VOLTAGE SENSING BY FLUORESCENCE RESONANCE ENERGY-TRANSFER IN SINGLE CELLS
VOLTAGE SENSING BY FLUORESCENCE RESONANCE ENERGY-TRANSFER IN SINGLE CELLS
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DOI：
10.1016/s0006-3495(95)80029-9
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发表时间：
1995-10-01
影响因子：
3.4
通讯作者：
TSIEN, RY
中科院分区：
文献类型：
--
作者：
GONZALEZ, JE;TSIEN, RY
一种新的机制已经开发实现快速的比率电压敏感的荧光变化在单细胞中使用荧光共振能量转移。该机制是基于疏水性荧光阴离子，迅速重新分配从一个面的质膜到其他根据Nemst方程。通过用第二荧光团标记细胞的细胞外表面来产生电压敏感的荧光读数，在此为荧光标记的凝集素，其可以与膜结合传感器进行能量转移。当膜电位去极化时，两个荧光团之间的荧光共振能量转移被破坏，因为阴离子被拉到远离凝集素的质膜的细胞内表面。双-（1，3-二烷基-2-硫代巴比妥酸酯）-三次甲基氧杂蒽酮醇（其中烷基为正己基和正癸基（分别为DiSBA-C-6-（3）和DiSBA-C-10-（3）可以作为德克萨斯红标记的小麦胚芽凝集素（TR-WGA）的供体和荧光素标记的凝集素（FI-WGA）的受体。在电压钳位成纤维细胞中，这些oxonols的易位被测量为位移电流，在20 ° C下对于100 mV去极化具有类似于2 ms的时间常数，其等于荧光变化的速度。在成纤维细胞、星形细胞瘤细胞、搏动心肌细胞和B104神经母细胞瘤细胞中，观察到100 mV去极化的荧光比率变化在4%和34%之间。大的荧光变化允许高速共焦成像。
A new mechanism has been developed for achieving fast ratiometric voltage-sensitive fluorescence changes in single cells using fluorescence resonance energy transfer. The mechanism is based on hydrophobic fluorescent anions that rapidly redistribute from one face of the plasma membrane to the other according to the Nemst equation. A voltage-sensitive fluorescent readout is created by labeling the extracellular surface of the cell with a second fluorophore, here a fluorescently labeled lectin, that can undergo energy transfer with the membrane-bound sensor. Fluorescence resonance energy transfer between the two fluorophores is disrupted when the membrane potential is depolarized, because the anion is pulled to the intracellular surface of the plasma membrane far from the lectin. Bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonols, where alkyl is n-hexyl and n-decyl (DiSBA-C-6-(3) and DiSBA-C-10-(3), respectively) can function as donors to Texas Red labeled wheat germ agglutinin (TR-WGA) and accepters from fluorescein-labeled lectin (FI-WGA). In voltage-clamped fibroblasts, the translocation of these oxonols is measured as a displacement current with a time constant of similar to 2 ms for 100 mV depolarization at 20 degrees C, which equals the speed of the fluorescence changes. Fluorescence ratio changes of between 4% and 34% were observed for a 100-mV depolarization in fibroblasts, astrocytoma cells, beating cardiac myocytes, and B104 neuroblastoma cells. The large fluorescence changes allow high-speed confocal imaging.