Multiphoton laser surgery at 100 um spatial resolution

我们从生物学(T.T.Takamatsu)和光学(O.Nakamura)的角度对第一年获得的实验和理论知识进行了全面评估。为了在生物组织中实现精确的纳米手术，我们优化了实验参数。我们成功地切割和加工了大鼠的红细胞-心肌细胞、毛细血管和其他亚微米分辨率的细胞。以此为标准，我们用基因操作的方法对大鼠胚胎和斑马鱼胚胎表面进行了处理，并评价了加工部分与功能组织表达的关系。探索了这些方法跟踪脑生长和校准胚胎结构与成人脑结构的可能性。此外，我们使用一些最新的高分辨率技术，包括4PI共聚焦光学系统，研究了操控细胞的总体行为。通过研究，我们新开发了一种显微镜光学系统和一种激光扫描技术，通过这种技术，我们可以以3D分辨率和精度操作细胞。最后，我们通过实验研究了纳米区域的细胞内局部破坏与功能组织生成的关系。

We totally evaluated the experimental and theoretical knowledge obtained in the first year, from biological (T. Takamatsu) and optical (O. Nakamura) points of view. We optimized the experimental parameters for achieving accurate nanosurgery in biological tissue. We successfully cut and processed red blood cells of rat-cardiomyocyto cells, capillary blood vessels, and others with submicron resolution. Using these results as criterion, we processed the surfaces of rat's and zebrafish's embryo doped with fluorescent protein (GFP) of aequorea coerulescens by genetic manipulation, and evaluated the relation between the processed part and expression of functioning tissue We explored the possibilities of these methods to track the brain growth and to calibrate the structure of embryo to that of an adult brain. Furthermore we investigated total behavior of the manipulated cells using some recent techniques for high resolution including a 4pi confocal optical system. From the investigation, we newly developed a microscope optical system and a laser scanning technique by which we could manipulate cells with 3D resolution and accuracy. Eventually we experimentally studied the relationship between intracellular local breakdown in a nanometer region and the generation of functioning tissue.