Improved Photon Yield from a Green Dye with a Reducing and Oxidizing System

Abstract

An optimized chemistry turns a small green dye into a useful label for single‐molecule experiments where steric hindrance is an issue. Thanks to a reducing and oxidizing system combined to oxygen depletion, a single Bodipy (boron‐dipyrromethene) FL fluorophore emits, on average, 20 times more photons around 510 nm (see picture) and its lifetime before photobleaching is increased by the same amount, reaching several seconds.

Mechanism of Intramolecular Photostabilization in Self‐Healing Cyanine Fluorophores

“…A plausible mechanism is that TX heals the fluorophore through intramolecular quenching of triplet‐related dark‐states, which is a mechanism that appears to be common for both triplet‐state quenchers (COT) and redox‐active compounds [TX, ascorbic acid (AA), methylviologen (MV)] and is in good agreement with published studies on the stabilization mechanism of fluorophores by redox‐active compounds in solution 6. 7, 10, 19–24 We further demonstrate the influence of cysteamine (MEA)25, 26 and procatechuic acid (PCA)27 on the self‐healing process in a Cy5 derivative. Our results suggest, instead of additional stabilization by MEA/PCA, the potential applicability of self‐healing fluorophores in stochastic optical reconstruction microscopy (STORM) with optical super‐resolution <250 nm 28–30.…”

“…10, 19, 21–23 All experiments were performed in phosphate‐buffered saline (PBS) and glucose‐oxidase catalase (GOC) was used for oxygen removal 6. 31 The GOC system is used preferentially over a combination of protocatechuic acid and protocatechuate‐3,4,dioxygenase (PCA/PCD),31 to avoid any unwanted convolution of solution‐based healing caused by millimolar concentrations of the reducer PCA,27 with intramolecular stabilization from TX.…”

Photophysics of Fluorescent Probes for Single-Molecule Biophysics and Super-Resolution Imaging

“…Simultaneous pulsed excitation, lead to increased photobleaching whereas alternating laser excitation had negligible effect indicating successive absorption of two photons in the singlet manifold leading to photobleaching (55–57). The reducing and oxidizing system works more efficiently for fluorophores with lower excitation energy, with some exceptions(58), probably because the higher energy photons of blue light are more capable of inducing photobleaching through higher excited states.…”

Photobleaching Lifetimes of Cyanine Fluorophores Used for Single‐Molecule Förster Resonance Energy Transfer in the Presence of Various Photoprotection Systems

“…[17,25] ROXS operates by introducing a reducing agent and an oxidizing agent to the solution, which opens up faster alternate pathways from the triplet state to the ground state. [17,36] TSQ is a combination of Trolox, nitrobenzyl alcohol, and cyclooctatetraene and works similarly to the ROXS system, by achieving triplet quenching by energy transfer with cyclooctatetraene. [25] The PCA solution can also act as a reducing agent and can serve as a partial photostabilizer.…”