Verveer Peter J, Rocks Oliver, Harpur Ailsa G, Bastiaens Philippe I H
CSH Protoc. 2006 Nov 1;2006(6):pdb.prot4597. doi: 10.1101/pdb.prot4597.
This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission. Conversely, fluorescence emission tends to tail into the red part of the spectrum, causing donor fluorescence bleed-through into the acceptor detection channel. Corrections for these effects involve the acquisition of fluorescence images of samples containing the donor, the acceptor, and both of these for three different filter settings. The result is an estimation of the sensitized emission, i.e., the emission induced by FRET from the donor to the acceptor alone. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor fluorescence and increased emission from the acceptor (sensitized emission). This can be normalized using the acceptor emission, measured after specific excitation of the acceptor, to define apparent energy transfer efficiency in each pixel of the image. It is also proportional to the fraction of acceptor molecules that is bound to a donor-tagged molecule. Alternatively, an apparent energy transfer efficiency can also be defined that is proportional to the bound fraction of donor-tagged molecules.
本方案描述了一种通过检测受体敏化发射来测量荧光共振能量转移（FRET）的方法。在供体强度较低和/或供体通道中存在高背景（自发）荧光污染的情况下，这种方法很有用。然而，吸收光谱在高能、短波长（蓝色）区域通常呈现长尾巴，这可能导致除了供体之外，受体分子也被直接激发，从而导致直接发射和敏化发射混合。相反，荧光发射往往会拖尾到光谱的红色部分，导致供体荧光渗漏到受体检测通道中。对这些效应的校正涉及获取包含供体、受体以及两者的样品在三种不同滤光片设置下的荧光图像。结果是对敏化发射的估计，即仅由从供体到受体的FRET诱导的发射。FRET对中供体荧光团的激发导致供体荧光猝灭和受体发射增加（敏化发射）。这可以使用在受体特定激发后测量的受体发射进行归一化，以定义图像每个像素中的表观能量转移效率。它还与与供体标记分子结合的受体分子分数成正比。或者，也可以定义一种表观能量转移效率，它与供体标记分子的结合分数成正比。
