Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores.

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

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 strategy and its application to the gamut of fluorophore species commonly used for biomedical imaging.