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Everlasting rhodamine dyes and true deciding factors in their STED microscopy performance

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Abstract

The authors took an independent and closer look at the family of red-emitting rhodamine dyes known for a decade due to their excellent performance in STED microscopy. After the family was further extended, the true grounds of this performance became clear. Small-molecule protective agents and/or auxiliary groups were attached at two different sites of the dye’s scaffold. Thus, a rhodamine core, which is already quite photostable as it is, and an intramolecular stabilizer–a 4-nitrobenzyl or a 4-nitrobenzylthio group were combined to give potentially “everlasting dyes”. The fluorescence quantum yields (Φf) and the fluorescence lifetimes (τ) of the modified dyes were thoroughly measured with comparison to those of the parent dyes. The correlation of their STED performance with photostability and fluorescence color stability under illumination in water were explored. Unexpectedly, the anaerobic GSDIM (GOC) buffer proved unhelpful with respect to STED performance. It was demonstrated that, even dyes with a Φf of only 14–17% allow STED imaging with a sufficient photon budget and good signal-to-noise ratio. For the dyes with photostabilizing groups (PSG) the Φf values are 4–5 times lower than in the reference dyes, and lifetimes τ are also strongly reduced. Noteworthy are very high fluorescence color stability and constant or even increasing fluorescence signal under photobleaching in bulk aqueous solutions, which suggests a sacrificing role of the 4-nitrobenzyl-containing moieties. Straightforward and improved recipes for “last-minute” modifications and preparations of “self-healing” red-emitting fluorescent tags are described.

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Authors and Affiliations

  1. glyXera GmbH, Brenneckestraße 20 * ZENIT II/Haus 66, D-39120 Magdeburg, Germany

    Kirill Kolmakov

  2. Abacus Laser GmbH, Hannah-Vogt-Str. 1, 37085, Göttingen, Germany

    Franziska R. Winter

  3. Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

    Maksim V. Sednev

  4. Drittes Physikalisches Institut–Biophysik, Georg-August-Universität Göttingen, Friedrich-Hund-Platz 1, D-37077 Göttingen, Germany

    Subhabrata Ghosh

  5. Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9/II, 8010 Graz, Austria

    Sergey M. Borisov

  6. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya str., Moscow, 117997, Russia

    Alexey V. Nizovtsev

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  1. Kirill Kolmakov
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  2. Franziska R. Winter
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Kolmakov, K., Winter, F.R., Sednev, M.V. et al. Everlasting rhodamine dyes and true deciding factors in their STED microscopy performance. Photochem Photobiol Sci 19, 1677–1689 (2020). https://doi.org/10.1039/d0pp00304b

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