Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system
Time-resolved long-lived luminescence imaging method employing luminescent lanthanide probes with a new microscopy system
复制标题
DOI：
10.1021/ja073392j
复制
发表时间：
2007-11-07
影响因子：
15
通讯作者：
Nagano, Tetsuo
中科院分区：
文献类型：
--
作者：
Hanaoka, Kenjiro;Kikuchi, Kazuya;Nagano, Tetsuo
对生物现象的详细研究需要上级荧光成像方法，并且允许精确分析的一种方法是时间分辨荧光测量，其提供高信噪比。在此，我们描述了一种新的荧光成像系统，通过时间分辨，长寿命发光显微镜（TRLLM）的方式来可视化活的生物样品内的生物分子。在TRLLM中，短寿命的背景荧光和散射光被选通，允许长寿命的发光被选择性地成像。荧光时间分辨荧光显微镜提供了纳秒分辨率的荧光图像，并已被用于蛋白质，蛋白质磷酸化，局部pH值，折射率，离子或氧浓度等之间的相互作用的图像发光镧系元素复合物（特别是铕和铽三价离子（Eu 3+和Tb 3+）），相比之下，具有毫秒级的长发光寿命。我们已经设计和合成了新的发光Eu 3+配合物的TRLLM，也开发了一个新的TRLLM系统，使用传统的荧光显微镜与图像增强器单元的门控信号采集和氙气闪光灯作为激发源。当新开发的发光Eu 3+配合物应用于活细胞时，用TRLLM系统获得清晰的荧光图像，并且完全排除了短寿命荧光。通过组合使用Eu 3+和Tb 3+发光配合物，时间分辨双色成像也是可能的。此外，我们监测细胞内的离子锌（Zn 2+）浓度的变化，通过使用Zn 2 +-选择性发光Eu 3+化学传感器，[Eu-7]。这种新的成像技术应促进与荧光显微镜的生物功能的调查，补充其他荧光成像方法。
Superior fluorescence imaging methods are needed for detailed studies on biological phenomena, and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy (TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides fluorescence images with nanosecond resolution and has been used to image interactions between proteins, protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent lanthanide complexes (especially europium and terbium trivalent ions (Eu3+ and Tb3+)), in contrast, have long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new luminescent Eu3+ complexes for TRLLM and also developed a new TRLLM system using a conventional fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp as the excitation source. When the newly developed luminescent Eu3+ complexes were applied to living cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was completely excluded. By using Eu3+ and Tb3+ luminescent complexes in combination, time-resolved dual-color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn2+) concentration by using a Zn2+-selective luminescent Eu3+ chemosensor, [Eu-7]. This new imaging technique should facilitate investigations of biological functions with fluorescence microscopy, complementing other fluorescence imaging methodologies.