Sensitized emission FRET method

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 ... 

Sensitized emission FRET (also called filter FRET or channel FRET)—a method that assesses the changes in donor and acceptor fluorescence intensity upon successful energy transfer [26, 48-50] (see Chapter 7) ... 

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... 

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. 

Additionally, since the acceptor is excited as a result of FRET, those acceptors that are fluorescent will emit photons (proportional to their quantum efficiency) also when FRET occurs. This is called sensitized emission and can also be a good measure of FRET (see Fig. 1). To quantitate FRET efficiency in practice, several approaches have been evolved so far. In flow cytometric FRET (7), we can obtain cell-averaged statistics for large cell populations, while the subcellular details can be investigated with various microscopic approaches. Jares-Erijman and Jovin have classified 22 different approaches that can be used to quantify energy transfer (8). Most of them are based on donor quenching and/or acceptor sensitization, and a few on measuring emission anisotropy of either the donor or the acceptor. Some of these methods can be combined to extend the information content of the measurement, for example two-sided FRET (9) involves both acceptor depletion (10) and... 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Fluorescence (or Forster) resonance energy transfer (FRET) (Forster, 1948 Perrin, 1932) provides a spectroscopic way of estimating distances over a size range that is appropriate for biological macromolecules. It is based upon fluorescence, one of the most sensitive spectroscopic methods. Fluorescence is the emission of light from an excited molecule, having lost... 

The binding of synthetic ion channels and pores to lipid bilayer membranes often causes a change in intra- or intermolecular self-organization that is visible in sufficiently sensitive methods such as fluorescence (e.g. tryptophan emission) [14] or circular dichroism spectroscopy and can be used to determine the partition coefficient. Convenient methods of detection under relevant conditions are fluorescence resonance energy transfer (FRET) or fluorescence depth quenching (FDQ) [3, 4, 6]. Many fluorescent probes for the labeling of both synthetic ion channels/... 

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