Scientists at Carnegie Mellon University's Department of Chemistry and Molecular Biosensor and Imaging Center (MBIC) are advancing the state-of-the-art in live cell fluorescent imaging by developing a new class of fluorescent probes that span the spectrum -- from violet to the near-infrared. The new technology, called fluoromodules, can be used to monitor biological activities of individual proteins in living cells in real time.
At the 239th national meeting of the American Chemical Society, Carnegie Mellon chemists and MBIC scientists will discuss recent advances in their fluoromodule technology that have produced diverse and photostable probes.
Fluoromodules, which consist of dye-protein complexes, provide alternatives to common fluorescent proteins, such as Green Fluorescent Protein (GFP), but with a wider selection of colors and the potential for significantly greater photostability, which allows scientists to image the dye for longer periods of time. This is made possible by the fact that the dye is noncovalently bound to the protein, which allows fresh dye to replace bleached dye.
"We initially isolated and characterized fluoromodules that generate fluorescence from the fluorogenic dyes thiazole orange and malachite green. We are now expanding our repertoire by synthesizing new dyes that emit in the orange and violet regions of the spectrum, and engineering proteins that bind to the new dyes with great affinity," said Chemistry Professor Bruce Armitage, co-director of the Center for Nucleic Acid Science and Technology at Carnegie Mellon and a member of the MBIC team developing the fluoromodules.
Fluoromodules are made of a fluorogen-activating protein (FAP) and a non-fluorescent dye called a fluorogen. The FAP, which is genetically expressed in a cell and tagged to a protein of interest, does not become fluorescent until it binds with its fluorogen. With the novel FAPs and associated fluorogens created by the MBIC team, the researchers can control when a target protein lights up, allowing them to track proteins on the cell surface and within living cells in very simple and direct ways, eliminating cumbersome experimental steps.
Recent advances in the MBIC fluoromodule technology being presented at the ACS meeting include:
The aforementioned work, funded by the Pennsylvania Department of Health and the National Institutes of Health (NIH), is part of the mission of the NIH National Technology Center for Networks and Pathways.
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