Spectral FRET

F4FlAsH-P12 61 at 544 nm expanded the spectral range of the biarsenical dyes. Furthermore, the fluorescence lifetime increased to a value of 5.2 ns. Owing to the results of this study, the authors suggested that the two compounds (57 and 58) would form an excellent FRET pair with a large critical distance. In addition, they envisage that the observed characteristics should facilitate improved structural and dynamic studies of proteins in living cells. 

While VFPs have boosted the applications of FRET-FLIM, chemical FRET probes should not be dismissed. The advantage of chemical probes is that they are much smaller in size and that they often have much better spectral readout than VFP probes. In Chapter 6, Amanda Cobos Correa and Carsten Schultz highlight the various small molecule-based FRET probes and their use in bioimaging. 

Chapter 8 written by Steve Vogel et al. also deals with sensitized emission based FRET methodology, but now using a spectral imaging detector device. Because a spectral detector and spectral unmixing software nowadays are standard options on the major commercial confocal microscopes, here a complete description is given how to quantify FRET from unmixed spectral components. 

Harpur, A. G., Wouters, F. S. and Bastiaens, P. I. (2001). Imaging FRET between spectrally similar GFP molecules in single cells. Nat. Biotechnol. 19, 167-9. 

Zimmermann, T., Rietdorf, J., Girod, A., Georget, V. and Pepperkok, R. (2002). Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531, 245-9. 

The term filterFRET here refers to intensity-based methods for calculating fluorescence resonance energy transfer (FRET) from sets of images of the preparation collected at different excitation and/or emission wavelength. The term is not intended to imply that interference filters are actually present in the setup very similar considerations apply when donor- and acceptor fluorophores are spectrally resolved by other means, such as monochromators or spectral detectors. 

Standard linear unmixing of a spectral image of a sample composed of two fluorophores yields a measure of the concentration of each fluorophore present for each pixel. If FRET is occurring, linear unmixing will produce an apparent donor concentration ( apparent) that underestimates the true donor concentration (d) by a factor of 1 -ED ... 

Strategies for measuring FRET using spectral imaging... 

One way to reduce the number of independent variables in the FRET-adjusted spectral equation is to use samples with a fixed donor-to-acceptor ratio. Under these conditions, the values of d and a are no longer independent, but rather the concentration of d is now a function of a and vice-versa. This approach is typical for the situation of FRET-based biosensor constructs. These sensors normally are designed to have a donor fluorophore attached to an acceptor by a domain whose structure is altered either as a result of a biological activity (such as proteolysis or phosphorylation), or by its interaction with a specific ligand with which it has high affinity. In general, FRET based biosensors have a stoichiometry of one... 

One criticism of this approach, however, is that in addition to requiring the specialized hardware for obtaining spectral images, additional instrumentation is often required to measure the FRET efficiency. Furthermore, the limitations specific to the FRET method used in conjunction with spectral imaging will also apply to this hybrid approach. 

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