj.2010.06.005. View

17.

Beliveau B, Joyce E, Apostolopoulos N, Yilmaz F, Fonseka C, McCole R . Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Acad Sci U S A. 2012; 109(52):21301-6. PMC: 3535588. DOI: 10.1073/pnas.1213818110. View

18.

Ries J, Kaplan C, Platonova E, Eghlidi H, Ewers H . A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods. 2012; 9(6):582-4. DOI: 10.1038/nmeth.1991. View

19.

Kao H, Verkman A . Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys J. 1994; 67(3):1291-300. PMC: 1225486. DOI: 10.1016/S0006-3495(94)80601-0. View

20.

Huang B, Wang W, Bates M, Zhuang X . Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science. 2008; 319(5864):810-3. PMC: 2633023. DOI: 10.1126/science.1153529. View