Evaluation of Fluorophores for Optimal Performance in Localization-based Super-resolution Imaging

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2011 Nov 8

PMID 22056676

Citations 558

Authors

Graham T Dempsey

Joshua C Vaughan

Kok Hao Chen

Mark Bates

Xiaowei Zhuang

Affiliations

Soon will be listed here.

Abstract

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.

Citing Articles

Protocol for performing 3D-STORM-based nanoscale organization of NMDA receptors in hippocampal brain tissue.

Ferreira J, Linares-Loyez J, Bon P, Cognet L, Groc L STAR Protoc. 2025; 6(1):103639.

PMID: 39998951 PMC: 11903812. DOI: 10.1016/j.xpro.2025.103639.

Electrochemically modulated single-molecule localization microscopy for imaging cytoskeletal protein structures.

Lei C, Hu D Nanophotonics. 2025; 14(4):459-470.

PMID: 39975638 PMC: 11834056. DOI: 10.1515/nanoph-2024-0559.

Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells.

Driouchi A, Bretan M, Davis B, Heckert A, Seeger M, Silva M Nat Methods. 2025; 22(3):559-568.

PMID: 39966678 PMC: 11903300. DOI: 10.1038/s41592-025-02594-6.

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.

Nanoscopy reveals integrin clustering reliant on kindlin-3 but not talin-1.

Wu Y, Cao Z, Liu W, Cahoon J, Wang K, Wang P Cell Commun Signal. 2025; 23(1):12.

PMID: 39773732 PMC: 11707915. DOI: 10.1186/s12964-024-02024-8.

References

Huang B, Babcock H, Zhuang X . Breaking the diffraction barrier: super-resolution imaging of cells. Cell. 2010; 143(7):1047-58. PMC: 3272504. DOI: 10.1016/j.cell.2010.12.002. View

Thompson R, Larson D, Webb W . Precise nanometer localization analysis for individual fluorescent probes. Biophys J. 2002; 82(5):2775-83. PMC: 1302065. DOI: 10.1016/S0006-3495(02)75618-X. View

Chudakov D, Verkhusha V, Staroverov D, Souslova E, Lukyanov S, Lukyanov K . Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol. 2004; 22(11):1435-9. DOI: 10.1038/nbt1025. View

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

Gurskaya N, Verkhusha V, Shcheglov A, Staroverov D, Chepurnykh T, Fradkov A . Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol. 2006; 24(4):461-5. DOI: 10.1038/nbt1191. View

Huang B, Jones S, Brandenburg B, Zhuang X . Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods. 2008; 5(12):1047-52. PMC: 2596623. DOI: 10.1038/nmeth.1274. View

Habuchi S, Ando R, Dedecker P, Verheijen W, Mizuno H, Miyawaki A . Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci U S A. 2005; 102(27):9511-6. PMC: 1157093. DOI: 10.1073/pnas.0500489102. View

Bates M, Huang B, Zhuang X . Super-resolution microscopy by nanoscale localization of photo-switchable fluorescent probes. Curr Opin Chem Biol. 2008; 12(5):505-14. PMC: 2642911. DOI: 10.1016/j.cbpa.2008.08.008. View

Conley N, Biteen J, Moerner W . Cy3-Cy5 covalent heterodimers for single-molecule photoswitching. J Phys Chem B. 2008; 112(38):11878-80. PMC: 2715847. DOI: 10.1021/jp806698p. View

10.

Dempsey G, Bates M, Kowtoniuk W, Liu D, Tsien R, Zhuang X . Photoswitching mechanism of cyanine dyes. J Am Chem Soc. 2009; 131(51):18192-3. PMC: 2797371. DOI: 10.1021/ja904588g. View

11.

Fernandez-Suarez M, Ting A . Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol. 2008; 9(12):929-43. DOI: 10.1038/nrm2531. View

12.

Folling J, Bossi M, Bock H, Medda R, Wurm C, Hein B . Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods. 2008; 5(11):943-5. DOI: 10.1038/nmeth.1257. View

13.

Laurence T, Chromy B . Efficient maximum likelihood estimator fitting of histograms. Nat Methods. 2010; 7(5):338-9. DOI: 10.1038/nmeth0510-338. View

14.

van de Linde S, Krstic I, Prisner T, Doose S, Heilemann M, Sauer M . Photoinduced formation of reversible dye radicals and their impact on super-resolution imaging. Photochem Photobiol Sci. 2010; 10(4):499-506. DOI: 10.1039/c0pp00317d. View

15.

Riddles P, Blakeley R, ZERNER B . Reassessment of Ellman's reagent. Methods Enzymol. 1983; 91:49-60. DOI: 10.1016/s0076-6879(83)91010-8. View

16.

Mortensen K, Churchman L, Spudich J, Flyvbjerg H . Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat Methods. 2010; 7(5):377-81. PMC: 3127582. DOI: 10.1038/nmeth.1447. View

17.

Bates M, Blosser T, Zhuang X . Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett. 2005; 94(10):108101. PMC: 2652517. DOI: 10.1103/PhysRevLett.94.108101. View

18.

Schwering M, Kiel A, Kurz A, Lymperopoulos K, Sprodefeld A, Kramer R . Far-field nanoscopy with reversible chemical reactions. Angew Chem Int Ed Engl. 2011; 50(13):2940-5. DOI: 10.1002/anie.201006013. View

19.

Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A . EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A. 2004; 101(45):15905-10. PMC: 528746. DOI: 10.1073/pnas.0403668101. View

20.

Folling J, Belov V, Kunetsky R, Medda R, Schonle A, Egner A . Photochromic rhodamines provide nanoscopy with optical sectioning. Angew Chem Int Ed Engl. 2007; 46(33):6266-70. DOI: 10.1002/anie.200702167. View