Identification, visualization, and quantitation of cardiolipin (CL) in biological membranes is of great interest because of the important structural and physiological roles of this lipid. Selective fluorescent detection of CL using noncovalently bound fluorophore 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenylethene (TTAPE-Me) has been recently proposed. However, this dye was only tested on wild-type mitochondria or liposomes containing negligible amounts of other anionic lipids, such as phosphatidylglycerol (PG) and phosphatidylserine (PS). No clear preference of TTAPE-Me for binding to CL compared to PG and PS was found in our experiments on artificial liposomes, Escherichia coli inside-out vesicles, or Saccharomyces cerevisiae mitochondria in vitro or in situ, respectively. The shapes of the emission spectra for these anionic phospholipids were also found to be indistinguishable. Thus, TTAPE-Me is not suitable for detection, visualization, and localization of CL in the presence of other anionic lipids present in substantial physiological amounts. Our experiments and complementary molecular dynamics simulations suggest that fluorescence intensity of TTAPE-Me is regulated by dynamic equilibrium between emitting dye aggregates, stabilized by unspecific but thermodynamically favorable electrostatic interactions with anionic lipids, and nonemitting dye monomers. These results should be taken into consideration when interpreting past and future results of CL detection and localization studies with this probe in vitro and in vivo. Provided methodology emphasizes minimal experimental requirements, which should be considered as a guideline during the development of novel lipid-specific probes.

由于这种脂质具有重要的结构和生理作用，因此生物膜中心磷脂 (CL) 的鉴定、可视化和定量具有重要意义。最近提出了使用非共价结合的荧光团 1,1,2,2-四[4-(2-三甲基氨基乙氧基)-苯基乙烯 (TTAPE-Me) 对 CL 进行选择性荧光检测。然而，这种染料仅在野生型线粒体或含有可忽略量的其他阴离子脂质（如磷脂酰甘油 (PG) 和磷脂酰丝氨酸 (PS)）的脂质体上进行了测试。在我们对人工脂质体、大肠杆菌内外囊泡或酿酒酵母的实验中，与 PG 和 PS 相比，没有发现 TTAPE-Me 与 CL 结合的明显偏好线粒体在体外或原位，分别。还发现这些阴离子磷脂的发射光谱形状难以区分。因此，在存在大量生理量的其他阴离子脂质的情况下，TTAPE-Me 不适用于 CL 的检测、可视化和定位。我们的实验和互补的分子动力学模拟表明，TTAPE-Me 的荧光强度受发光染料聚集体之间的动态平衡调节，通过与阴离子脂质和非发光染料单体的非特异性但热力学有利的静电相互作用来稳定。在解释使用该探针在体外和体内进行 CL 检测和定位研究的过去和未来结果时，应考虑这些结果。提供的方法强调最小的实验要求，在开发新的脂质特异性探针期间应将其视为指导方针。