MICROBEAM ABLATION & CELLULAR MECHANISMS

光学显微镜的复兴带来了新的 在亚细胞水平上可视化分子事件的能力。 显微镜不再提供仅用于观察形态的工具 单元及其子组件，它现在作为一个平台 用工具对离散的分子活动进行成像 操纵这些事件。利用激光微束来 灭活活细胞中的特定细胞成分 用于监测基因表达和其他方面的单细胞分析的发展 识别功能的细胞活动将在本课程中重点介绍 一节。我们的具体目标是a)开发体内标记 带有绿色荧光蛋白(GFP)的亚细胞结构 促进微束介导的体内靶点的消融 随后评估细胞中所产生的结构改变， B)将激光灭活与特定的细胞/分子分析相结合 为了阐明烧蚀靶的功能，c)开发 利用绿色荧光共振能量转移(FRET) 荧光蛋白(GFP)与蓝色荧光蛋白(BFP)的融合 蛋白质，并使用FRET作为一种分析方法来获得 微束烧蚀离散的亚细胞结构或结构域 细胞内相互作用，d)将激光镊子与亚消融术相结合 通过微束将生物试剂导入细胞 Ropto-porationS。这将扩大微束的曲目 将技术扩展到以前未探索的生物领域，即悬浮物 细胞模型。

The renaissance of optical microscopy brings with it new capabilities to visualize molecular events at the subcellular level. No longer does microscopy provide the tools only to see the morphology of the cell and its subcomponents, it now serves as a platform for imaging discrete molecular activities with tools to perturb and manipulate these events. Utilization of laser microbeams to inactivate specific cellular components in a living cell and the development of single cell assays to monitor gene expression and other cellular activities to discern function will be highlighted in this section. Our specific aims are to a) Develop in vivo labeling of subcellular structures with green fluorescent protein (GFP) to facilitate microbeam-mediated ablation of an in vivo target and to subsequently assess the resulting structural alteration in the cell, b) Couple laser inactivation with specific cellular/molecular assays in order to elucidate the function of the ablated targets, c) Develop Fluorescence Resonance Energy Transfer (FRET) utilizing green fluorescent protein (GFP)- and blue fluorescent protein (BFP)- fusion proteins and employ FRET as an assay to access the effects of microbeam ablation of discrete subcellular structures or domains of interaction within a cell, d) Combine laser tweezers with sub-ablative microbeams to introduce biological reagents into cells via Ropto-porationS. This will expand the repertoire of microbeam technologies to previously unexplored biological areas i.e. suspension cell models.