Interactive Tutorials - Spinning Disk Fundamentals

Interactive Tutorials

Spectral Imaging

Spectral Imaging FRET with 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.

In FRET applications, spectral imaging can be considered a variation of the sensitized emission technique that relies on excitation of the donor alone, followed by acquisition of the entire emission spectrum of both the donor and acceptor fluorescence instead of capturing data in two independent channels. Until the introduction of laser scanning confocal microscopes designed for spectral imaging, the technique was largely limited to spectroscopy experiments using cuvettes and purified fluorophores. Spectral imaging FRET assumes that gathering of the entire fluorescence spectrum will enable overlapping spectral profiles to be separated according to the distinct shapes of the spectra rather than simply monitoring emission intensity in a limited bandwidth region using a filter. Thus, by collecting the entire spectrum from both the donor and acceptor, it is possible with spectral imaging to determine the levels of donor and acceptor fluorescence, and from that information (combined with controls), to calculate the FRET efficiency.

Contributing Authors

Adam M. Rainey and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

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