title: Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging.
abstract: 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-foldin vitroand 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 strategy and its application to the gamut of fluorophore species commonly used for biomedical imaging.
authors.affiliation: Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medicine, New York, NY.
authors.forename: Qinsi
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Department of Chemistry, Columbia University, New York, NY.
authors.forename: Steffen
authors.initials: S
authors.lastname: Jockusch
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY.
authors.forename: Roger B
authors.initials: RB
authors.lastname: Altman
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Departments of Psychiatry and Pharmacology, Columbia University College of Physicians & Surgeons.
authors.forename: Wesley
authors.initials: W
authors.lastname: Asher
authors.affiliation: Departments of Psychiatry and Pharmacology, Columbia University College of Physicians & Surgeons.
authors.forename: Michael
authors.initials: M
authors.lastname: Holsey
authors.affiliation: Departments of Psychiatry and Pharmacology, Columbia University College of Physicians & Surgeons.
authors.forename: Signe
authors.initials: S
authors.lastname: Mathiasen
authors.affiliation: Departments of Psychiatry and Pharmacology, Columbia University College of Physicians & Surgeons.
authors.forename: Peter
authors.initials: P
authors.lastname: Geggier
authors.affiliation: Departments of Psychiatry and Pharmacology, Columbia University College of Physicians & Surgeons; Division of Molecular Therapeutics, New York State Psychiatric Institute.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.affiliation: Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medicine, New York, NY; Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard