Researchers have determined the mechanism by which one of the most popular families of organic dyes—the cyanines—shift their absorbance spectra to shorter wavelengths over time. This process, known as “photoblueing” can confound experimental results. The new findings could help manipulate reaction conditions to avoid the process or to harness it.
Alexander Greer of Brooklyn College, Markus Sauer of Julius Maximilian University of Würzburg, Martin J. Schnermann of the US National Institutes of Health, and coworkers have shown that photoblueing in cyanines results from a photoreaction mediated by singlet oxygen (ACS Cent. Sci. 2021, DOI: 10.1021/acscentsci.1c00483). Cyanine dye irradiation leads to generation of singlet oxygen, which then reacts with the dye. This reaction causes the loss of an ethene group and truncation to form another cyanine with a shorter alkenyl chain.
Microscopists had seen signs of the photoconversions before, Schnermann says, but they had attributed them to impurities. “It has been ignored because nobody knew the mechanism,” Sauer says. “Without having any knowledge about what’s behind it, it’s all rough speculation, so you’d rather ignore it.”
Under normal conditions, only about 1% of a pentamethine cyanine converts into a trimethine one. “But modern microscopes are incredibly sensitive,” Schnermann says. Even 1% can cause a problem in an experiment intended to determine the distribution of different biomolecules labeled with the two dyes.
The researchers show that they can use buffer conditions to either suppress or enhance the truncation reaction. Enhancing the reaction could open up experimental possibilities that take advantage of the dye shift, by, for instance, detecting the photoshifted molecule at a wavelength with lower background signal than that of the original dye.
Blue-shifted cyanine dyes can end up in the same wavelength region as fluorescent proteins that are used in superresolution microscopy experiments, Schuyler van Engelenburg, a microscopy expert at the University of Denver, says. “This unfortunate consequence has precluded my laboratory and others who recognize the problem from using these two powerful probes in conjunction for our experiments,” he says. “This publication, however, points to potential additives in buffers that could be used to prevent this shifting and enable these two probes to be used in combination for [superresolution microscopy] experiments.”