Live Cell Imaging
Probes and Sensors
Mining Imaging Data
Single Molecule Imaging
The development of new classes of probes and sensors has played a significant role in the now widespread use of optical imaging in biology. Since the advent of green fluorescent protein (GFP) in 1994, researchers from across biology have exploited genetically-encoded fluorescent probes for their studies. Recognition of the value of such designer probes has led to an increased interest in identifying new types of dyes, optically-active materials, labeling strategies, fluorescent proteins and nanoparticles for characterizing cellular structures and processes. Since 2000, NIGMS initiatives have been under way to create “brighter,” more sensitive probes, with the goal of using them to facilitate real-time imaging of individual molecules in living cells. Here are examples of projects to develop or improve probes for optical microscopy.
Researchers created novel chromophores by derivatizing the naturally occurring fluorescent proteins green fluorescent protein (GFP) and red fluorescent protein (DsRed). Systematically analyzing the structural elements that underlie the emission and excitation maxima as well as their brightness and photostability led to this array of new genetically encodable fluorescent proteins, which each have unique spectral characteristics. This photo is a time exposure of fluorescence excited at different wavelengths and viewed through different cutoff filters used to produce accurate color images. Credit: Nathan Shaner, Lei Wang, Paul Steinbach, Roger Tsien, University of California, San Diego.
Brighter Molecular Beacons
Researchers have turned up the brightness of a group of fluorescent probes called “dyedrons,” which represent a new class of fluorescent detection reagent. Up to 7 times brighter than green fluorescent proteins (GFP), dyedrons are made up of a fluorogen and a fluorogen-activating protein (FAP). The FAP is genetically expressed in a cell and linked to a protein of interest, where it remains dark until it comes into contact with its associated fluorogen. The latest advance, shown in this image, has been to create dyedrons that are brighter (compare top and bottom panels) when bound to the surface of yeast cells. Credit: Christopher Szent-Gyorgyi, Brigitte Schmidt, James Fitzpatrick and Marcel Bruchez, Carnegie Mellon University.
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