SPECTRAL CONFOCAL MICROSCOPE

描述（由申请人提供）：我们建议购买Leica TCS SP2-MP 光谱共聚焦显微镜，以增加现有的设施在光学 赵氏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年以来，UCI的共享设施使研究组织成为可能， 固定组织中特异性分子的亚细胞定位 荧光标记，通常不需要组织切片。最近 基于荧光标记的基因编码荧光标记的设计进展 水母绿色荧光蛋白（GFP）现在可以检查 蛋白质在活的而不是固定的组织中的定位。延长 这项技术的一个特点是使用成对的突变GFP标签， 不同波长检测荧光共振能量转移（FRET） 这两个标签之间，揭示了重要的分子信息 固定或活细胞中标记蛋白质之间的相互作用。类似 方法使得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， 在分子中的不同位置。FRET显微镜的其他应用 允许监测钙水平、蛋白酶活性、膜 潜力，以及活细胞和组织中的神经元活动模式。这些 技术有望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，该方法将 需要在一定的波长下区分激发波长和发射波长的能力。 比现有的共聚焦激光器更高的选择性 显微镜.所提出的显微镜通过使用棱镜而不是 而不是过滤器来选择发射波长。初步数据包括在 应用表明，双光子激发提供了显着的改善， FRET信号与背景的分离，因此该提议包括 双光子升级该仪器不仅将大大促进FRET 分析以及需要多个标签的应用，特别是在生活中， 细胞，双光子激发将提供许多优点 包括比可能的更深地穿透到组织中的能力 传统的激励。该仪器将安装在光学 生物核心设施，并将提供给任何合格的用户充电 基础准备使用这一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，信号转导蛋白之间的相互作用， 控制胚胎模式的转录因子之间， 钾和钙通道，T细胞受体的运输，关联 谷氨酸受体亚基，以及许多其他蛋白质相互作用， 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.