Fluorescence donors

Day RN, Booker CF, Periasamy A (2008) Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. J Biomed Opt 13 031203. doi 10.1117/1111.2939094... 

It is convenient, but not necessary, if the intensity of excitation light is the same in both cases. If this is true and if the concentration of donor molecules is also the same for both measurements, then the number of initially excited donor molecules is the same. If these conditions are not met, then the value of detected donor fluorescence can be corrected so the measured values can be compared. Once these corrections have been made (if they are necessary), we can form the following ratio ... 

It is very difficult to excite the donor without also exciting some of the acceptor population. This is because the absorption spectra of dyes extend significantly into the blue side of their absorption maxima, so the absorption spectra of the donor and acceptor usually overlap. The donor fluorescence can typically be observed without acceptor fluorescence interference therefore, when measuring FRET efficiency by observing the donor fluorescence, this overlap is not important. However, when observing the acceptor fluorescence the overlap of the donor and acceptor absorption must be taken into account. The total steady-state fluorescence of the acceptor, assuming that [A] = [D (i.e., a equal donor and acceptor concentrations, and 100% labeling) is... 

Maximal overlap between the fluorescent emission spectra of the donor and the fluorescent absorption spectra of the acceptor is important for efficient FRET. Since the donor can be directly excited, even fluorophores with mediocre quantum yield may be used. However, the success of a ratiometric FRET approach will strongly depend on the quantum yield of the acceptor. While an acceptor with a lousy quantum yield may still be a good quencher of the donor fluorescence, it will not yield acceptor fluorescence to an appreciable extent. When calculating the ratio between donor and acceptor emission values, acceptors with a good quantum yield will therefore be preferred. 

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. 

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). 

The fluorescent components are denoted by / (intensity) followed by a capitalized subscript (D A or s, for respectively Donors, Acceptors, or s.e.) to indicate the particular population of molecules responsible for emission and a lower-case superscript ( " or ) that indicates the detection channel (or filter cube). For example, I denotes the intensity of the donors as detected in the donor channel and reads as Intensity of donors in the donor channel, etc. Notes (1) The excitation in the s.e. channel is generally set up to be equal to that in the donor channel. In case a separate filter cube is used, slight differences may occur, which is denoted by Don(S). See the text and appendix for further details. (2) The s.e. emission filter is usually the same as the acceptor emission filter in confocal determinations. We here designate a different filter to accommodate those wide-field/digital camera experiments that employ different filters for A and S. (3) Here the notation D-S indicates the residual (quenched) donor fluorescence in the presence of the acceptor. In the other chapters this is indicated as DA. Hence ... 

Note that this equation is identical to the expression for unquenched donor fluorescence of van Rheenen et al. (Eq. (A 17)). 

A second approach also considers three populations free (unquenched) donors No, free acceptors NA, and a population engaged in FRET pairs Ns that transfer energy with characteristic efficiency E (between 0 and 1). However, in this case, the Ns population emits both donor fluorescence (quenched by a fraction (1 - E)) and sensitized emission (proportional to ENS). To keep in line with the treatment and terminology in other chapters in this volume, this latter approach will be followed here. Note, however, that in other chapters the population of FRET pairs is indicated by the subscript DA whereas we stick to the notation Ns to indicate that this quantity is based on photons emitted from sensitized emission (S image) and to keep the close synonymy with the former approach. Thus, our Io-s equals Ida and Is +1 a equals IAo- Both ways yield essentially identical results. 

D is the output gray scale value after amplification8 in the donor channel (gd) of the sum of the fraction (F ) of donor fluorescence in the donor channel and the fraction (i ) of acceptor fluorescence in the donor channel. The fluorescence of donors depends on the number of donor molecules (No) diminished by... 

S is the output gray value after the s.e. channel detector scaling (gs) of the sum of the fractions of donor fluorescence in the s.e. channel (FJ,) and of acceptor fluorescence in the s.e. channel (F) ). The donor fluorescence depends on Qd, the excitation efficiency at lfx (that is, Fe ), the number of donors (ND), and the population of donors that lose their energy by FRET (ENs-). The fluorescence of acceptors depends on QA, the amount of acceptor molecules (NA) excited with 2dx ( szsA) and on the amount of acceptor molecules excited by FRET (ENs, which is linear to Fefj). 

Finally, A is the output gray value after the acceptor channel9 scaling (g3) of the fraction of acceptor fluorescence in the acceptor channel (FJ), which depends on the acceptor quantum yield (Qa) and on the amount of acceptors NA excited at a x (t il A ), of (usually very minor) contributions of donor fluorescence cross-excited at and leaking into the acceptor channel ((No — ENs)fa< -DQdF d F) and of sensitized emission resulting from cross-excitation at 2 x (ENs dQaF 3) However, as... 

Because FRET results in a decrease in donor fluorescence intensity and excited state lifetime with a corresponding increase in the acceptor fluorescence (sensitized emission), various methods for measuring FRET have been based on assessing one of the above photophysical consequences [22], The most commonly employed methods for measuring FRET in living systems (described elsewhere in more detail in this volume) are ... 

Donor fluorescence recovery after acceptor photobleaching (also called acceptor photobleaching or acceptor depletion FRET) [22, 23, 26, 28, 30, 48, 53-56] (see Chapters 1 and 7) and... 

CFP-YFP donor-acceptor pair, YFP is several times brighter than CFP [62]. Lastly, for studying dynamic protein associations in plants, the presence of chlorophyll pigments in leaf and stem cells is an additional limitation. These pigments directly absorb the fluorescence, which decreases blue fluorescence intensity for BFP and CFP donors that can be erroneously interpreted as reduced donor fluorescence quantum yield caused by FRET [18]. If sensitized emission or FSPIM is the only available method for quantifying FRET, then it is very important to restrict measurements to chlorophyll free areas within the cells. 

Fig. 11.2. A donor dequenching experiment that demonstrates that extracellular domains of APP and LRP closely associated with one another. H4 cells were transfected with APP770 and LRP and immunolabeled with Fluorescein (A) and Cy3 (B), respectively. When the Cy3 is photobleached in the area marked by the white rectangle, the signal from fluorescein increases (C), while the signal from Cy3 disappears (D). The increase in donor fluorescence is calculated to be 51%, which shows that the ectodomains of APP770 and LRP, are in close proximity and therefore likely to be interacting. This result has implications for the field of Alzheimer s disease research as it helps elucidate the nature of APP processing into amyloid-/ [50],...

FRET manifests itself through the quenching of donor fluorescence and a reduction of the fluorescence lifetime, accompanied by an increase in acceptor fluorescence emission. The efficiency of the energy-transfer process varies in proportion to the inverse sixth power of the distance separating the donor and acceptor molecules. Consequently, FRET measurements can be utilised as an effective molecular ruler for determining distances between molecules labelled with an appropriate donor and acceptor fluorophore, provided they are within lOnm of each other. 

Radiative transfer results in a decrease of the donor fluorescence intensity in the region of spectral overlap. Such a distortion of the fluorescence spectrum is called the inner filter effect (see Chapter 6). 

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