Ultra-Precise Laser Surgery to study Cell Biomechanics

描述(申请人提供)：我们开发了一种用于超高精度激光加工的通用技术，该技术使用紧密聚焦的飞秒激光脉冲来烧蚀材料中清晰定义的纳米级区域。有了这项技术，有可能选择性地烧蚀直径甚至小于20纳米的区域。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地破坏细胞内结构的工具更令人兴奋的了。创造有选择地破坏细胞内成分的结构性“敲除”的能力，对于阐明结构-功能关系有着巨大的希望，就像分子遗传敲除对于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，这项提案有两个目标：向更广泛的生物界展示这种方法的有效性，以及解决与细胞分裂有关的基本问题。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。这些实验的特点是：1)允许染色体结合并沿微管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；3)中心粒在建立有丝分裂结构中的作用。结构敲除技术的终极极限也将被调查，以探索未来有趣的应用的潜力，例如定向破坏单分子或基因。

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.