High-resolution nonlinear optical imaging of live cells by second harmonic generation
High-resolution nonlinear optical imaging of live cells by second harmonic generation
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DOI：
10.1016/s0006-3495(99)77165-1
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发表时间：
1999-12-01
影响因子：
3.4
通讯作者：
Loew, LM
中科院分区：
文献类型：
--
作者：
Campagnola, PJ;Wei, MD;Loew, LM
利用钛蓝宝石飞秒脉冲激光和透射光学器件的激光扫描显微镜，利用二次谐波产生（SHG）的非线性光学现象，制备了活细胞图像。二次谐波成像（SHIM）是探测活细胞膜的理想方法，因为它提供了非线性光学显微镜的高分辨率，并且几乎完全避免了光漂白和光毒性。该技术已在三种具有大非线性光学系数的膜染色染料标记的细胞系上实施。这些图像可以在生理相关的时间尺度内获得。用非手性和手性染料对单叶和双叶染色的成像进行比较，发现手性在造影剂的形成机制中起重要作用。研究还表明，SHIM对膜电位高度敏感，25mv的去极化导致信号强度损失约为原来的两倍。
By adapting a laser scanning microscope with a titanium sapphire femtosecond pulsed laser and transmission optics, we are able to produce live cell images based on the nonlinear optical phenomenon of second harmonic generation (SHG). Second harmonic imaging (SHIM) is an ideal method for probing membranes of living cells because it offers the high resolution of nonlinear optical microscopy with the potential for near-total avoidance of photobleaching and phototoxicity. The technique has been implemented on three cell lines labeled with membrane-staining dyes that have large nonlinear optical coefficients. The images can be obtained within physiologically relevant time scales. Both achiral and chiral dyes were used to compare image formation for the case of single- and double-leaflet staining, and it was found that chirality plays a significant role in the mechanism of contrast generation. It is also shown that SHIM is highly sensitive to membrane potential, with a depolarization of 25 mV resulting in an approximately twofold loss of signal intensity.