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首页> 外文会议>Multiphoton Microscopy in the Biomedical Sciences V; Progress in Biomedical Optics and Imaging; vol.6 no.15 >Membrane protein dynamics measured by two-photon ring correlation spectroscopy: theory and application to living cells
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Membrane protein dynamics measured by two-photon ring correlation spectroscopy: theory and application to living cells

机译：双光子环相关光谱法测量膜蛋白动力学：理论和对活细胞的应用

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摘要

Many biochemical reactions and processes are regulated by proteins associated with cellular membranes. Trans-membrane proteins play an important role in many aspects of cellular development, cellular migration and signaling, and many diseases. Quantitative measurement of protein dynamics under various experimental conditions can give insights into the mechanisms of interaction and the functionality of the protein. Fluctuation techniques, such as fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS), have been used for such dynamic measurements in membranes. However, FCS is limited to fast dynamics, and ICS works best on a flat 2-dimensional area. We present an alternative way to measure protein transport in spherical (non flat) living cells that combines laser scanning microscopy and image correlation methods: ring correlation spectroscopy (RCS). The RCS analysis is performed on CLSM or two-photon cross-sectional images of labeled proteins in the cell membrane, where the optical sectioning gives a "ring" of fluorescence in the images. We present computer simulations of two dimensional diffusion confined to the surface of a spherical shell, where the RCS analysis can extract the set input parameters from the simulation. As well, we present RCS analysis of two-photon microscopy images of Pre-B leukocytes cells expressing CD44 labeled with EGFP.
机译：许多生物化学反应和过程受与细胞膜相关的蛋白质的调节。跨膜蛋白在细胞发育，细胞迁移和信号传导以及许多疾病的许多方面起着重要作用。在各种实验条件下对蛋白质动力学的定量测量可以深入了解相互作用的机制和蛋白质的功能。诸如荧光相关光谱法（FCS）和图像相关光谱法（ICS）的波动技术已经用于膜中的这种动态测量。但是，FCS仅限于快速动态变化，而ICS在平面二维区域上效果最佳。我们提出了一种测量蛋白质在球形（非平面）活细胞中转运的替代方法，该方法结合了激光扫描显微镜和图像相关方法：环相关光谱（RCS）。 RCS分析是在细胞膜中标记蛋白质的CLSM或两光子横截面图像上进行的，其中光学切片会在图像中产生荧光“环”。我们提出了局限于球形壳表面的二维扩散的计算机仿真，其中RCS分析可以从仿真中提取设置的输入参数。同样，我们目前对前E-白细胞表达CD44标记EGFP的前B白细胞的两光子显微镜图像进行RCS分析。

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