Welsher Kevin, Yang Haw
Department of Chemistry, Princeton University, New Jersey, USA.
Faraday Discuss. 2015;184:359-79. doi: 10.1039/c5fd00090d. Epub 2015 Oct 1.
The overwhelming effort in the development of new microscopy methods has been focused on increasing the spatial and temporal resolution in all three dimensions to enable the measurement of the molecular scale phenomena at the heart of biological processes. However, there exists a significant speed barrier to existing 3D imaging methods, which is associated with the overhead required to image large volumes. This overhead can be overcome to provide nearly unlimited temporal precision by simply focusing on a single molecule or particle via real-time 3D single-particle tracking and the newly developed 3D Multi-resolution Microscopy (3D-MM). Here, we investigate the optical and mechanical limits of real-time 3D single-particle tracking in the context of other methods. In particular, we investigate the use of an optical cantilever for position sensitive detection, finding that this method yields system magnifications of over 3000×. We also investigate the ideal PID control parameters and their effect on the power spectrum of simulated trajectories. Taken together, these data suggest that the speed limit in real-time 3D single particle-tracking is a result of slow piezoelectric stage response as opposed to optical sensitivity or PID control.
在开发新的显微镜方法方面，绝大多数努力都集中在提高三维空间和时间分辨率上，以便能够测量生物过程核心的分子尺度现象。然而，现有的三维成像方法存在显著的速度障碍，这与对大体积成像所需的额外开销有关。通过实时三维单粒子跟踪和新开发的三维多分辨率显微镜（3D-MM）简单地聚焦于单个分子或粒子，可以克服这种额外开销，从而提供几乎无限的时间精度。在这里，我们在其他方法的背景下研究实时三维单粒子跟踪的光学和机械极限。特别是，我们研究了使用光学悬臂进行位置敏感检测，发现这种方法产生的系统放大倍数超过3000倍。我们还研究了理想的PID控制参数及其对模拟轨迹功率谱的影响。综合这些数据表明，实时三维单粒子跟踪中的速度限制是压电平台响应缓慢的结果，而不是光学灵敏度或PID控制的结果。
