Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores
Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores
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DOI：
10.1364/oe.20.018525
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发表时间：
2012-08-13
期刊：
影响因子：
3.8
通讯作者：
Min, Wei
中科院分区：
文献类型：
--
作者：
Chen, Zhixing;Wei, Lu;Min, Wei
人们非常希望能够光学探测活体内部的生物活动。通过非线性跃迁实现空间受限激发，多光子荧光显微镜已成为成像散射样品不可缺少的手段。然而，由于散射损耗，入射激光功率随成像深度呈指数下降，焦外荧光最终会淹没焦内信号。由此产生的成像对比度损失定义了成像深度的基本限制，这不是通过增加激发强度就能克服的。在这里，我们建议通过多光子激活和成像(MPAI)光可激活荧光团来显著扩展这一深度限制。由于在空间中产生了明暗量子态的视差，成像对比度大大提高。我们通过分析理论和用人工合成的笼状荧光素染料或基因可编码的可光激活绿色荧光蛋白标记的组织模体的实验证明了这一新原理。(C)2012年美国光学学会
It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP. (C) 2012 Optical Society of America