Li Jiayao, Kim Yeonuk, Liu Boyin, Qin Ruwen, Li Jian, Fu Jing
Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia.
Department of Mechanical and Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia.
Micron. 2017 Oct;101:132-137. doi: 10.1016/j.micron.2017.06.002. Epub 2017 Jul 31.
The nano-manipulation approach that combines Focused Ion Beam (FIB) milling and various imaging and probing techniques enables researchers to investigate the cellular structures in three dimensions. Such fusion approach, however, requires extensive effort on locating and examining randomly-distributed targets due to limited Field of View (FOV) when high magnification is desired. In the present study, we present the development that automates 'pattern and probe' particularly for single-cell analysis, achieved by computer aided tools including feature recognition and geometric planning algorithms. Scheduling of serial FOVs for imaging and probing of multiple cells was considered as a rectangle covering problem, and optimal or near-optimal solutions were obtained with the heuristics developed. FIB milling was then employed automatically followed by downstream analysis using Atomic Force Microscopy (AFM) to probe the cellular interior. Our strategy was applied to examine bacterial cells (Klebsiella pneumoniae) and achieved high efficiency with limited human interference. The developed algorithms can be easily adapted and integrated with different imaging platforms towards high-throughput imaging analysis of single cells.
结合聚焦离子束（FIB）铣削与各种成像和探测技术的纳米操纵方法，使研究人员能够在三维空间中研究细胞结构。然而，由于在需要高放大倍数时视野（FOV）有限，这种融合方法在定位和检查随机分布的目标时需要付出大量努力。在本研究中，我们展示了通过包括特征识别和几何规划算法在内的计算机辅助工具实现的、特别针对单细胞分析的“模式与探测”自动化进展。将用于多个细胞成像和探测的连续视野调度视为一个矩形覆盖问题，并使用所开发的启发式方法获得了最优或接近最优的解决方案。然后自动进行FIB铣削，随后使用原子力显微镜（AFM）进行下游分析以探测细胞内部。我们的策略应用于检查细菌细胞（肺炎克雷伯菌），并在有限的人工干预下实现了高效率。所开发的算法可以很容易地进行调整，并与不同的成像平台集成，以实现单细胞的高通量成像分析。
