DFG Research Center for the Molecular Physiology of the Brain, Göttingen, Germany.
Biophys J. 2010 Oct 6;99(7):2344-54. doi: 10.1016/j.bpj.2010.07.053.
Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected.
基于分子内Förster 共振能量转移（FRET）的分子传感器已成为监测活组织中调节分子的多功能工具。然而，它们的使用常常受到信号强度低和噪声过大的限制。我们分析了光谱 FRET 分析方法的信号/噪声（SNR）方面，得出以下结论：最常用的方法（在单次短波长激发后测量发射比）在信号/噪声方面是最优的，如果只对该未经校准的比值的相对变化感兴趣的话。在需要定量 FRET 效率数据的情况下，可以从发射比和一些校准参数计算得出，但 SNR 会降低。最近描述的用于 FRET 数据光谱分析的 Lux-FRET 方法允许以三种不同的方式做到这一点，每种方式都基于三个测量荧光信号中的两个比值（短波长激发期间的供体和受体信号以及长波长激发期间的受体信号）。Lux-FRET 还允许计算供体和受体荧光团的总丰度。由于不利的误差传播，所有这些数量的 SNR 都低于纯发射比的 SNR。然而，如果配体浓度是根据 lux-FRET 值计算的，或者在经过校准后，根据发射比计算的，那么这两种分析模式的 SNR 非常相似。同样，如果这些数量的噪声与预期的动态范围有关，则 SNR 值也相似。我们基于基于 Epac 的 cAMP 传感器的数据证明了这些关系，并讨论了 SNR 如何随 FRET 效率和收集的光子数而变化。
