Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Anal Chem. 2010 Mar 15;82(6):2441-7. doi: 10.1021/ac902789z.
An automatic calibration and scanning-angle prism-type total internal reflection fluorescence microscope (TIRFM) was constructed and tested for the highest vertical resolution. The angle of the incident laser beam can be changed automatically and reliably from subcritical angles to nearly 90 degrees with intervals smaller than 0.2 degrees, and the laser illumination spot in the sample can be calibrated to automatically overlap with the center of the microscope's field of view. By scanning through a wide range of incident angles with different evanescent-field layer thicknesses, the fluorescence intensity decay curves of randomly distributed fluorescent nanospheres in agarose gel were obtained and fitted with the theoretical decay functions to determine their vertical positions. The best axial resolution was demonstrated to be better than 10 nm under the rigorous statistical analysis of confidence levels and by the Monte Carlo simulation. The new setup was further utilized to determine the tilting angle of the microtubules buried in agarose gel and to find the precise surface plasmon resonance (SPR) angle for gold film enhanced TIRFM. We demonstrate the new microscope's unique capability to find the best illumination configuration for complex systems automatically and reproducibly.
构建并测试了一种自动校准和扫描角棱锥型全内反射荧光显微镜（TIRFM），以实现最高的垂直分辨率。入射激光束的角度可以自动且可靠地从亚临界角度改变到近 90 度，角度间隔小于 0.2 度，并且可以将样品中的激光照明光斑自动校准到与显微镜视场的中心重合。通过在不同消逝场层厚度范围内扫描宽角度范围，获得了琼脂糖凝胶中随机分布的荧光纳米球的荧光强度衰减曲线，并通过理论衰减函数进行拟合，以确定其垂直位置。通过置信水平的严格统计分析和蒙特卡罗模拟，证明最佳轴向分辨率优于 10nm。新的设置进一步用于确定埋在琼脂糖凝胶中的微管的倾斜角度，并找到金膜增强 TIRFM 的精确表面等离子体共振（SPR）角度。我们展示了新显微镜自动且可重复地为复杂系统找到最佳照明配置的独特能力。
