Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging

Qinsi Zheng, Anthony X. Ayala, Inhee Chung, Aubrey V. Weigel, Anand Ranjan, Natalie Falco, Jonathan B. Grimm, Ariana N. Tkachuk, Carl Wu, Jennifer Lippincott-Schwartz, Robert H. Singer, Luke D. Lavis

Research output: Contribution to journalArticlepeer-review

87 Scopus citations


Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds - ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant (KL-Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore - Janelia Fluor 526 (JF526) - with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Original languageEnglish (US)
Pages (from-to)1602-1613
Number of pages12
JournalACS Central Science
Issue number9
StatePublished - Sep 25 2019

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)


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