Assessing the sensitivity of commercially available fluorophores to the intracellular environment.
Assessing the sensitivity of commercially available fluorophores to the intracellular environment.
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DOI：
10.1021/ac8011347
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发表时间：
2008-10-01
影响因子：
7.4
通讯作者：
Tsourkas, Andrew
中科院分区：
文献类型：
--
作者：
Chen, Antony K.;Cheng, Zhiliang;Behlke, Mark A.;Tsourkas, Andrew
荧光的使用在生物科学中已经变得司空见惯，许多研究利用基于市售荧光团的探针来提供对细胞功能和行为的洞察。随着这些成像应用变得越来越先进，获取荧光信号的精确定量测量变得越来越重要。荧光的绝对定量，但是，需要荧光团本身是不敏感的环境因素，如非特异性蛋白质相互作用和pH值。在这里，我们提出了一种方法，通过比较它们的荧光强度的环境不敏感的参考信号之前和之后的细胞内交付的荧光团的细胞溶质环境的敏感性进行表征。结果表明，虽然一些荧光团的荧光强度，如荧光素，是高度敏感的细胞内环境，其他荧光团，如Dylight 649，Alexa 647，和Alexa 750，是不敏感的细胞内环境。还观察到荧光团的灵敏度可能取决于其所连接的生物分子。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。总的来说，本研究提供了一种方法来选择荧光团的研究，需要在细胞内环境中的荧光的绝对定量。
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 non-specific 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.
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影响因子：
14.9
作者：
Chen AK;Behlke MA;Tsourkas A
通讯作者：
Tsourkas A
影响因子：
7.4
作者：
Kato, N;Pontier, D;Lam, E
通讯作者：
Lam, E
DOI：
10.1073/pnas.2233244100
发表时间：
2003-11-11
影响因子：
11.1
作者：
Bratu, DP;Cha, BJ;Tyagi, S
通讯作者：
Tyagi, S
影响因子：
2.9
作者：
GRABER, ML;DILILLO, DC;PASTORIZAMUNOZ, E
通讯作者：
PASTORIZAMUNOZ, E
影响因子：
2.9
作者：
LEE, GM;THORNTHWAITE, JT;RASCH, EM
通讯作者：
RASCH, EM