Aside from their ability to selectively label specific epitope targets for sub-cellular localization and trafficking investigations, as well as the unique optical highlighter behavior displayed by a specialized subset of the fluorescent protein family, this ubiquitous class of genetically-encoded probes has also demonstrated significant potential as an effective biosensor for reporting on a wide array of intracellular processes. By creatively fusing fluorescent proteins to biopolymers that perform critical functions involved in various aspects of physiological signaling, research scientists have developed a host of new molecular probes that are useful for optical live-cell imaging of important processes such as calcium wave induction, cyclic nucleotide messenger effects, membrane potential fluctuations, phosphorylation, and intracellular protease action.
The tutorial initializes with an inositol phosphate biosensor composed of cyan and yellow fluorescent proteins sandwiching a phosphate binding site (PBD) appearing in the tutorial window. An image of the specimen (a single cell) expressing the biosensor appears in the Specimen Image window. The CFP is being excited by 440-nanometer light and is emitting cyan fluorescence. To operate the tutorial, use the FRET slider to increase the inositol triphosphate concentration, thus evoking a conformational change in the phosphate binding domain to bring the CFP and YFP moieties closer together. At high phosphate concentrations, FRET occurs and excitation with 440-nanometer light produces yellow fluorescence (527 nanometer peak). Note how the emission color of the specimen changes as FRET begins to occur. To choose a new specimen, use the Choose a Biosensor pull-down menu.
Tony Gines, Adam Rainey, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310