Chapman C F, Liu Y, Sonek G J, Tromberg B J
Beckman Laser Institute and Medical Clinic, University of California, Irvine 92717, USA.
Photochem Photobiol. 1995 Sep;62(3):416-25. doi: 10.1111/j.1751-1097.1995.tb02362.x.
The fluorescent membrane probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) and 6-dodecanoyl-2-dimethylamino-naphthalene (laurdan) have been studied for use as optical thermometers in living cells. The thermal sensitivity of NBD is primarily a consequence of rapid, heat-induced electronic changes, which increase the observed fluorescence decay rate. As a result, fluorescence intensity and lifetime variations of membrane-bound NBD-conjugated phospholipids and fatty acids can be directly correlated with cellular temperature. In contrast, laurdan fluorescence undergoes a dramatic temperature-dependent Stokes shift as the membrane undergoes a gel-to-liquid-crystalline phase transition. This facilitates the use of fluorescence spectra to record the indirect effect of microenvironmental changes, which occur during bilayer heating. Microscope and suspension measurements of cells and phospholipid vesicles are compared for both probes using steady-state and fluorescence lifetime (suspension only) data. Our results show that NBD fluorescence lifetime recordings can provide reasonable temperature resolution (approximately 2 degrees C) over a broad temperature range. Laurdan's microenvironmental sensitivity permits better temperature resolution (0.1-1 degree C) at the expense of a more limited dynamic range that is determined solely by bilayer properties. The temperature sensitivity of NBD is based on rapid intramolecular rotations and vibrations, while laurdan relies on a slower, multistep mechanism involving bilayer rearrangement, water penetration and intermolecular processes. Because of these differences in time scale, NBD appears to be more suitable for monitoring ultrafast phenomena, such as the impact of short-pulse microirradiation on single cells.
荧光膜探针7-硝基苯并-2-恶唑-1,3-二氮杂萘-4-基（NBD）和6-十二烷酰基-2-二甲基氨基萘（laurdan）已被研究用作活细胞中的光学温度计。NBD的热敏感性主要是快速的热诱导电子变化的结果，这种变化会增加观察到的荧光衰减率。因此，膜结合的NBD共轭磷脂和脂肪酸的荧光强度和寿命变化可以直接与细胞温度相关联。相比之下，随着膜经历凝胶到液晶相转变，laurdan荧光会发生显著的温度依赖性斯托克斯位移。这便于利用荧光光谱记录双层加热过程中发生的微环境变化的间接影响。使用稳态和荧光寿命（仅适用于悬浮液）数据，对两种探针的细胞和磷脂囊泡的显微镜和悬浮液测量进行了比较。我们的结果表明，NBD荧光寿命记录在很宽的温度范围内可以提供合理的温度分辨率（约2摄氏度）。laurdan的微环境敏感性允许更好的温度分辨率（0.1 - 1摄氏度），但代价是动态范围更有限，这完全由双层性质决定。NBD的温度敏感性基于快速的分子内旋转和振动，而laurdan则依赖于较慢的多步机制，涉及双层重排、水渗透和分子间过程。由于时间尺度上的这些差异，NBD似乎更适合监测超快现象，例如短脉冲微辐照对单细胞的影响。
