Donor emission

In these dye-functionalized dendrimers, light absorbed by the numerous peripheral coumarin-2 units is funneled to the coumarin-343 core with remarkably high efficiency (toluene solution 98% for the first three generations 93% for compound 8). Given the large transition moments and the good overlap between donor emission and acceptor absorption, energy transfer takes place by Forster mechanism [34]. 

This can be an important process if the acceptor A absorbs in the wavelength region in which the donor D emits. The efficiency of the process is determined by the quantum yield of D emission and by the optical density of A at the donor emission wavelength. The probability that an acceptor molecule will reabsorb the light varies as R 2, where R is the donor-acceptor separation. 

Energy transfer by the trivial mechanism is characterized by (a) change in the donor emission spectrum (inner filter effect), (b) invariance of the donor emission lifetime, and (c) lack of dependence upon viscosity of the medium. 

Inasmuch as K and L are constants not readily available from experimental data, only the form of Eq. (6.22) is of interest. For example, the rate constant should decrease exponentially with increasing separation R between the donor and acceptor. Also, because donor and acceptor multiplicities can change during the transfer, the overlap integral is calculated with both the donor emission and acceptor absorption normalized to unity. 

If the energy is transferred by trivial emission/reabsorption, it will lengthen the measured lifetime of the donor emission, not shorten it as happens in resonance energy transfer. This comes about because intervening absorption and emission processes take place prior to the final fluorescence emission (the reabsorption cannot take place until the photon has been emitted) the two processes do not compete dynamically, but follow in a serial fashion. In FRET, such an emission/reabsorption process does not occur, and the fluorescence lifetime of the donor decreases. This is an experimental check for reabsorption/reemission. 

Every time an excited molecule exits the excited state region by the fluorescence pathway it emits a photon. We can either count the number of photons in a longer time interval (by a steady-state measurement of the fluorescence intensity) or make a time-resolved measurement of the fluorescence decay. These measurements can be done in an ensemble mode or on single molecules—the basic process is the same. The number of photons collected from the donor emission will be depicted by IDA and ID, where we mean the fluorescence intensity of D in the presence (Ida) and absence (ID) of acceptor. All other conditions, other than the presence or absence of acceptor, remain the same. During the same time of the experiment where we have measured the photons emitted by D, many of the excited D molecules have exited from the excited state by a pathway other than fluorescence. Obviously, the number of times a pathway has been chosen as an exit pathway is proportional to the... 

D Don Don raw donor image collected at with donor emission filter (donor channel)... 

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

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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],... 

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. 

It is the emission spectrum of the acceptor multiplied by the abundance of the donor (attenuated by the fraction of donor emissions that actually result in FRET), then multiplied by a factor... 

Qa/Qd k that equates how the emission intensity increase of the acceptor corresponds to the attenuation of the donors emission. This factor is equal to the extinction coefficient ratio of the donor and acceptor at the used excitation wavelength, A[,x, see appendix. If FRET is not occurring in a sample (i.e., ED = 0), the whole equation reduces to ... 

Thus, E is defined as the product of the energy transfer rate constant, ku and the fluorescence lifetime, xDA, of the donor experiencing quenching by the acceptor. The other quantities in Eq. (12.1) are the DA separation, rDA the DA overlap integral, / the refractive index of the transfer medium, n the orientation factor, k2 the normalized (to unit area) donor emission spectrum, (2) the acceptor extinction coefficient, eA(k) and the unperturbed donor quantum yield, QD. 

In Eq. (4.5) the donor emission spectrum/ and the acceptor absorption spectrum eA are separately normalized to unity, so that the transfer rate is independent of the oscillator strength of either transition. Unfortunately, the constants W and L are not easily determined by experiment. Nevertheless, an exponential dependence on the distance is expected. It should be noted that this type of transfer involves extensive orbital overlap and is guided by Wigner s (1927) spin rule. 

Thus, this mechanism requires that A must be capable of absorbing the photon emitted by D that is, the acceptor absorption spectrum must overlap with the donor emission spectrum. Radiative energy transfer can operate over very large distances because a photon can travel a long way and A simply intercepts the photon emitted by D. ... 

Characteristics of the donor emission Radiative transfer Non-radiative transfer... 

For the design of complexing bifluorophores, much attention should be paid to the Forster critical radius of the donor-acceptor pair as compared to the interchromophoric distance (Figure 2.13). This critical radius depends on the donor quantum yield and on the spectral overlap between donor emission and acceptor emission. Complex-... 

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