Herman P, Maliwal B P, Lin H J, Lakowicz J R
University of Maryland School of Medicine, Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, Maryland 21201, USA.
J Microsc. 2001 Aug;203(Pt 2):176-81. doi: 10.1046/j.1365-2818.2001.00943.x.
We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz--250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10--15 microm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C(12),with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.
我们描述了一种基于显微镜和调制发光二极管（LED）激发源（370/460纳米）的频域寿命荧光计，其工作频率范围为120赫兹至250兆赫。我们从直径小至10 - 15微米的细胞区域收集了多频相位和调制荧光响应。我们还从用亲脂性香豆素敏化铕荧光团（Coum-Eu，寿命为毫秒级）和Ru(bpy)(2)phe-C(12)（寿命为微秒级）染色的细胞中收集了荧光寿命数据。还用SYTO 14和SYTO 16探针测量了天然细胞核的纳秒级寿命。我们证明，对于许多应用而言，简单的LED激发源能够成功替代用于细胞频域寿命测量的复杂且昂贵的激光系统。由于LED非常稳定且噪声低，使用更亮的LED将有可能对更小的样品区域进行成像。鉴于有多种波长的调制LED可供使用，几乎覆盖了整个可见光谱，因此很容易组装一个用于所选荧光团的系统。能够为给定的荧光团选择激发源以及价格低廉使得这样的激发源更加实用。
