用于单分子水平DNA研究的近场光学显微镜

M F Garcia-Parajo, J-A Veerman, S J T van Noort, B G de Grooth, J Greve, N F van Hulst
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引用次数: 35

摘要

采用双偏振检测通道的孔径型近场光学显微镜(NSOM)对荧光标记DNA进行了高空间分辨率和单分子荧光灵敏度的成像。这种样品经过改造,每条DNA链上只有一种罗丹明染料。剪切力图像中DNA的横向和纵向尺寸分别为14±2 nm和1.4±0.2 nm。在我们的成像条件下没有观察到样品变形。单个荧光团的近场荧光成像显示在全宽半最大值处的光学分辨率为70 nm。从NSOM图像中可以观察到附着在DNA上的单个罗丹明分子之间的巨大强度差异。罗丹明染料在不同环境下(附着在玻璃上,嵌入在聚合物层中,附着在DNA上)的漂白率为10−5。总强度线轮廓与面内角取向一起用于表征单个染料。罗丹明染料表现出与特定环境无关的强强度波动。这些结果与嵌入在聚合物基质中的碳菁分子所观察到的更稳定的光物理行为形成对比。罗丹明的移动性——横向的和旋转的——明显受到其周围环境和附着在表面的影响。
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Near-field optical microscopy for DNA studies at the single molecular level
An aperture-type near-field optical microscope (NSOM) with two polarization detection channels has been used to image fluorescently labelled DNA with high spatial resolution and single molecule fluorescence sensitivity. The sample has been engineered such that there is o­nly o­ne rhodamine dye per DNA strand. Lateral and vertical DNA dimensions in the shear-force image are 14 +/- 2 nm and 1.4 +/- 0.2 nm, respectively. No sample deformation was observed under our imaging conditions. Near-field fluorescence imaging of individual fluorophores shows an optical resolution of 70 nm at full-width at half- maximum. Large intensity differences between individual rhodamine molecules attached to DNA are observed from the NSOM images. Statistics o­n rhodamine dyes in different environments (attached to glass, embedded in a polymer layer and attached to DNA) show bleaching rates of 10(-5). Total intensity line profiles together with in-plane angle orientation are used to characterize individual dyes. Rhodamine dyes show strong intensity fluctuations independent of the particular environment. These results are in contrast with the more stable photophysical behaviour as observed for carbocyanine molecules embedded in polymer matrices. The mobility of rhodamine-both lateral and rotational-is clearly influenced by its immediate surrounding and attachment to the surface.
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