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New coumarin- and phenoxazine-based fluorescent probes for live-cell STED nanoscopy

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Abstract

The potential of live-cell stimulated emission depletion (STED) nanoscopy has not yet been fully exploited. Currently, the main limitation is the small number of fluorophores and probes that can sustain high light intensity/high dose employed in STED. Namely, fluorophores suitable for STED nanoscopy must be bright and highly photostable and exhibit a large Stokes shift. To expand the list of available probes, we synthesized and evaluated several new membrane probes for live-cell STED nanoscopy. Of the tested probes, probes MePyr500, ThiaCN545 and NB640 not only allow high-resolution STED images, but also partition into the intracellular membranes relatively quickly, thus lacking the selectivity of labelling solely the plasma membrane. During experiments, cytotoxicity was observed merely with the probe ThiaCN545, which blebs the plasma membrane. In comparison with commercially available CellMask Orange and STAR RED (KK114) DPPE, all our tested probes exhibited better photostability with the exception of NB640, which had the fastest bleaching rate of all tested probes. The best overall results can be assigned to the probe MePyr500, providing high-resolution STED images as well as high photostability with no noticeable cytotoxicity, making it an excellent candidate for further development.

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References

  • Dyba M, Hell SW (2003) Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission. Appl Opt 42:5123–5129

    Article  PubMed  Google Scholar 

  • Eggeling C, Willig KI, Barrantes FJ (2013) STED microscopy of living cells—new frontiers in membrane and neurobiology. J Neurochem 126:203–212

    Article  CAS  PubMed  Google Scholar 

  • Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Normanno D, Singer RH et al (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12:244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grimm JB, Muthusamy AK, Liang Y, Brown TA, Lemon WC, Patel R, Lu R, Macklin JJ, Keller PJ, Ji N et al (2017) A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat Methods 14:987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782

    Article  CAS  PubMed  Google Scholar 

  • Klymchenko AS, Kreder R (2014) Fluorescent probes for lipid rafts: from model membranes to living cells. Chem Biol 21:97–113

    Article  CAS  PubMed  Google Scholar 

  • Kolmakov K, Belov VN, Wurm CA, Harke B, Leutenegger M, Eggeling C, Hell SW (2010) A versatile route to red-emitting carbopyronine dyes for optical microscopy and nanoscopy. Eur J Org Chem 2010:3593–3610

    Article  CAS  Google Scholar 

  • Luo X, Naiyun X, Cheng L, Huang D (2001) Synthesis of coumarin dyes containing N-alkylsulfonamide groups. Dyes Pigm 51:153–159

    Article  CAS  Google Scholar 

  • Maksim VS, Vladimir NB, Stefan WH (2015) Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review. Methods Appl Fluoresc 3:042004

    Article  CAS  Google Scholar 

  • Nizamov S, Willig KI, Sednev MV, Belov VN, Hell SW (2012) Phosphorylated 3-heteroarylcoumarins and their use in fluorescence microscopy and nanoscopy. Chem Eur J 18:16339–16348

    Article  CAS  PubMed  Google Scholar 

  • Nizamov S, Sednev MV, Bossi ML, Hebisch E, Frauendorf H, Lehnart SE, Belov VN, Hell SW (2016) “Reduced” Coumarin dyes with an O-phosphorylated 2,2-dimethyl-4-(hydroxymethyl)-1,2,3,4-tetrahydroquinoline fragment: synthesis, spectra, and STED microscopy. Chem Eur J 22:11631–11642

    Article  CAS  PubMed  Google Scholar 

  • Pajk S, Živec M, Šink R, Sosič I, Neu M, C-w Chung, Martínez-Hoyos M, Pérez-Herrán E, Álvarez-Gómez D, Álvarez-Ruíz E et al (2016) New direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold.”. Eur J Med Chem 112:252–257

    Article  CAS  PubMed  Google Scholar 

  • Schill H, Nizamov S, Bottanelli F, Bierwagen J, Belov VN, Hell SW (2013) 4-Trifluoromethyl-substituted coumarins with large stokes shifts: synthesis, bioconjugates, and their use in super-resolution fluorescence microscopy. Chem Eur J 19:16556–16565

    Article  CAS  PubMed  Google Scholar 

  • Sezgin E, Schneider F, Zilles V, Urbančič I, Garcia E, Waithe D, Klymchenko AS, Eggeling C (2017) Polarity-sensitive probes for superresolution stimulated emission depletion microscopy. Biophys J 113:1321–1330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW (2011) Sharper low-power STED nanoscopy by time gating. Nat Methods 8:571

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Funding was provided by Horizon 2020 Framework Programme (Grant No. 686098).

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Authors and Affiliations

  1. Faculty of Pharmacy, University of Ljubljana, Aškerčeva Cesta 7, 1000, Ljubljana, Slovenia

    Stane Pajk & Matej Novak

  2. Jožef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia

    Stane Pajk, Hana Majaron, Boštjan Kokot & Janez Štrancar

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  1. Stane Pajk
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  2. Hana Majaron
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  3. Matej Novak
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  4. Boštjan Kokot
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  5. Janez Štrancar
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Correspondence to Janez Štrancar.

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Special Issue: Regional Biophysics Conference 2018.

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Pajk, S., Majaron, H., Novak, M. et al. New coumarin- and phenoxazine-based fluorescent probes for live-cell STED nanoscopy. Eur Biophys J 48, 485–490 (2019). https://doi.org/10.1007/s00249-019-01354-7

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  • DOI: https://doi.org/10.1007/s00249-019-01354-7

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