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Rhodamines are important fluorescent dyes, chromophores, for spectral calibration in fluorometers, single-molecule detection, as imaging agents for biomolecules, for scanning confocal microscopy, in fluorescence correlation spectroscopy (FCS), and in high-throughput screening. However, extending their stability further could improve all of these techniques and perhaps lead to novel applications. Jyotirmayee Mohanty and Werner Nau at the International University Bremen, Germany, explain that a combination of useful properties make rhodamines, and the archetypal member of the group rhodamine 6G (Rh6G) almost perfect for numerous analytical techniques. The compounds are soluble in water, produce quantum yields close to unity, have high extinction coefficients, and are quite stable to light. However, all these properties could be improved on, in particular the stability and fluorescence lifetime. Nau points out that various efforts have been undertaken to get the best out of rhodamines, but these have generally compromised one or more of the compounds' properties in preference for another. Now, Nau and Mohanty have exploited the cage-like structure of cucurbit{7}uril (CB7), a macrocyclic molecule which can act as a protective enclosure for smaller molecules within. They have found that harbouring Rh6G within CB7 has little effect on the useful properties of the rhodamine, but has some very important advantages. The team demonstrated that the Rh6G molecules are at least partially held within the CB7 cages, and that binding is very tight (as shown by UV-Vis titration). Proton NMR was also used to show that the dye was trapped inside the cage. Importantly, the team found that the fluorescence lifetime of Rh6G increases from about 4 nanoseconds to almost 5 ns (4.76 ± 0.04 ns) inside CB7. This is the longest lifetime value reported for Rh6G, claim the researchers. "This special effect is related to the very low polarizability/refractive index (close to the gas phase) which a guest molecule experiences inside the CB7 cavity", explains Nau. This enhanced fluorescence lifetime by an additive is unique, say the researchers, and suggest that it could be useful in recently emerging assays based on fluorescence lifetimes, as well as fluorescence lifetime imaging microscopy. The CB7 also provides one more important effect. It acts as a substantial bunker for the rhodamine dye protecting it from aggregation, adsorption to material surfaces, photobleaching. Photobleaching is the irreversible destruction of a chromophore's capacity to fluoresce caused by incident light. Resistance to photobleaching and the observed increase in storage life of the cage-stabilized solutions is critical in many applications and solving the problem could allow new techniques in the areas of single-molecule detection and scanning confocal microscopy to be developed. Chromophore stability even at high incident light levels is "quintessential" to these techniques, adds Nau. "The approach should be transferable to many other cationic dyes, e.g., rhodamines, coumarins, pyronines, oxazines, cyanines, etc," Nau told Spectral Lines, "In fact, we have already successfully tested additional fluorescent dyes." Related links: Article by David Bradley |
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