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

The breakthroughs in single molecule spectroscopy of the last decade and therecent advances in super resolution microscopy have boosted the popularity of cyanine dyesin biophysical research. These applications have motivated the investigation of the reactionsand relaxation processes that cyanines undergo in their electronically excited states. Studiesshow that the triplet state is a key intermediate in the photochemical reactions that limit thephotostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, butinduces rapid intensity fluctuations (blinking). The existence of non-fluorescent stateslasting from milliseconds to seconds was early identified as a limitation in single-moleculespectroscopy and a potential source of artifacts. Recent studies demonstrate that acombination of oxidizing and reducing agents is the most efficient way of guaranteeing that theground state is recovered rapidly and efficiently. Thiol-containing reducing agents have beenidentified as the source of long-lived dark states in some cyanines that can be photochemicallyswitched back to the emissive state. The mechanism of this process is the reversible additionof the thiol-containing compound to a double bond in the polymethine chain resulting in anon-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–transisomerization 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 efficiencythat depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes hasbeen shown to depend dramatically on their molecular environment within the biomolecule.For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends onthe type of linkage used for attachment, DNA sequence and secondary structure. Cyanineslinked 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 ormajor grooves. These interactions not only affect the photophysical properties of theprobes but also create a large uncertainty in their orientation