https://www.leica-microsystems.com/science-lab/foerster-resonance-energy-transfer-fret/

Tutorial教程

Page title页面标题

Förster Resonance Energy Transfer (FRET)

Subtitle副标题

An introduction
一个介绍

FRET

Fluorescence

Confocal Microscopy

F-Techniques

FLIM (Fluorescence Lifetime Imaging Microscopy)

Quantitative Imaging

New trends in today’s fluorescence microscopy aim to overcome the limitations of optical resolution.
今天荧光显微镜的新趋势旨在克服光学分辨率的限制。

Direct methods like STED or GSD create images that show resolution far below the diffraction limit. They employ properties of light-matter interactions with optical focusing patterns or fluorescence state phenomena in combination with reconstruction of stochastic single-molecule events.
STED 或 GSD 等直接方法创建的图像显示出远低于衍射极限的分辨率。它们利用光与物质相互作用的特性，结合光学聚焦模式或荧光态现象的重建以及随机单分子事件的重建。

Indirect methods like FCS , FRAP and FRET measure modifications of fluorescence parameters caused by interactions or movement of molecules. FRET has sensitivity for molecule-molecule interactions in the range of a few nanometers1
像 FCS，FRAP 和 FRET 这样的间接方法测量由分子相互作用或运动引起的荧光参数的修改。FRET 对几纳米范围内的分子间相互作用具有敏感性1.

The FRET phenomenon

Fluorescence describes the spontaneous emission of a photon from a molecule or atom after excitation of the electronic system by absorption of light. The emitted photon usually has less energy as compared to the excitation photon and consequently has a longer wavelength (Stokes shift). Blue excitation may for example cause green emission. If a second fluorescent molecule can absorb the green photon, the emission of that molecule is again Stokes-shifted, e.g. into red color. This re-absorption is partially responsible for errors in measuring fluorescence (part of "inner filter" effects) in comparably densely concentrated samples. In weakly concentrated samples, re-absorption is a very rare event.
荧光描述了分子或原子在吸收光后电子系统被激发后发射光子的自发辐射。发射的光子通常比激发光子能量低，并且因此具有较长的波长（斯托克斯位移）。例如，蓝色激发可能导致绿色发射。如果第二个荧光分子可以吸收绿光子，则该分子的发射再次斯托克斯位移，例如变成红色。这种再吸收在测量荧光时（“内部滤光器”效应的一部分）在相对浓缩的样品中部分负责误差。在浓度较低的样品中，再吸收是一个非常罕见的事件。

If the two molecules are spatially very close (a few nanometers), the energy can “hop” directly from the "donor" to the "acceptor" without occurrence of light. This direct exchange of energy is called "Förster Resonance Energy Transfer", FRET. "Resonance" indicates that the two molecules have to share energetic windows. The windows are the emission spectrum of the donor and the absorption spectrum of the acceptor. Although photons are neither emitted nor re-absorbed, the energetic considerations still hold. The probability for FRET rises with the overlap area of the two spectra – more energy wins through if the windows fit better. Also, the orientation of the molecules has an influence on the transfer efficiency.
如果两个分子在空间上非常接近（几纳米），能量可以直接从“给体”跃迁到“受体”，而无需发生光的作用。这种能量的直接交换被称为“Förster 共振能量转移”，简称 FRET。 “共振”表示两个分子必须共享能量窗口。这些窗口是给体的发射光谱和受体的吸收光谱。虽然光子既不会被发射也不会被重新吸收，但能量考虑仍然成立。FRET 的概率随着两个光谱的重叠区域增加而增加 - 如果窗口更匹配，更多的能量就会通过。此外，分子的方向也会影响传输效率。

For the example given above, the collected emission is normally green if the sample is illuminated with blue light. If FRET occurs, red photons will also be emitted – and fewer green photons accordingly. The extreme case would be a complete transfer of all energy and no green emission at all. The FRET efficiency, defined by the number of absorbed blue photons divided by the number of emitted red photons, can consequently assume values from 0 to 1. For a given FRET pair, the FRET efficiency indicates the spatial distance between the two fluorescent species – this is the goal of FRET measurements [2].
对于上面给出的例子，如果样品被蓝光照射，收集到的发射通常是绿色的。如果发生 FRET，红光子也会被发射 - 因此绿光子会减少。极端情况是所有能量完全转移，没有任何绿色发射。FRET 效率由吸收的蓝光子数除以发射的红光子数定义，因此可以假设值从 0 到 1。对于给定的 FRET 对，FRET 效率指示两种荧光物种之间的空间距离 - 这是 FRET 测量的目标。

As FRET requires a very short distance between fluorochromes, it is a tool for verifying interactions of cellular components. Although a biological interaction cannot be proved by measuring the sheer distance, FRET increases the colocalization accuracy down to a few nanometers. Dimerization processes and conformational changes of proteins are also targets of this method [3]. An area of indirect applications is provided by FRET biosensors that change their FRET efficiency upon interaction with the perceived target. A very prominent example is the Cameleon biosensor for probing Ca2+ concentration changes [4].
由于 FRET 需要荧光染料之间非常短的距离，因此它是验证细胞组分相互作用的工具。尽管通过测量纯粹距离无法证明生物相互作用，但 FRET 可以将共定位的准确性提高到几纳米。蛋白质的二聚化过程和构象变化也是这种方法的目标[3]。FRET 生物传感器提供了间接应用领域，这些传感器在与感知目标相互作用时会改变其 FRET 效率。一个非常著名的例子是用于探测 Ca2+浓度变化的 Cameleon 生物传感器[4]。

Fig. 1: Jablonski diagram for Alexa 488 and Cy3, a commonly used FRET pair.
Fig. 1: Alexa 488 和 Cy3 的 Jablonski 图，这是一对常用的 FRET 对。

Förster Radius and FRET pairs

The Förster radius R0 is the distance between a pair of fluorochromes at which the FRET efficiency reaches ½ (i.e. 50 %). Typically, this distance is in the range of 20 …60 Å (2 … 6 nm). The Förster radius R0 depends on the photophysical parameters of the FRET pair [5]:
弗斯特半径 R0 是一对荧光染料之间的距离，其中 FRET 效率达到 ½（即 50%）。通常，这个距离在 20 到 60 Å（2 到 6 nm）的范围内。弗斯特半径 R0 取决于 FRET 对的光物理参数[5]：

with
kappa2: orientation factor
J(λ): overlap integral
n: refractive index
QD: quantum yield of donor
QD：受体的量子产率

FRET efficiency E as depending on the distance between the fluorochromes is expressed by the following formula:
依赖于荧光染料之间距离的 FRET 效率 E 由以下公式表示：

with
R0: Förster radius
r: Donor-acceptor distance
受体-给体距离

If the Förster radius for a given FRET pair is known, the measured FRET efficiency reveals the distance r between the two fluorochromes. Obviously, the measurement is very critical, due to the 6th power involved in the dependence of efficiency on distance. In turn, all calculations of distances should not be overstated. Nevertheless, measuring FRET efficiency changes in living samples can reveal direct indications for changes in interactivity, transport phenomena and other cellular events.
如果对于给定的 FRET 双体已知 Förster 半径，则测得的 FRET 效率揭示了两种荧光染料之间的距离 r。显然，由于效率与距离依赖关系中涉及到六次幂，测量非常关键。反过来，所有距离的计算都不应夸大。然而，在活体样本中测量 FRET 效率的变化可以揭示出与相互作用、传输现象和其他细胞事件变化直接相关的迹象。

In living cells the dyes of choice are usually GFP variants, whereas in fixed preparations the dyes used range from antibody-tagged dyes to GFP variants or a combination of both. Figure 2 shows a couple of commonly used FRET pairs. There are many more options, and with the daily increasing number of new dyes and fluorescent proteins, this area is becoming increasingly crowded. Dyes of similar spectral properties are interchangeable and all kinds of permutations are possible.
在活细胞中，通常选择的染料是 GFP 变体，而在固定制备中使用的染料范围从标记抗体染料到 GFP 变体或两者的组合。图 2 展示了一些常用的 FRET 双体。还有许多其他选择，随着新染料和荧光蛋白数量日益增加，这一领域变得越来越拥挤。具有类似光谱特性的染料是可以互换的，各种排列组合都是可能的。

Methods for measuring FRET efficiencies

The most direct way to measure FRET effects is the FRET-AP method. Here, the sample that is supposed to show FRET is imaged for the donor. In the example given before, this refers to the experiment with blue excitation and collecting green emission. After collecting an image, an area of interest is intensely illuminated by light that can photobleach the acceptor (hence “AP”, acceptor photobleaching), but does not damage the donor. Then, a second image of the donor is taken. If there was FRET, the donor should show an increased intensity in the bleached region, as there are more molecules ready for donor fluorescence and not drained into the FRET channel. The FRET efficiency E is estimated by the following formula:
用于测量 FRET 效应的最直接方法是 FRET-AP 方法。在这里，应该展示 FRET 的样品被用于给体。在之前给出的例子中，这指的是使用蓝色激发并收集绿色发射的实验。拍摄图像后，感兴趣的区域被强烈照射，可以光漂白受体（因此“AP”，受体光漂白），但不会损坏给体。然后，拍摄给体的第二张图像。如果存在 FRET，给体应该在漂白区域显示增加的强度，因为有更多的分子准备用于给体荧光而不是流入 FRET 通道。FRET 效率 E 由以下公式估算：

with
用
• DA: donor-intensity before bleaching
• DΩ: donor-intensity after bleaching
• DΩ：漂白后的给体强度

The AP method is restricted to fixed samples, as images are taken serially. This takes time and living samples would change structurally or move away. A method that improves results in live sample experiments is called FRET-SE, for "sensitized" emission. Sensitized refers to the fact that acceptor molecules start to emit upon donor excitation as soon as FRET is possible. In this method, all fluorescence channels are collected simultaneously. Based on previously collected correction factors, FRET images are calculated from the different parallel images. Although this method enables inspection of living material, the correction is prone to errors and solid results require very meticulous experimenting [1, 5].
AP 方法仅限于固定样本，因为图像是串行拍摄的。这需要时间，活样本会在结构上发生变化或移开。一种改善活样本实验结果的方法称为 FRET-SE，即“敏感”发射。敏感指的是当 FRET 可能时，受体分子在供体激发后开始发射。在这种方法中，所有荧光通道同时收集。根据先前收集的校正因子，FRET 图像从不同的并行图像中计算出来。尽管这种方法能够检查活体材料，但校正容易出错，获得可靠结果需要非常细致的实验。

Besides measuring intensity changes, measurements of fluorescent lifetime can also reveal FRET occurrence. The excited state of the donor decays by emitting a photon back to the ground state. If no FRET occurs, the fluorescence lifetime describes these single-path decay kinetics. If the excited state can also decay into a FRET channel, the lifetime shortens – because there is a second option speeding up the clearance of the excited state. Measuring the altered lifetime and extraction of the kinetic contributions also allows estimation of FRET efficiencies. This method is referred to as "FLIM-FRET" [6].
除了测量强度变化外，荧光寿命的测量也可以揭示 FRET 的发生。给体的激发态通过向基态发射光子而衰减。如果没有发生 FRET，荧光寿命描述这些单路径衰减动力学。如果激发态还可以衰减到 FRET 通道，寿命会缩短，因为有第二个选项加快激发态的清除。测量改变的寿命并提取动力学贡献还允许估计 FRET 效率。这种方法被称为“FLIM-FRET” [6]。

Fig. 3: Example of FRET Acceptor Photobleaching. On the left, the donor image is shown before photobleaching. 
图 3：FRET 受体光漂白示例。左侧显示了光漂白前的给体图像。

The middle image shows the same field after bleaching the acceptor in a small rectangular region. In this region, the donor emission is increased, as all excited molecules can only decay by emitting a fluorescence photon. The darker fluorescence in the surrounding area indicates “leakage” of fluorescence into the FRET channel.
中间图像显示了在一个小矩形区域中漂白受体后的同一区域。在这个区域内，给体发射增加，因为所有激发的分子只能通过发射荧光光子来衰减。周围区域的较暗荧光表明荧光“泄漏”到 FRET 通道中。

The false-color image on the right side is calculated according to the formula for FRET efficiency. The higher intensity in the rectangular region accounts for the FRET efficiency that occurred before the area was bleached. The background does not contain any FRET efficiency information.
右侧的伪彩色图像是根据 FRET 效率的公式计算的。矩形区域中较高的强度代表了在漂白之前发生的 FRET 效率。背景不包含任何 FRET 效率信息。