Vickery S A, Dunn R C
Department of Chemistry, University of Kansas, Malott Hall, Lawrence, Kansas 66045, USA.
Biophys J. 1999 Apr;76(4):1812-8. doi: 10.1016/S0006-3495(99)77341-8.
A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.
展示了一种新的显微技术，它结合了近场扫描光学显微镜（NSOM）和荧光共振能量转移（FRET）的特性。该方法依赖于将FRET对的受体染料连接到近场光纤探针的末端。从与受体染料非共振的NSOM探针射出的光激发引入样品中的供体染料。当探针尖端靠近含有供体染料的样品时，从激发的供体到尖端结合受体的能量转移产生红移荧光。通过监测这种红移的受体发射，可以观察到未涂覆的NSOM尖端探测的样品体积显著减小。通过对使用朗缪尔-布洛杰特（LB）技术制备的多层膜的荧光成像来证明该技术。该膜由含有供体染料荧光素的L-α-二棕榈酰磷脂酰胆碱（DPPC）单层组成，由三层花生酸层的间隔基团隔开。使用LB技术还将含有受体染料罗丹明的DPPC单层转移到NSOM尖端上。使用这种改进的探针，多层膜的荧光图像显示了在监测供体或受体发射时收集的图像之间的明显差异。后者是由于从样品到NSOM探针的能量转移。该方法在荧光测量中显示出增强的深度敏感性，这在对厚标本如细胞的研究中可能特别有用。该技术还提供了一种无需在NSOM探针周围进行金属涂层即可获得高空间分辨率的机制，并且对于非波导探针如原子力显微镜尖端也同样适用。这可能会显著提高荧光成像的空间分辨率。
