Kolodny L A, Willard D M, Carillo L L, Nelson M W, Van Orden A
Department of Chemistry, Colorado State University, Fort Collins 80523, USA.
Anal Chem. 2001 May 1;73(9):1959-66. doi: 10.1021/ac001472z.
Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.
将单个荧光聚苯乙烯纳米球（直径<10 - 100纳米）和单个荧光标记的DNA分子分散在云母上，并使用时间分辨荧光光谱和原子力显微镜（AFM）进行分析。荧光测量与AFM测量的空间相关性通过以下方式实现：（1）将单个荧光颗粒定位到光学显微镜的近衍射极限共聚焦激发区域内；（2）记录时间分辨荧光发射；（3）测量从AFM探针尖端顶点散射的激发激光光强度以及AFM形貌，作为尖端相对于样品基底横向位置的函数。后一种测量产生了共聚焦显微镜激光激发轮廓和样品形貌的同时高分辨率（横向约10 - 20纳米）图像。这些光学图像和形貌图像的叠加使得能够明确识别位于光学显微镜激发区域内的样品形貌，有助于识别和结构表征在步骤2中观察到的产生荧光信号的纳米颗粒或生物分子。这些测量还提供了颗粒相对于激光激发轮廓和周围形貌的横向位置，精度达到纳米级，以及颗粒的光谱性质和结构性质之间的关系。讨论了将这些方法扩展到研究其他类型纳米结构材料的情况。
