A Membrane-Bound FRET-Based Caspase Sensor for Detection of Apoptosis Using Fluorescence Lifetime and Total Internal Reflection Microscopy
A Membrane-Bound FRET-Based Caspase Sensor for Detection of Apoptosis Using Fluorescence Lifetime and Total Internal Reflection Microscopy
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DOI：
10.1002/cyto.a.20698
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发表时间：
2009-05-01
期刊：
影响因子：
3.7
通讯作者：
Schneckenburger, Herbert
中科院分区：
文献类型：
--
作者：
Angres, Brigitte;Steuer, Heiko;Schneckenburger, Herbert
报道了一种基于荧光蛋白间Forster共振能量转移的半胱天冬酶传感器。锚定到活细胞质膜的内小叶的增强型青色荧光蛋白被瞬逝电磁场光学激发，并经由间隔物（DEVD）将其激发能量转移到增强型黄色荧光蛋白。在细胞凋亡时，DEVD被切割并且能量转移被破坏，如通过荧光光谱和衰减时间的显著变化所证明的。荧光光谱和寿命成像（FLIM）与全内反射荧光显微镜（TIRFM）相结合，用于选择性检测这种膜结合的半胱天冬酶传感器。因此，细胞质的荧光团被排除在外，并且信号与背景比显著增加。与传统或激光扫描显微镜相比，这允许使用非常低的吸收率长期观察活细胞培养物中的细胞凋亡，并避免样品的光诱导损伤。(c)2008年国际细胞计数促进学会
A caspase sensor based on Forster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane-bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal-to-background ratio is increased considerably. In comparison with conventional or laser scanning microscopy, this permits long-term observation of apoptosis in live cell cultures using very low absorption and avoiding light-induced damages of the samples. (c) 2008 International Society for Advancement of Cytometry