Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes
Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes
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DOI：
10.1152/jn.00087.2004
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发表时间：
2004-07-01
影响因子：
2.5
通讯作者：
Miesenböck, G
中科院分区：
文献类型：
--
作者：
Roorda, RD;Hohl, TM;Miesenböck, G
用合适的造影剂修饰的生物膜在用超短、高强度的红外激光脉冲照射时产生非线性光学信号，例如双光子荧光和谐波上转换。基于这些非线性对比度的显微图像通过在神经组织上快速扫描聚焦的照明点以视频或更高的帧速率获得。扫描引擎依靠声光偏转器（AOD）产生快速水平光栅，并依靠校正棱镜抵消AOD引起的超短激发光脉冲在空间和时间上的色散。两个膜结合衍生物的绿色荧光蛋白（GFP）进行了测试，作为非线性造影剂。Synapto-pHluorin是一种与突触囊泡膜蛋白融合的pH敏感的GFP变体，它提供了完整大脑中遗传指定的突触末端神经递质释放的时间分辨荧光读数。在质膜上的双重脂化GFP分子阵列产生强烈的双光子荧光，但没有检测到的二次谐波功率。用合成苯乙烯基染料染色的膜与二次谐波产生的比较表明，遗传编码的发色团的安排缺乏入射光场的有效相干散射所需的取向各向异性和/或偶极密度。
Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.