Genetically encoded reporters provide dynamic imaging of proteins in live cells, but researchers increasingly are interested in biomolecules such as glycans and lipids that cannot be probed with the methods developed for proteins. Studies of both in vitro and static systems have not revealed much information about how these biomolecules behave in live cells.Researchers have described a technique that enables dynamic in vivo imaging of biomolecules that are not accessible with methods developed for imaging proteins. Shown here are carbohydrates in living cells 0, 15, 30, 45 and 60 min after the chemistry reaction has finished. The green shows the carbohydrates, which, initially, are on the cell surface. Over the next hour, however, some of the carbohydrates are trafficked to the inside of the cell. The nucleus is shown in blue. Image courtesy of Jeremy M. Baskin.Carolyn R. Bertozzi’s team at the University of California, Berkeley, wanted to study the role of carbohydrates in intercellular communication but could not access the carbohydrates in living systems. Therefore, the group developed a technique that allows dynamic imaging of these and other biomolecules.Previously, two types of chemical reactions were available for imaging the biomolecules. Metabolic labeling with the chemical reporter azide allows dynamic imaging through the subsequent covalent attachment of an imaging probe via the Staudinger ligation, a reaction developed by Bertozzi’s group in 2000. However, although this reaction is highly biocompatible in cells and living animals, it does not occur quickly enough to visualize all of the biological processes in question or to detect species present in low abundance.The other type of reaction, the copper-catalyzed azide-alkyne cycloaddition, commonly known as click chemistry, is faster and enables much more sensitive detection. However, the copper catalyst is cytotoxic, prohibiting use of the reaction for dynamic imaging in live cells.The researchers described a copper-free and hence nontoxic form of click chemistry that effectively combines the advantages of these two types of reactions. In a previous study, they achieved click chemistry with no toxicity by using ring strain, the destabilization of a cyclic molecule. However, this approach proved to be no more sensitive than the biocompatible method already in use. Therefore, in the present study, they boosted the sensitivity by adding fluorine atoms to the reagent. “Essentially, this lowered the energetic hill that everything has to go over,” explained Jeremy M. Baskin, the first author of the study.Completing the synthesis, with its nine steps, was a bit of a challenge. “A couple of times I wasn’t sure if it was worth it,” Baskin added. “Persevering with the synthesis was the hardest part for me.”The investigators demonstrated the new type of reaction by using it to image the process of glycosylation in Chinese hamster ovary cells — glycosylation is an especially complex posttranslational modification and therefore a suitable candidate for testing this approach to dynamic in vivo imaging. They incubated the cells with difluorinated cyclooctyne (DIFO) conjugated with Alexa Fluor 488 and then visualized them using a Zeiss epifluorescence microscope for various time spans and concentrations. The measurements showed that the process is imaged readily with the copper-free click chemistry technique.The team is looking forward to applying the technique to the imaging of carbohydrates, Baskin said. They intend to explore the patterns of carbohydrates on cell surfaces, particularly in models of embryonic development and cancer. They are planning also to measure intracellular trafficking using the method.PNAS, Oct. 23, 2007, pp. 16793-16797.