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.

Fluorescence provides a mechanism for achieving contrast in biological imaging that enables investigations of molecular structure, dynamics, and function at high spatial and temporal resolution. Small-molecule organic fluorophores have proven essential for such efforts and are widely used in advanced applications such as single-molecule and super-resolution microscopy. Yet, organic fluorophores, like all fluorescent species, exhibit instabilities in their emission characteristics, including blinking and photobleaching that limit their utility and performance. Here, we review the photophysics and photochemistry of organic fluorophores as they pertain to mitigating such instabilities, with a specific focus on the development of stabilized fluorophores through derivatization. Self-healing organic fluorophores, wherein the triplet state is intramolecularly quenched by a covalently attached protective agent, exhibit markedly improved photostabilities. We discuss the potential for further enhancements towards the goal of developing “ultra-stable” fluorophores spanning the visible spectrum and how such fluorophores are likely to impact the future of single-molecule research.

SUMMARY Homeostasis and wound-healing rely on stem cells (SCs) whose activity and directed migration are often governed by Wnt signaling. In dissecting how this pathway integrates with the necessary downstream cytoskeletal dynamics, we discovered that GSK3β directly phosphorylates ACF7, a >500kd microtubule-actin crosslinking protein abundant in hair follicle stem cells (HF-SCs). We map ACF7’s GSK3β sites to the microtubule-binding domain and show that phosphorylation uncouples ACF7 from microtubules. Phosphorylation-refractile ACF7 rescues overall microtubule architecture, but phosphorylation-constitutive mutants do not. Neither mutant rescues polarized movement, revealing that phospho-regulation must be dynamic. This circuitry is physiologically relevant, depending upon polarized GSK3β inhibition at the migrating front of SCs/progeny streaming from HFs during wound-repair. Moreover, only ACF7 and not GSKβ-refractile-ACF7 restore polarized microtubule-growth and SC-migration to ACF7-null skin. Our findings provide insights into how this conserved spectraplakin integrates signaling, cytoskeletal dynamics and polarized locomotion of somatic SCs.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of “fluorogenic” compounds—ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone–zwitterion equilibrium constant (KL–Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore—Janelia Fluor 526 (JF526)—with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore due to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups.

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Photo-excitation 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.

Cyanine fluorophores exhibit greatly improved photostability when covalently linked to stabilizers, such as cyclooctatetraene (COT), nitrobenzyl alcohol (NBA) or Trolox. However, the mechanism by which photostabilization is mediated has yet to be determined. Here we present spectroscopic evidence that COT, when covalently linked to Cy5, substantially reduces the lifetime of the Cy5 triplet state, and that the degree of triplet state quenching correlates with enhancements in photostability observed in single-molecule fluorescence measurements. By contrast, NBA and Trolox did not quench the Cy5 triplet state under our conditions suggesting that their mechanism of photostabilization is different from COT and does not target the fluorophore triplet state directly. These findings provide insights into the mechanisms of fluorophore photostabilization that may lead to improved fluorophore designs for biological imaging applications.