Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging

Research output: Contribution to journalJournal articleResearchpeer-review

  • Qinsi Zheng
  • Steffen Jockusch
  • Zhou Zhou
  • Roger B. Altman
  • Hong Zhao
  • Wesley Asher
  • Michael Holsey
  • Mathiasen, Signe
  • Peter Geggier
  • Jonathan A. Javitch
  • Scott C. Blanchard

Bright, long-lasting organic fluorophores enable a broad range of imaging applications. “Self-healing” fluorophores, in which intra-molecularly linked protective agents quench photo-induced reactive species, exhibit both enhanced photostability and biological compatibility. However, the self-healing strategy has yet to achieve its predicted potential, particularly in the presence of ambient oxygen where live-cell imaging studies must often be performed. To identify key bottlenecks in this technology that can be used to guide further engineering developments, we synthesized a series of Cy5 derivatives linked to the protective agent cyclooctatetraene (COT) and examined the photophysical mechanisms curtailing their performance. The data obtained reveal that the photostability of self-healing fluorophores is limited by reactivity of the COT protective agent. The addition of electron withdrawing substituents to COT reduced its susceptibility to reactions with molecular oxygen and the fluorophore to which it is attached and increased its capacity to participate in triplet energy transfer. Exploiting these insights, we designed and synthesized a suite of modified COT-fluorophores spanning the visible spectrum that exhibited markedly increased intra-molecular photostabilization. Under ambient oxygen conditions, the photostability of Cy3 and Cy5 fluorophore derivatives increased by 3- and 9-fold in vitro and by 2- and 6-fold in living cells, respectively. We further show that this approach can improve a silicon rhodamine fluorophore. These findings offer a clear strategy for achieving the full potential of the self-healing approach and its application to the gamut of fluorophore species commonly used for biomedical imaging.

Original languageEnglish
JournalChemical Science
Issue number1
Pages (from-to)755-762
Number of pages8
Publication statusPublished - 2016

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health (GM098859-01A1 to SCB; MH054137 and DA022413 to JAJ) and the National Science Foundation (CHE 11-11392 to SJ

Publisher Copyright:
© The Royal Society of Chemistry.

ID: 311722736