SPECTRAL CONFOCAL MICROSCOPE

描述(申请人提供)：我们建议收购徕卡TCS SP2-MP 光谱共焦显微镜增加了现有的光学设备 赵氏家族综合癌症中心生物核心设施 加州大学欧文分校。共聚焦显微镜已经作为一种 自1987年起在UCI共享设施，使研究组织和 固定组织中特定分子的亚细胞定位 荧光标记，通常不需要组织切片。近期 基于基因编码的荧光标记设计的研究进展 水母绿色荧光蛋白(GFP)现在使检测 蛋白质在活组织中的定位，而不是固定组织中的定位。延期 这项技术的关键是使用一对突变的GFP标签，这些标签在 检测荧光共振能量转移(FRET)的不同波长 在两个标签之间，揭示了关于分子的重要信息 固定细胞或活细胞中标记蛋白之间的相互作用。类似 方法使研究个体的构象变化成为可能 通过检测荧光标记之间FRET的变化来检测细胞内的蛋白质 在分子中的不同位置。FRET显微镜的其他应用 允许监测膜的钙水平、蛋白酶活性和 以及活细胞和组织中的神经元活动模式。这些 技术有望在以下领域引领全新的研究方向 发育和细胞生物学以及神经生物学。然而，这种方法将 需要能够区分激发波长和发射波长 比现有共焦的选择性更高 显微镜。所提出的显微镜通过使用棱镜来解决这个问题 而不是滤光片来选择发射波长。初步数据载于 应用表明，双光子激发大大改善了 FRET信号与背景的分离，因此该建议包括一种 双光子升级。该仪器不仅将极大地方便烦躁 分析，以及需要多个标签的应用，尤其是在生活中 电池，而双光子激发将提供许多优势 包括比可能的更深入组织的能力 用常规的激发。该仪器将安装在光学系统中 生物核心设施，将提供给任何有资格的用户充值 基础。准备使用该工具的项目包括研究 支架蛋白与信号转导通路的相互作用 控制生长，信号转导蛋白和蛋白之间的相互作用 在控制胚胎模式的转录因子之间，监测 钾和钙通道，T细胞受体的运输，关联 谷氨酸受体亚基和许多其他蛋白质的相互作用 发展和疾病。

DESCRIPTION (provided by applicant): We propose to acquire a Leica TCS SP2-MP Spectral Confocal Microscope to add to the facilities available in the Optical Biology Core Facility of the Chao Family Comprehensive Cancer Center at the University of California, Irvine. Confocal microscopy has been available as a shared facility at UCI since 1987, making it possible to study the tissue and subcellular localization of specific molecules in fixed tissues using fluorescent labels, often without the need for tissue sectioning. Recent advances in the design of genetically encoded fluorescent tags based on the jellyfish Green Fluorescent Protein (GFP) now make it possible to examine the localization of proteins in living, rather than fixed, tissue. An extension ofthis technology is to use pairs of mutant GFP tags that fluoresce at different wavelengths to detect fluorescence resonance energy transfer (FRET) between the two tags, revealing important informnation on molecular interactions between the tagged proteins in either fixed or live cells. Similar methods make it possible to study conformational changes in an individual protein within a cell by detecting changes in FRET between fluorescent labels at different locations in the molecule. Other applications of FRET microscopy allow monitoring of calcium levels, of protease activity, of membrane potential, and of neuronal activity patterns in live cells and tissues. These technologies promise to lead to entirely new research directions in developmental and cell biology and neurobiology. However, the approach will require the ability to discriminate excitation and emission wavelengths at a higher level of selectivity than is possible with available confocal microscopes. The proposed microscope solves this problem by using prisms rather than filters to select emission wavelengths. Preliminary data included in the application shows that two-photon excitation provides a dramatic improvement in separation of FRET signal from background, so this proposal includes a two-photon upgrade. The instrumnent will greatly facilitate not only FRET analysis but also applications requiring multiple labels, especially in living cells, and the two-photon excitation will provide a number of advantages including the ability to penetrate more deeply into tissue than is possible with conventional excitation. The instrument will be installed in the Optical Biology Core Facility and will be available to any qualified user on a recharge basis. Projects that are ready to use this instrument include studies of interactions between scaffolding proteins and signal transduction pathways controlling growth, interactions between signal transduction proteins and between transcription factors controlling patterning in embryos, monitoring of potassium and calcium channels, trafficking of the T cell receptor, association of glutamate receptor subunits, and many other protein interactions in development and disease.