Mechanism of Intramolecular Photostabilization in Self‐Healing Cyanine Fluorophores

Abstract

Organic fluorophores, which are popular labels for microscopy applications, intrinsically suffer from transient and irreversible excursions to dark-states. An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood. In this contribution, we investigate the mechanism of intramolecular photostabilization in self-healing cyanines, in which photodamage is automatically repaired. Experimental evidence is provided to demonstrate that a single photostabilizer, that is, the vitamin E derivative Trolox, efficiently heals the cyanine fluorophore Cy5 in the absence of any photostabilizers in solution. A plausible mechanism is that Trolox interacts with the fluorophore through intramolecular quenching of triplet-related dark-states, which is a mechanism that appears to be common for both triplet-state quenchers (cyclooctatetraene) and redox-active compounds (Trolox, ascorbic acid, methylviologen). Additionally, the influence of solution-additives, such as cysteamine and procatechuic acid, on the self-healing process are studied. The results suggest the potential applicability of self-healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super-resolution. The presented data contributes to an improved understanding of the mechanism involved in intramolecular photostabilization and has high relevance for the future development of self-healing fluorophores, including their applications in various research fields.

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

“…Labeled McjD was further diluted in imaging buffer to a final concentration of 25 pM. McjD was studied using a custom‐built confocal FRET microscope (van der Velde et al , 2013) at room temperature. Excitation light at 532 and 640 nm was used in accordance with the fluorophore wavelengths (SuperK Extreme, NKT Photonics, Denmark).…”

Cyanine polyene reactivity: scope and biomedical applications

“…These triplet quenchers both reduce dark states and decrease the rate of photobleaching. 130,131 8C).…”

Photostabilizing Efficiency of PVC in the Presence of Schiff Bases as Photostabilizers

“…An alternative to adding photostabilizers at high concentrations to the imaging buffer relies on the direct linkage to the fluorophore. However, the working principles of this approach are not yet fully understood [15]. In the continuation of previous studies [16][17][18][19][20][21][22][23], we focused on the photostabilization of PVC using 1,3,4-thiadiazole compounds.…”