Chen Antony K, Cheng Zhiliang, Behlke Mark A, Tsourkas Andrew
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Anal Chem. 2008 Oct 1;80(19):7437-44. doi: 10.1021/ac8011347. Epub 2008 Aug 14.
The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute quantification of fluorescence, however, requires the fluorophores themselves to be insensitive to environmental factors such as nonspecific protein interactions and pH. Here, we present a method for characterizing the sensitivity of fluorophores to the cytosolic environment by comparing their fluorescent intensity to an environment-insensitive reference signal before and after intracellular delivery. Results indicated that although the fluorescent intensity of a few fluorophores, e.g., fluorescein, were highly susceptible to the intracellular environment, other fluorophores, e.g., Dylight 649, Alexa647, and Alexa750, were insensitive to the intracellular environment. It was also observed that the sensitivity of the fluorophore could be dependent on the biomolecule to which it was attached. In addition to assessing the environmental sensitivity of fluorophores, a method for quantifying the amount of fluorophores within living cells is also introduced. Overall, the present study provides a means to select fluorophores for studies that require an absolute quantification of fluorescence in the intracellular environment.
荧光的应用在生物科学领域已变得十分普遍，许多研究都利用基于市售荧光团的探针来深入了解细胞功能和行为。随着这些成像应用变得越来越先进，获取准确的荧光信号定量测量结果变得愈发重要。然而，荧光的绝对定量要求荧光团本身对诸如非特异性蛋白质相互作用和pH等环境因素不敏感。在此，我们提出一种方法，通过比较细胞内递送前后荧光团的荧光强度与对环境不敏感的参考信号，来表征荧光团对细胞质环境的敏感性。结果表明，尽管一些荧光团（如荧光素）的荧光强度对细胞内环境高度敏感，但其他荧光团（如DyLight 649、Alexa647和Alexa750）对细胞内环境不敏感。还观察到荧光团的敏感性可能取决于其连接的生物分子。除了评估荧光团的环境敏感性外，还介绍了一种定量活细胞内荧光团数量的方法。总体而言，本研究提供了一种手段，可为需要在细胞内环境中进行荧光绝对定量的研究选择荧光团。
