FRET efficiencies

FRET sensing based on protic equilibrium in the acceptor that changes its absorption spectrum and thus modulates the overlap integral [35]. There are many fluorescent pH indicators that display pH-dependent absorption spectra in the visible with their different positions depending on ionization state. Thus, the change in pH can be translated into the change of FRET efficiency. 

FRET efficiency can be quantitatively measured over D-A separations of 0.5-10 nm. For shorter distances the assumption of point dipoles may not be valid (although this does not seem to be noticeable until very short distances). Very short distances comparable to the spatial extension of electron orbitals can lead to energy transfer by electron transfer (Section 1.11). 

Simple depiction of basic fluorescence how you determine the FRET efficiency... 

Measuring any de-excitation process in order to determine FRET efficiency the acronym FRET... 

Measuring photobleaching to determine FRET efficiency 1.8.1. The rate of donor photobleaching... 

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... 

Another method to detect energy transfer directly is to measure the concentration or amount of acceptor that has undergone an excited state reaction by means other than detecting its fluorescence. For instance, by chemical analysis or chromatographic analysis of the product of a reaction involving excited A [117, 118]. An early application of this determined the photolyzed A molecules by absorption spectroscopic analysis. [119-121], This can be a powerful method, because it does not depend on expensive instrumentation however, it lacks real-time observation, and requires subsequent manipulation. For this reason, fluorescence is the usual method of detection of the sensitized excitation of the acceptor. If it is possible to excite the donor without exciting the acceptor, then the rate of photolysis of the acceptor (which is an excited state reaction) can be used to calculate the FRET efficiency [122],... 

The presence of the acceptor, lower row of images, results in a clear reduction in the lifetime to about 2.05 ns. The reduction corresponds to a 6% FRET efficiency. Experiments on more cells (.N = 4, not shown) confirms that this reduction is indeed significant and that the two lipid raft markers colocalize in the plasma membrane. 

A closer look at the data shows the lifetime distributions are comparatively broad, about 0.25 ns for both distributions. This is in fact much broader than what one would expect from photon statistics alone. Based on realistic / -values (1.2-1.5) lifetime images recorded with this many counts are expected to yield distributions with widths on the order of 0.1 ns. The broadening is therefore not because of photon statistics. Variations in the microenvironment of the GFP are the most likely source of the lifetime heterogeneities. Importantly, such sensitivity for local microenvironment may be the source of apparent FRET signals. In this particular FRET-FLIM experiment, we found that the presence of CTB itself without the acceptor dye already introduced a noticeable shift of the donor lifetime. Therefore, in this experiment the donor-only lifetime image was recorded after unlabeled CTB was added to the cells. The low FRET efficiency and broadened lifetime distribution call for careful control experiments and repeatability checks. 

The FRET efficiency (E) is highly dependent on the distance between donor and acceptor and is defined by Forsters theory [105] ... 

Fig. 5.4. Correlation between R0 and FRET efficiency. Exchanging ECFP/ EYFP (R0 = 4.72 nm, dashed line) for the red-shifted VFP FRET-pair mKO/ mCherry (R0 = 6.37 nm, solid line) will increase the measured FRET efficiency, since the distance between donor and acceptor is expected to remain unchanged. A FRET pair with increased R0 yields detectable FRET over longer distances and can be used to measure protein-protein interaction between larger proteins.

The fluorescence intensity of fluorescent proteins is pH dependent and most fluorescent proteins are less fluorescent at lower pH mainly because of a reduction in absorbance. Since the absorbance of the acceptor determines the FRET efficiency, changes in the acceptor absorbance spectrum due to pH variations can be wrongly interpreted as changes in FRET efficiency. Thus, a pKa well below physiological pH is recommended to prevent artifacts due to pH changes inside cells. This is especially challenging if the fluorescent proteins are to be targeted to acid cellular compartments, for example, endosomes, lysosomes, or plant vacuoles. 

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. 

Finally, a nonfluorescent YFP variant has been constructed for use as a FRET acceptor for GFP [89]. This allows the detection of the complete emission spectrum of GFP, while the FRET efficiency is high (R0 = 5.9 nm) due to strong overlap of the GFP fluorescence and YFP absorbance band. The occurrence of FRET was detected by a reduction in the excited state lifetime of the GFP by FLIM. The main disadvantage is that the presence of the acceptor cannot be detected in living cells. 

Importantly, in most applications the measured (change in) FRET efficiency cannot be translated directly into an average distance between donor and acceptor fluorophore because the fraction of donor molecules involved in FRET is unknown (i.e., all molecules display 25% FRET or 50% of the molecules display 50% FRET), and the orientation factor (k2) is unknown (see also Chapter 7). 

Since FRET is sensitive to the dipole orientation of the donor and acceptor, changes in orientation of either the donor fluorophore or the acceptor fluorophore may lead to changes in FRET efficiency. Therefore conformational changes within a protein can be detected when the protein of interest is tagged with both a donor and an... 

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. 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

S depends on FRET efficiency but also on fluorophore concentration, donor excitation, and detector sensitivity. 

Luckily, a mathematical framework to solve these problems has been worked out by several groups [1-6] who showed that from just three acquired images S, D, and A quantitative FRET efficiency images can be calculated. This framework relies on calibrations taken from cells expressing either donors only or acceptors only and it allows direct comparison of results obtained around the world. 

In general, ratio imaging is not quantitative nor is it, strictly spoken, normalized because the acquired data do not permit Problems 1-4 (see Sect. 7.1.1) to be properly addressed. One important exception is the case where donors and acceptors are present at a fixed stoichiometry. Examples of that are the popular single-polypeptide FRET sensors. In this case, the normalization problem (2) is inherently solved and the overlap- and reference-image problems (1 and 3) simplify considerably. It can be shown [1 and Appendix 7.A.6] that in that case FRET efficiency ( ) can be calculated from D and S images. 

Fig. 7.3. Fret efficiency. The unquenched donor image (top row, middle panel), as calculated according to Eq. (7.13), and the acceptor image (top right panel) are used to normalize the s.e. image. The resulting images ED and EA (Eqs. (7.10) and (7.11), respectively) are quantitative, as detailed in the text. Unfiltered, raw data are shown. Scale bar is 12 /mi.

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],... 

The FRET efficiency Ed as determined above is the fraction of energy quanta absorbed by all donor molecules that is transferred to acceptors. For a given pixel, Ed effectively reflects both the efficiency with which paired donor-acceptors transfer energy (E) and the fraction of molecules in that pixel that pair up (/)>). This means, for example, that a pixel with ED = 0.2 may result from 100% of donors having =0.2, or from 20% of donors having E = 1, or anything in between. The FRET efficiency E of a donor/acceptor pair (termed characteristic FRET efficiency, Ec in some literature [2, 3]) is most often unknown. 

Fig. 7.7. Effects of Poisson photon noise on calculated SE and FRET values. (A) Statistical distribution of number of incoming photons for the mean fluorescence intensities of 5,10, 20, 50, and 100 photons/pixel, respectively. For n = 100 (rightmost curve), the SD is 10 thus the relative coefficient of variation (RCV this is SD/mean) is 10 %. In this case, 95% of observations are between 80 and 120. For example, n — 10 the RCY has increased to 33%. (B) To visualize the spread in s.e. caused by the Poisson distribution of pixel intensities that averaged 100 photons for each A, D, and S (right-most curve), s.e. was calculated repeatedly using a Monte Carlo simulation approach. Realistic correction factors were used (a = 0.0023,/ = 0.59, y = 0.15, <5 = 0.0015) that determine 25% FRET efficiency. Note that spread in s.e. based on a population of pixels with RCY = 10 % amounts to RCV = 60 % for these particular settings Other curves for photon counts decreasing as in (A), the uncertainty further grows and an increasing fraction of calculated s.e. values are actually below zero. (C) Spread in Ed values for photon counts as in (A). Note that whereas the value of the mean remains the same, the spread (RCV) increases to several hundred percent. (D) Spread depends not only on photon counts but also on values of the correction...

Unbiased cleaning up Mixing FRET efficiency with image intensity information... 

Fig. 7.9. Further FRET efficiency analysis. (A) Unbiased display of FRET efficiency. The ED image (upper left panel) is modulated with an intensity picture (in this case, s.e., upper right panel) to yield the lower left image. See text for further details. Lower right panel, example with several cells... 

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