Department of Physiology and Neurobiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Microsc Microanal. 2011 Apr;17(2):176-90. doi: 10.1017/S1431927610094493. Epub 2011 Feb 21.
Contemporary research aims to understand biological processes not only by identifying participating proteins, but also by characterizing the dynamics of their interactions. Because Förster's Resonance Energy Transfer (FRET) is invaluable for the latter undertaking, its usage is steadily increasing. However, FRET measurements are notoriously error-prone, especially when its inherent efficiency is low, a not uncommon situation. Furthermore, many FRET methods are either difficult to implement, are not appropriate for observation of cellular dynamics, or report instrument-specific indices that hamper communication of results within the scientific community. We present here a novel comprehensive spectral methodology, SpRET, which substantially increases both the reliability and sensitivity of FRET microscopy, even under unfavorable conditions such as weak fluorescence or the presence of noise. While SpRET overcomes common pitfalls such as interchannel crosstalk and direct excitation of the acceptor, it also excels in removal of autofluorescence or background contaminations and in correcting chromatic aberrations, often overlooked factors that severely undermine FRET experiments. Finally, SpRET quantitatively reports absolute rather than relative FRET efficiency values, as well as the acceptor-to-donor molar ratio, which is critical for full and proper interpretation of FRET experiments. Thus, SpRET serves as an advanced, improved, and powerful tool in the cell biologist's toolbox.
当代研究的目的不仅在于识别参与的蛋白质，而且在于描述它们相互作用的动态，因此旨在理解生物过程。由于Förster 共振能量转移（FRET）对于后者的研究是非常宝贵的，所以它的使用正在稳步增加。然而，FRET 测量通常是容易出错的，尤其是当它的固有效率较低时，这种情况并不少见。此外，许多 FRET 方法要么难以实施，要么不适合观察细胞动力学，要么报告特定于仪器的指数，从而阻碍了科学界内结果的交流。我们在这里提出了一种新颖的综合光谱方法 SpRET，即使在荧光较弱或存在噪声等不利条件下，它也能显著提高 FRET 显微镜的可靠性和灵敏度。虽然 SpRET 克服了常见的陷阱，如通道间串扰和供体的直接激发，但它在去除自发荧光或背景污染以及校正色差方面也很出色，这些通常被忽视的因素严重破坏了 FRET 实验。最后，SpRET 定量报告绝对而非相对的 FRET 效率值以及供体-受体摩尔比，这对于充分和正确解释 FRET 实验至关重要。因此，SpRET 是细胞生物学家工具包中的一个高级、改进和强大的工具。
