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。 显微镜不再仅仅提供观察形态的工具 细胞及其子组件，它现在作为一个平台， 利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 活细胞中的特定细胞成分， 开发单细胞测定以监测基因表达和其他 细胞的活动，以辨别功能将突出在这一点， 科. 我们的具体目标是a）开发以下的体内标记： 亚细胞结构与绿色荧光蛋白（GFP）， 促进体内靶的微波束介导的消融， 随后评估细胞中产生的结构改变， B）将激光灭活与特异性细胞/分子测定结合 为了阐明烧蚀目标的功能，c）开发 利用绿色的荧光共振能量转移（FRET） 荧光蛋白（GFP）和蓝色荧光蛋白（BFP）融合 蛋白质，并采用FRET作为测定，以获得的影响， 微束烧蚀离散的亚细胞结构或结构域 d）将联合收割机激光镊子与亚烧蚀 通过微束将生物试剂引入细胞 Ropto-porationS. 这将扩大微束的功能 技术到以前未开发的生物领域，即暂停 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.