Ultra-Precise Laser Surgery to study Cell Biomechanics

描述（由申请人提供）：我们开发了一种用于超高精度激光加工的多功能技术，该技术使用紧密聚焦的飞秒激光脉冲来烧蚀材料中精确定义的纳米级区域。利用这种技术，可以选择性地烧蚀甚至小于20 nm的区域。这一里程碑式的成就为广泛的应用带来了巨大的希望，也许没有什么比作为一种高度选择性破坏细胞内结构的工具更令人兴奋的了。创造结构“敲除”的能力，其中细胞内成分被选择性地破坏，为阐明结构-功能关系带来了巨大的希望，就像分子遗传敲除对理解基因及其编码的蛋白质的功能至关重要一样。为了实现这一潜力，该提案具有双重目标，即展示这种方法对更广泛的生物界的实用性，并解决有关细胞分裂的基本问题。为此，我们建议应用结构敲除技术来研究细胞骨架和有丝分裂的生物力学。这些实验将表征：1）允许染色体结合并沿沿着移动的机械和力产生特性，2）化疗药物紫杉醇的抗有丝分裂活性，以及3）中心粒在建立有丝分裂结构中的作用。还将研究结构敲除技术的极限，以探索未来有趣应用的潜力，例如单分子或基因的靶向破坏。

DESCRIPTION (provided by applicant): We have developed a versatile technique for ultra-high-precision laser machining that uses tightly focused femtosecond laser pulses to ablate sharply defined nanometer-scale regions in materials. With this technology it is possible to selectively ablate regions even smaller than 20 nm across. This milestone achievement holds great promise for a wide range of applications, perhaps none more exciting than as a tool to highly selectively destroy intracellular structures. The ability to create structural "knockouts" in which intracellular components are selectively destroyed holds enormous promise for elucidating structure-function relationships, just as molecular-genetic knockouts have been crucial to understanding the function of genes and the proteins they encode. Toward fulfilling this potential, this proposal has dual goals of demonstrating the utility of this approach to the broader biological community, and addressing fundamental questions concerning cell division. To these ends we propose to apply structural knockout technology to study the biomechanics of the cytoskeleton and mitosis. These experiments will characterize: 1) the mechanical and force generating properties that allow chromosomes to bind and move along microtubules, 2) the antimitotic activity of the chemotherapy drug taxol, and 3) the role of centrioles in establishing mitotic architecture. The ultimate limits of structural knockout technology will also be investigated to explore the potential for intriguing future applications such as targeted disruption of single molecules or genes.