The Power of Two: Covalent Coupling of Photostabilizers for Fluorescence Applications

Abstract

Fluorescence is a versatile tool for spectroscopic investigations and imaging of dynamic processes and structures across various scientific disciplines. The photophysical performance, that is, signal stability and signal duration, of the employed fluorophores is a major limiting factor. In this Letter, we propose a general concept to covalently link molecules, which are known for their positive effect in photostabilization, to form a combined photostabilizer with new properties. The direct linkage of two (or more) photostabilizers will allow one to obtain combined or synergetic effects in fluorophore stabilization and can simplify the preparation of imaging buffers that would otherwise require a mixture of photostabilizers for optimal performance. This concept was explored by synthesizing a molecule with a reducing and oxidizing moiety that is referred to as internal ROXS or "iROXS". Using single-molecule fluorescence microscopy, inter- and intramolecular healing of iROXS was observed, that is, strongly reduced blinking and increased photostability of the cyanine fluorophore Cy5. Moreover, it is shown that a covalently coupled photostabilizer can replace a mixture of molecules needed to make a functional photostabilizing ROXS buffer and might hence represent the new standard for defined and reproducible imaging conditions in single-molecule experiments. In self-healing fluorophores with intramolecular triplet-state quenching, an unprecedented photostability increase of >100-fold was obtained when using iROXS, which is even competitive with solution-based healing. Control experiments show that the oxidizing part of the iROXS molecule, an aromatic nitro group, dominates the healing process. The suggested synthetic concept and the proof-of-concept experiments represent the starting point for the quest to identify optimal combinations of linked photostabilizers for various fluorescence applications.

On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

“…The GOX-buffer system in Figure 2 and 3 contains 200 µM TCEP, which was added for enzyme stability. In agreement with previously published results 15,16,19 , we found an improvement of photophysical parameters in the presence of solution additives or when comparing unmodified fluorophores with their NPA-or COT-conjugates as shown in Figure 3. This included increased photobleaching lifetimes, signal-to-noise ratio, count-rates and total number of collected photons, and can be attributed to the depopulation of the dark triplet-state (Figure 3a-c and Figure S1-S17).…”

“…These are generated by either addition of the photostabilizer to the imaging buffer at millimolar concentrations 2,3,10,13 to allow diffusional intermolecular quenching (Figure 1a), or via covalent linkage, i.e., to create high local concentrations of the photostabilizer (Figure 1b). [14][15][16][17][18][19][20] Figure 1. Methods to create collisions between photostabilizer and reactive fluorophore state (here triplet-state T1) using intermolecular diffusional quenching (a) or intramolecular processes (b).…”

Cyanine polyene reactivity: scope and biomedical applications

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

Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

“…Vogelsang et al [20] and others [22] carried out their experiments in aqueous solution using a fluorescence microscope. This study aims to apply their findings in DNA microarray experiments, where the cyanine dye is bound to the DNA which itself is fixated on a modified glass slide and is scanned using a microarray slide scanner.…”