Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Spectral Imaging

Fluorescent Protein FRET Biosensors

Among the most useful FRET applications in cell biology is a technique that involves the fusion of two fluorescent proteins to the ends of an environmentally sensitive protein or peptide to act as a biosensor of specific cellular functions. Fluorescent protein biosensors have found widespread utility in reporting on a diverse array of intracellular processes. By creatively fusing FRET-capable pairs of 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, pH, membrane potential fluctuations, phosphorylation, and intracellular protease action.

Spectral imaging has been very useful for the examination of fluorescent protein biosensors to determine the presence or absence of FRET in response to a biological stimulus. The methodology requires obtaining a spectrum of the biosensor in the presence and absence of the stimulator to determine whether FRET is observed or eliminated and to monitor the dynamic range of the response. Thus, by creatively fusing pairs of fluorescent proteins to biopolymers that perform critical functions involved in various aspects of physiological signaling or other biological activities, a number of investigators have developed a host of new molecular probes that are useful for optical live-cell imaging of important metabolic and signaling processes. Typically, two fluorescent proteins (usually a cyan and yellow variant) are inserted on opposing ends of a sensor protein or peptide sequence, as briefly described above. Changes in the conformation of the sensor protein produce corresponding tertiary alterations in the structural organization of the complex, and thus, the level of FRET that can be observed between the flanking fluorescent proteins. A ratiometric change in the fluorescence output of the donor and acceptor using a single excitation parameter coupled with spectral imaging is becoming a popular methodology to examine these biosensors.

Contributing Authors

Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

We use cookies on this site. Cookies are small text files that are stored on your computer by websites. Cookies are widely used and help to optimize the pages that you view. By using this site, you agree to their use. more