通过二次谐波产生对活细胞进行高分辨率非线性光学成像。
High-resolution nonlinear optical imaging of live cells by second harmonic generation.
作者信息
Campagnola P J, Wei M D, Lewis A, Loew L M
机构信息
Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030 USA.
出版信息
Biophys J. 1999 Dec;77(6):3341-9. doi: 10.1016/S0006-3495(99)77165-1.
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.
通过采用配备钛宝石飞秒脉冲激光器和透射光学元件的激光扫描显微镜，我们能够基于二次谐波产生（SHG）这一非线性光学现象生成活细胞图像。二次谐波成像（SHIM）是探测活细胞膜的理想方法，因为它兼具非线性光学显微镜的高分辨率，且有可能几乎完全避免光漂白和光毒性。该技术已应用于三种用具有大非线性光学系数的膜染色染料标记的细胞系。图像可在生理相关的时间尺度内获取。使用了非手性和手性染料来比较单叶和双叶染色情况下的成像，结果发现手性在对比度产生机制中起着重要作用。研究还表明，SHIM对膜电位高度敏感，去极化25 mV会导致信号强度损失约两倍。
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