Emission, sensitized

Self-assembly of functionalized carboxylate-core dendrons around Er +, Tb +, or Eu + ions leads to the formation of dendrimers [19]. Experiments carried out in toluene solution showed that UV excitation of the chromophoric groups contained in the branches caused the sensitized emission of the lanthanide ion, presumably by an energy transfer Forster mechanism. The much lower sensitization effect found for Eu + compared with Tb + was ascribed to a weaker spectral overlap, but it could be related to the fact that Eu + can quench the donor excited state by electron transfer [20]. 

Figure 4 Cartoon illustrating sensitized emission from a lanthanide ion.

An alternative approach is that adopted by Horrocks and co-workers, where the aromatic residues in metal-binding proteins are used as sensitizers. Since the distance between the metal and the donor is effectively fixed, this provides a rigid scaffold for the experiment, and the absence of a directly conjugated pathway between the metals means that Forster (through space) energy transfer can be assumed. The r-6 distance-dependence of this means that the extent of sensitized emission from the lanthanide ion provides information on the spatial relationship between the metal-ion binding site (lanthanide ions often bind at Ca2+ sites) and nearby aromatic residues. 58-60... 

Lanthanides also have potential as DEFRET energy donors. Selvin et al. have reported the use of carbostyril-124 complexes (53) with europium and terbium as sensitizers for cyanine dyes (e.g., (54)) in a variety of immunoassays and DNA hybridization assays.138-140 The advantage of this is that the long lifetime of the lanthanide excited state means than it can transfer its excitation energy to the acceptor over a long distance (up to 100 A) sensitized emission from the acceptor, which occurs at a wavelength where there is minimal interference from residual lanthanide emission, is then measured. 

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. 

In previous chapters it was shown that FRET can be reliably detected by donor fluorescence lifetime imaging. Here, we will focus on what is perhaps the most intuitive and straightforward way to record FRET imaging of sensitized emission (s.e., that is, the amount of acceptor emission that results from energy transferred by the donor through resonance) by filterFRET. While simple in principle, determinations of s.e. are complicated by overlap of excitation and emission spectra of the donors and acceptors, and by several imperfections of the recording optics, light sources and detectors. 

Even if we forget, for a moment, the overlap problem and assume that we obtained a pure sensitized emission image, interpretation of this image is still ambiguous. That is because first, the intensity of S varies linearly with the excitation intensity and with the detector sensitivity. The exact same preparation will, when measured on a different microscope, yield different s.e. intensities. In fact, as much as renewing the arc lamp would impede comparison of results obtained on the same microscope. Second, the interpretation... 

D is the sum of the remaining donor fluorescence in the donor channel (/p s) > and of leak-through components of sensitized emission back into the donor channel (/ ) and of cross-excited acceptors back into the donor channel (/ J). 

Finally, A contains acceptor fluorescence (/jj) and two usually very minor leak-through components that of the (partially quenched) donor population inappropriately excited at acceptor wavelength and leaking into the acceptor channel (lf) s), and the small amount of sensitized emission that stems from FRET after inappropriate excitation of donors at acceptor wavelength (/ ). 

S Don s.e. raw sensitized emission image collected at with the sensitized emission filter (s.e. channel)... 

Sensitized emission (/ ), as defined in Eq. (7.8), reliably measures the relative amount of energy transfer occurring in each pixel (Fig. 7.2, lower right panel). Iss is corrected for spectral overlap (i.e., Problem 1 has been taken care of) however, unlike E, it is not a normalized measure for interaction nor is it quantitative in absolute terms. It depends on the specific biological question which of the two yields the most relevant information. 

In order to obtain the desired quantitative measure of FRET (Fig. 7.3), an additional correction factor must scale the nominator to the denominator in Eq. (7.9) [1-3, 6], In other words, we must relate the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel as in ... 

Note that the Loss in donor emission due to FRET (Eq. (7.11)) is just a constant times the sensitized emission (Eq. (7.8)) for given acquisition settings, or /,d(lss = 4>LS. Thus (noting that both and

[Pg.318]

Several other approaches to solve the quantitation problem have been proposed. Hoppe et al. [2] determined y/ by calibrating it against constructs with known FRET efficiency. We and others [3, 6] have used data from a cell before and after acceptor photobleaching to relate the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel by factors termed or G, respectively. For the CFP/YFP pair this works very well on confocal microscopes with a 514-nm Argon ion laser line, but on wide-held systems, selective acceptor photobleaching reportedly causes problems [ 14]. F inally, G can also be determined by comparison of several constructs that differ in FRET efficiency, a bit analogous to the Yellow Cameleon calibration described above [10,14],... 

Unlike donor-based FRET methods like FLIM, filterFRET also yields spatial information on the acceptor population. This means that in addition to querying donor-FRET (by solving for Ed or / )), we can also assess the relationship between sensitized emission and the acceptor population. At 1 1 stoichiometry obviously Ed should equal the acceptor-normalized efficiency EA. In other cases, EA deviates from E but sometimes can yield biologically more relevant information than Ed or E. For example, dislocation of 50% of the... 

In this chapter, it was shown that filterFRET is an easy, intuitive and quantitative alternative to record sensitized emission and FRET efficiency. The major advantages of filterFRET over donor-based FRET detection methods (FLIM) are that it can be carried out with standard wide-held or confocal fluorescence microscopes that are available in most laboratories, and that it yields additional data on the acceptor population. FilterFRET is also fast, requiring just two confocal scans (if need be on a line-by-line basis) which minimizes the risk of artifacts due to, for example, organelle movement in living cells, and acquisition can be optimized for each channel independently. However, quantitative... 

Before proceeding, an important note must be made. In literature, two different but fully equivalent approaches have been taken in s.e. The first approach considers a cell that contains (unknown) numbers of donors and acceptors No and NA. When energy transfer takes place (be it from collisional encounters or because a stable population of FRET pairs exist with FRET efficiency E) this diminishes the effective number of emitting donors with Ns [3] that is, the FRET efficiency for this population is unity. Thus, the residual donor emission results from (No — Ns) unquenched donor molecules, and the Ns population emits only sensitized emission. This approach is intuitive in case no assumptions are being made on the presence of a stable population of FRET pairs or on the magnitude of E in a donor-acceptor complex. 

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