Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
J Microbiol Methods. 2012 Feb;88(2):189-96. doi: 10.1016/j.mimet.2011.10.008. Epub 2011 Oct 26.
Nile red (NR) is a popular fluorescent indicator to visualize lipid bodies in intact cells and has been extensively utilized to monitor triglyceride accumulation in microalgae. Typically, addition of NR to algae results in a rapid fluorescence enhancement followed by fluorescence quenching. NR fluorescence rise can be resolved into two kinetic phases: a fast phase (P₁, sec), monitored at 525 nm/630 nm, followed by a slower phase (P₂, min), monitored at 488 nm/575 nm. Studies with isolated plasma membrane (PM) and lipid globule (LG) preparations, suggest that P₁ and P₂ represent entry to the PM and transfer to LG, respectively. High NaCl slows down the interactions of NR with algae and with lipid globules. The onset of NR fluorescence quenching varies in different algae species between 5 min to 1h, and is observed in intact cells and in isolated LG. NR fluorescence quenching depends on NR concentration and is almost eliminated at low NR/cell ratios, indicating that it results from self-interactions of LG-associated dye. Glycerol has a dual effect on NR fluorescence: it eliminates kinetic anomalies resulting from limited solubility and self-interactions, but it also quenches NR fluorescence. NR fluorescence quenching by glycerol, as well as NR fluorescence enhancement by iodide anions, was observed only at high NR/LG ratios. These findings suggest that lipid-associated NR is more exposed to hydrophilic quenchers at high than at low NR concentrations. The results emphasize the importance of defining the optimal time window and NR concentrations for monitoring lipid accumulation in microalgae by NR fluorescence and clarify the origin of spectral anomalies resulting from self-interactions of dye molecules.
尼罗红（NR）是一种常用的荧光指示剂，可用于可视化完整细胞中的脂滴，广泛用于监测微藻中的甘油三酯积累。通常，NR 加入藻类后会迅速增强荧光，随后荧光猝灭。NR 荧光上升可分为两个动力学相：快速相（P₁，秒），在 525nm/630nm 处监测，随后是较慢相（P₂，分钟），在 488nm/575nm 处监测。与分离的质膜（PM）和脂滴（LG）制剂的研究表明，P₁和 P₂分别代表进入 PM 和转移到 LG。高 NaCl 会减缓 NR 与藻类和脂滴的相互作用。NR 荧光猝灭在不同藻类物种中的起始时间在 5 分钟到 1 小时之间变化，并且在完整细胞和分离的 LG 中都观察到。NR 荧光猝灭取决于 NR 浓度，在低 NR/细胞比下几乎消除，表明它是由与 LG 相关的染料的自相互作用引起的。甘油对 NR 荧光有双重影响：它消除了由于溶解度有限和自相互作用引起的动力学异常，但也猝灭了 NR 荧光。仅在高 NR/LG 比下才观察到甘油对 NR 荧光的猝灭以及碘阴离子对 NR 荧光的增强。这些发现表明，在高 NR 浓度下，与脂质相关的 NR 比在低 NR 浓度下更容易受到亲水性猝灭剂的影响。这些结果强调了定义监测微藻中脂质积累的最佳时间窗口和 NR 浓度的重要性，并阐明了由于染料分子自相互作用而导致光谱异常的原因。
