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Ultra high-resolution scanning near-field optical microscope for direct gene reading

用于直接基因读取的超高分辨率扫描近场光学显微镜

基本信息

批准号：
15360132
负责人：
HAMAGUCHI Tetsuya
金额：
$ 9.86万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15360132/
关键词：
scanning near-field optical microscope scattering probe focused ion beam electron beam deposition fluorescent observation crystalline carbon

项目摘要

The objective of this research is developing a scanning near-field optical microscope (SNOM), to realize ultra high-resolution observation of fluorescent molecules on a specific gene of a DNA. The outline of the results is shown below.(1)Fundamental fabrication technique which produces micro SNOM probe for scattering near-field light around a specimen. Electron beam deposition method has been newly developed for fabricate a micro SNOM probe with a high aspect ratio tip end with 1 micrometer length and 20 nanometer diameter. And we have prototyped a micro SNOM probe with 500 nanometer square aperture, fabricating an AFM cantilever using focused ion beam.(2)For further miniaturization of the SNOM probe tip, we develop a new electron beam deposition method that produces not only amorphous carbon but also crystalline carbon. A examination result shows the optimum condition for producing crystalline carbon was 900 degrees temperature and 1 torr pressure. To realize the optimum condition in a scanning electron microscope (SEM) chamber, we developed a local heating and compression equipment, and confirmed that crystalline carbon was produced in the SEM camber. And it was proposed to control the direction and length of the crystalline carbon by applying magnetic field, to produce crystalline carbon with a desired shape at a desired position.(3)Through a fluorescent observation using prototyped SNOM probe, the optimum probe shape to get the highest S/N ratio and resolution at each wavelength. We optimized the parameters of excitation light strength, irradiation area, transmissivity of the filter, and reduce the noise. As the result, it was confirmed that reducing excitation light strength improved the S/N ratio, and reduced excitation light made a fluorescent image. These results showed feasibility of the observation of only fluorescent particle.

本研究的目的是发展一种扫描近场光学显微镜（SNOM），以实现对DNA特定基因上荧光分子的超高分辨率观察。结果概要如下所示。（1）制造用于在样品周围散射近场光的微型SNOM探针的基本制造技术。采用电子束沉积法制备了一种长1 μ m、直径20 nm的高深宽比微SNOM探针。我们制作了一个孔径为500纳米的微型SNOM探针，并利用聚焦离子束制作了AFM悬臂梁。（2）为了进一步实现SNOM探针针尖的小型化，我们开发了一种新的电子束沉积方法，该方法不仅可以生成非晶碳，还可以生成结晶碳。试验结果表明，制备结晶炭的最佳条件为温度900 ℃，压力1 ℃。为了实现扫描电子显微镜（SEM）室中的最佳条件，我们开发了一种局部加热和压缩设备，并证实了结晶碳在SEM室中产生。并且提出了通过施加磁场来控制结晶碳的方向和长度，以在期望的位置产生具有期望形状的结晶碳。（3）通过对原型SNOM探针的荧光观察，确定了在各波长处获得最高信噪比和分辨率的最佳探针形状。优化了激发光强度、照射面积、滤光片透过率等参数，降低了噪声。结果，证实了降低激发光强度改善了S/N比，并且降低的激发光产生荧光图像。这些结果表明了仅观察荧光颗粒的可行性。