IMAGING BARRIERS TO MOLECULAR DIFFUSION BY PAIR CORRELATION FUNCTIONS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我使用一种新的、基于对相关函数的非常规荧光相关技术来研究模型底物(NLS-GFP)的细胞内扩散系数。我通过测量单个分子从细胞内一个特定位置移动到另一个位置所需的时间来检测分子运输事件。这是与传统的FCS方法相比的新奇之处，该方法测量分子穿过激光光束所需的时间。通过这种方法，扩散障碍既可以在细胞隔间内检测到(对自由扩散的限制)，也可以跨越核膜屏障(由于存在核孔通道而受到限制)。在这两种情况下，测量提供了NLS-GFP在单分子水平和活细胞中转运的动力学步骤的定量描述，而不需要复杂的标记程序和细胞操作。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. I study the intracellular diffusivity of a model substrate (NLS-GFP) by using a novel and unconventional fluorescence correlation technique, based on pair correlation functions. I detect molecular transport events by measuring the time a single molecule takes to move from a specific location to another within the cell. Here is the novelty with respect to the traditional FCS approach that measures the time a molecule takes to cross the laser beam. By this approach obstacle to diffusion can be detected both within cellular compartments (restrictions to free diffusion) and across the nuclear envelope barrier (restrictions due to the presence of nuclear pore channels). In both cases the measurement affords a quantitative description of the kinetic steps of NLS-GFP transport at the single molecule level, and in living cells, without the need for complex labeling procedures and cell manipulations.