Qinsi Zheng

Cyanine fluorophore derivatives with enhanced photostability

Fluorescence applications requiring high photostability often depend on the use of solution additives to enhance fluorophore performance. Here we demonstrate that the direct or proximal conjugation of cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA) or Trolox to the cyanine fluorophore Cy5 dramatically enhanced fluorophore photostability without otherwise affecting its native spectral characteristics. Such conjugation is a powerful means of improving the robustness of fluorescence-based applications demanding long-lived, nonblinking fluorescence emission.

Enhanced photostability of cyanine fluorophores across the visible spectrum

protective agents. Notably, these positive effects varied substantially for each fluorophore type. For instance, each of the COT-linked fluorophores showed dramatic enhancements in overall performance. For Cy2-COT, we observed an ~25-fold increase in the number of photons emitted before a dark-state transition. For Cy3-COT and Cy3.5-COT, this parameter changed very little, but the SNR of fluorescence for both molecules increased approximately fourto fivefold. Cy5.5-COT and Cy7-COT showed 50and 70-fold increases, respectively, in the number of photons emitted before entering a dark state. Consequently, the average Cy5.5-COT and Cy7-COT molecule could be continuously imaged for ~3 min at an SNR >7:1 (Fig. 1c). Consistent with our previous work1, we observed only modest improvements for most NBA-linked fluorophores. However, we observed an almost 70-fold enhancement of photon count for Cy7-NBA. Trolox, which shows the most favorable impact on the performance of the Cy5 fluorophore1, also had output before blinking or photobleaching. However, a quantitative understanding of the distinct mechanisms for enhancing fluorophore performance can only be delineated with confidence when married with bulk electrochemical and spectroscopic investigations. These combined approaches, although requiring large quantities of material, could provide grounded insights into the impact and relative weighting of ‘self-healing’ and ‘self-protecting’ mechanisms for distinct fluorophore types. Progress on this front will enable both additional improvements in the performances of known protective agents and the search for new compounds with similar or improved properties. Developments of this kind may ultimately facilitate the design and synthesis of new classes of fluorophores spanning the visible spectrum with enhancements in performance even greater than those observed for the cyanine class4 and tailored properties for distinct experimental demands.

The bright future of single-molecule fluorescence imaging.

Single-molecule Förster resonance energy transfer (smFRET) is an essential and maturing tool to probe biomolecular interactions and conformational dynamics in vitro and, increasingly, in living cells. Multi-color smFRET enables the correlation of multiple such events and the precise dissection of their order and timing. However, the requirements for good spectral separation, high time resolution, and extended observation times place extraordinary demands on the fluorescent labels used in such experiments. Together with advanced experimental designs and data analysis, the development of long-lasting, non-fluctuating fluorophores is therefore proving key to progress in the field. Recently developed strategies for obtaining ultra-stable organic fluorophores spanning the visible spectrum are underway that will enable multi-color smFRET studies to deliver on their promise of previously unachievable biological insights.

The Contribution of Reactive Oxygen Species to the Photobleaching of Organic Fluorophores

Photoexcitation of fluorophores commonly used for biological imaging applications generates reactive oxygen species (ROS) which can cause bleaching of the fluorophore and damage to the biological system under investigation. In this study, we show that singlet oxygen contributes relatively little to Cy5 and ATTO 647N photobleaching at low concentrations in aqueous solution. We also show that Cy5 generates significantly less ROS when covalently linked to the protective agents, cyclooctatetraene (COT), nitrobenzyl alcohol (NBA) or Trolox. Such fluorophores exhibit enhanced photostability both in bulk solutions and in single‐molecule fluorescence measurements. While the fluorophores ATTO 647N and ATTO 655 showed greater photostability than Cy5 and the protective–agent‐linked Cy5 derivatives investigated here, both of ATTO 647N and ATTO 655 generated singlet oxygen and hydroxyl radicals at relatively rapid rates, suggesting that they may be substantially more phototoxic than Cy5 and its derivatives.

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

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.