TY - JOUR

T1 - Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging

AU - Zheng, Qinsi

AU - Jockusch, Steffen

AU - Zhou, Zhou

AU - Altman, Roger B.

AU - Zhao, Hong

AU - Asher, Wesley

AU - Holsey, Michael

AU - Mathiasen, Signe

AU - Geggier, Peter

AU - Javitch, Jonathan A.

AU - Blanchard, Scott C.

N1 - 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.

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85007227194&partnerID=8YFLogxK

U2 - 10.1039/C6SC02976K

DO - 10.1039/C6SC02976K

M3 - Journal article

AN - SCOPUS:85007227194

VL - 8

SP - 755

EP - 762

JO - Chemical Science

JF - Chemical Science

SN - 2041-6520

IS - 1

ER -