Fluorescence decay, FRET

This toy model depicts the basic statistical ideas of fluorescence decay that is critical for understanding FRET. I apologize to all who already know all this. You can skip it, or just read it over for fun. 

FRET applications employing CFP and YFP are complicated due to considerable bleed-through between CFP and YFP fluorescence (Figs. 5.5B and 5.6B). Direct excitation of YFP and bleed-through of CFP fluorescence into the YFP detection channel have to be corrected for as shown in Chapters 7 and 8. The multiexponential fluorescence decay of all CFP variants complicates the quantification of FRET by donor lifetime methods. Altogether these factors make quantitative analysis of the FRET efficiency relatively difficult. 

There remain challenges to overcome in the use of FLIM and FRET in the general laboratory environment. A clear understanding of the nature of fluorescence decay is necessary for the correct interpretation of FLIM data and care must be taken to use negative controls appropriately. The contamination of lifetime data by... 

Selected entries from Methods in Enzymology [vol, page(s)] Analysis of GTP-binding/GTPase cycle of G protein, 237, 411-412 applications, 240, 216-217, 247 246, 301-302 [diffusion rates, 246, 303 distance of closest approach, 246, 303 DNA (Holliday junctions, 246, 325-326 hybridization, 246, 324 structure, 246, 322-324) dye development, 246, 303, 328 reaction kinetics, 246, 18, 302-303, 322] computer programs for testing, 240, 243-247 conformational distribution determination, 240, 247-253 decay evaluation [donor fluorescence decay, 240, 230-234, 249-250, 252 exponential approximation of exact theoretical decay, 240, 222-229 linked systems, 240, 234-237, 249-253 randomly distributed fluorophores, 240, 237-243] diffusion coefficient determination, 240, 248, 250-251 diffusion-enhanced FRET, 246, 326-328 distance measurement [accuracy, 246, 330 effect of dye orientation, 246, 305, 312-313 limitations, 246,... 

Cha et al. (1999) used a variant of FRET called LRET for lanthanide-based fluorescence energy transfer. In this technique (Selvin, 1996) the donor is terbium or europium which, in fact, is luminescent. There are several advantages of this technique over regular FRET. It has been found that terbium emits isotropically, which means that the uncertainty due to the dipole orientation is decreased to a maximum error of 10%. This error can be decreased even further if the anisotropy of the acceptor is also known. The second advantage is that the fluorescence decay has a time constant of about 1.5 ms, making it easily measurable with conventional recording techniques. The third advantage is that the emission of terbium is peaked and one can find fluorophores that emit in between peaks. This means that the fluorescence of the acceptor can be measured with little or no contamination from the donor. In addition, as the acceptor has a fast decay, any measurement of the acceptor fluorescence with decays comparable to the donor will exclude any possible direct... 

The analysis of the histograms of photon arrival times is equivalent in both cases and relies on fitting appropriate model functions to the measured decay. The selection of the fitting model depends on the investigated system and on practical considerations such as noise. For instance, when a cyan fluorescent protein (CFP) is used, a multi-exponential decay is expected furthermore, when CFP is used in FRET experiments more components should be considered for molecules exhibiting FRET. Several thousands of photons per pixel would be required to separate just two unknown fluorescent... 

Donor and acceptors can be covalently linked using a chemical spacer. Assume that we have the same D-A pair Eosin-Phenol Red. In this case we will have a mixture of two linked donor-acceptor species (Eosin-Phenol Red protonated and Eosin-Phenol Red unprotonated) characterized by the same distance distribution and different critical distances (ftoi = 28.3 A and Rm = 52.5 A) for FRET. A distribution of D- to -A distances will be present because the linker is typically flexible. The fractional intensity of the first species at time t = 0 is gi and that of the second species is (1 - 1). The fractional intensity at time t = 0 is equal to fractional concentration of each form, which can be in case of pH indicator (Phenol Red) calculated using Eq. (10.31). The donor fluorescence intensity decay of the mixture is described by the equation... 

In the microscopic technique, photobleaching FRET (5), the intensity of fluorescence excitation is increased to cause photobleaching of the donor fluorochrome, and the decay kinetics are measured in the absence or presence of an acceptor fluorochrome. If the acceptor is in close proximity to the donor, then the availability of excited-state donors for photobleaching is reduced, thus making the photobleaching process slower. 

Another example of improved sensitivity due to modulation of lanthanide photophysics by ancillary ligands can be found in the europium and terbiiun chelates used in time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays (100,101). Due to their line-type emissions and long decay times, the lanthanide chelate is used as a donor, with some visible-absorbing dye such as Alexa 647 or a rhodamine derivative as the acceptor. Without the helper ligand, the lanthanides would be unprotected from solvent and have much shorter decay times, making them unsuitable for such an assay. 

Volkmer A, Subramaniam V, Birch DJS, Jovin TM. One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins. Biophys. J. 2000 78 1589-1598. Subramaniam V, Hanley QS, Clayton AHA, Jovin TM. Photophysics of green and red fluorescent proteins implications for quantitative microscopy. Methods Enzymol. 2003 360 178-201. Rizzo MA, Springer GH, Granada B, Fdston DW. An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnol. 2004 22 445-449. 

Because the electron transfer rate decays rapidly over a few angstroms, the fluorescence quenching via electron transfer approach is sensitive to subtle distance changes on the angstrom scale. This distance range is complementary to that of the widely used FRET-based techniques, such as FRET and fluorescence quenching... 

The second method for measuring FRET involves time-resolved fluorescence measurements. This method provides a way of obtaining average lifetimes without a precise knowledge of donor concentrations. The technique enables the quantitative determination of donor-acceptor separation distances, and is based on the measurements of the donor lifetime in the presence and absence of the acceptor. Measuring fluorescence intensity decay as a function of time elucidates the emission dynamics of... 

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