Fluorescence correlation spectroscopy in a reverse Kretchmann surface plasmon assisted microscope.
Fluorescence correlation spectroscopy in a reverse Kretchmann surface plasmon assisted microscope.
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DOI：
10.1364/oe.16.013381
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发表时间：
2008-08
期刊：
影响因子：
3.8
通讯作者：
N. Calander;P. Muthu;Z. Gryczynski;I. Gryczynski;J. Borejdo
中科院分区：
文献类型：
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作者：
N. Calander;P. Muthu;Z. Gryczynski;I. Gryczynski;J. Borejdo
荧光相关光谱（FCS）要求每个分子的光子检测率高，背景低，与平移运动相关的荧光波动大。这里提出的新方法，表面等离子体辅助显微镜（SPAM），满足这些要求，大大限制了观察体积。在这种方法中，观测层非常薄，以至于波动主要是由于分子的轴向运动。这可以通过将样品放置在薄金属膜上并用激光束通过水介质照射来方便地实现。靠近表面的激发荧光团耦合（通过近场相互作用）到金属中的表面等离子体。增强的表面等离子体激元在金属膜的相对侧上解耦为远场辐射并且以定向方式发射。使用高数值孔径物镜收集荧光。共焦孔径插入其共轭图像平面减少检测体积的横向尺寸的衍射极限。检测层的厚度通过激发的荧光团在表面附近（约30 nm）的金属淬灭而进一步减小。我们使用的荧光微球悬浮液，表明FCS-SPAM是一种有效的方法来测量分子扩散。
Fluorescence Correlation Spectroscopy (FCS) demands a high rate of photon detection per molecule, low background, and large fluctuations of fluorescence associated with translational motion. The new approach presented here, Surface Plasmon Assisted Microscope (SPAM), meets these requirements by drastically limiting the observation volume. In this method, the observational layer is made so thin that fluctuations are mostly due to the axial motion of molecules. This is conveniently realized by placing a sample on a thin metal film and illuminating it with a laser beam through an aqueous medium. The excited fluorophores close to the surface couple (via near-field interactions) to surface plasmons in the metal. Propagated surface plasmons decouple on opposite side of the metal film as a far-field radiation and emit in directional manner. Fluorescence is collected with a high Numerical Aperture objective. A confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a diffraction limit. The thickness of the detection layer is reduced further by metal quenching of excited fluorophores at a close proximity (about 30 nm) to the surface. We used a suspension of fluorescent microspheres to show that FCS-SPAM is an efficient method to measure molecular diffusion.