Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

Abstract

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

“…1C). Such changes are speculated to arise from an environment-specific increase in Cy3 quantum yield upon ternary complex formation, stemming from reductions in solvent-mediated, non-radiative relaxation pathways and/or cis-trans isomerization rates (53,54). Consistent with previous studies (16) and the determinants of ternary complex formation as outlined in Reaction 3, the observed increase in Cy3 fluorescence intensity was strictly dependent upon the presence of GTP as well as the acylation of tRNA Phe with the Phe amino acid (Fig.…”

Protein–DNA force assay in a microfluidic format

“…Furthermore, the fluorescence dyes are expected to show excitation and emission characteristics dependant on the local environment, which differ at the glass and the PDMS surfaces. 33 This very good agreement underlines the equivalency of the two analysis methods. The top-vs.-bottom analysis route is possibly less prone to errors.…”

New Trends in Near-Infrared Fluorophores for Bioimaging

“…12 Despite the long-wavelength absorption/fluorescence of cyanine dyes, their fluorescence quantum yields and photostabilities are in some cases not satisfactory, especially with increasing length of the polymethine chain. 13 Both the strategy to improve the photophysical properties of cyanine fluorophores by chemical modification, 14 and the search for NIR fluorophores alternative to cyanines, are of great interest. Indeed, the investigation of novel NIR fluorophores has been increasing year by year.…”