2-photon microscope

描述（申请人提供）：每个细胞和组织生物学研究项目都需要研究标本的显微镜可视化。虽然这些需求中的一些是最好的解决了传统的，常规的光和共聚焦显微镜薄，固定的样品，近几十年来已经看到了非常复杂的和苛刻的新的显微镜程序在体内成像（如活细胞和深层组织成像，同时表达多种荧光蛋白，FRET和FRAP），现在是当代细胞和组织研究的一个必要部分的爆炸。要成功应用，这些方法需要一个多功能的，先进的成像系统，结合双光子激发，快速成像和环境控制。这种最先进的显微镜方法对专业知识和专业设备的需求往往超过了单个研究实验室可用的资源，因此最好作为共享成像设施中的共享仪器资源来满足。该提案要求提供资金，用于购买配备相干变色龙超II超快脉冲激光器的Prairie Technologies Ultima双光子显微镜，以高空间和时间分辨率对活生物样本进行成像。这种双光子活细胞成像系统将是一种共享资源，位于斯坦福大学的一个完善的多用户光学和电子显微镜设施：细胞科学成像设施（http：taltos.stanford.edu）。这个设施是访问斯坦福大学的整个研究社区以及周围的生物技术公司。所要求的双光子活细胞成像系统将支持来自8个主要用户的NIH资助项目。这些项目调查了NIH支持的广泛主题和模型系统，包括：1）发育过程中细胞表达模式的分析（C。elegans，Kim）; 2）肿瘤的血管生成和抗血管生成治疗（小鼠、斑马鱼、Kuo）; 3）皮肤基底细胞癌的发病机制（果蝇，奥罗）; 4）病毒复制、组装和从感染细胞中排出的分子机制（小鼠，人组织，阿尔文）; 5，6）表征和建模癫痫发展的神经元机制（大鼠，Huguenard，Prince）; 7）哺乳动物视网膜和脑中神经胶质细胞的发育和功能特异性（Barres）和8）调节胰腺发育的细胞动力学（小鼠，Seung Kim）。这些研究调查了各种模式生物和人体组织中的关键功能和结构问题，并涵盖了对人类健康和疾病的各个方面产生影响的生物医学研究领域，从癌症和病毒发病机制到了解癫痫的细胞和分子基础，感觉生物学和器官发育。所有这些都需要双光子仪器来对活样本进行成像，这是由所要求的Prairie Technologies的Ultima双光子成像系统最有效地提供的。公共卫生相关性：美国国立卫生研究院用户组研究人员目前正在进行的研究，调查各种模式生物和人体组织中的关键功能和结构问题，并涵盖生物医学研究领域，对人类健康和疾病的各个方面产生影响，从癌症和病毒发病机制到了解癫痫的细胞和分子基础，感觉生物学和器官发育。所有这些都需要双光子仪器，用于在延长的时间段内对活样本进行成像，这是由所请求的Prairie Technologies Inc.最有效地提供的，配备Coherent Inc.的Ultima双光子显微镜。Chameleon Ultra II超快脉冲激光。

DESCRIPTION (provided by applicant): Every cell and tissue biology research program has needs for microscopic visualization of research specimens. While some of these needs are best addressed by traditional, routine light and confocal microscopy of thin, fixed samples, recent decades have seen an explosion of very sophisticated and demanding new microscopy procedures for in vivo imaging (e.g. live cell and deep tissue imaging, simultaneous expression of multiple fluorescent proteins, FRET and FRAP) that are now a required part of contemporary cell and tissue research. To be applied successfully, these methods require a versatile, advanced imaging system that incorporates 2-photon excitation, rapid imaging and environmental control. The demands of such state-of-the-art microscopy methods for both specialized expertise and specialized equipment often outstrip the resources available to individual research laboratories and as such are best met as a shared instrument resource housed in a shared imaging facility. This proposal requests funds to purchase a Prairie Technologies Ultima, 2-photon microscope equipped with the Coherent Chameleon Ultra II ultra-fast pulsed laser, for imaging, with high spatial and temporal resolution, live biological samples. This 2-photon live cell imaging system will be a shared resource, located in a well-established, multi-user, light and electron microscopy facility at Stanford University: the Cell Sciences Imaging Facility (http://taltos.stanford.edu). This facility is accessible to Stanford University's entire research community as well as surrounding biotech companies. The requested 2-photon live cell imaging system will support NIH funded projects from 8 major users. These projects investigate a wide range of NIH supported topics and model systems including, 1) analysis of cellular expression patterns during development (C. elegans, Kim); 2) angiogenesis and anti-angiogenic therapy of cancer (mice, zebra fish, Kuo); 3) pathogenesis of basal cell carcinoma of the skin (Drosophila, Oro); 4) molecular mechanisms of viral replication, assembly and egress from infected cells (mice, human tissue, Arvin); 5,6) characterization and modeling of the neuronal mechanisms underlying the development of epilepsy (rat, Huguenard, Prince); 7) development and functional specificity of glial cells in the mammalian retina and brain (Barres) and 8) cellular dynamics regulating pancreatic development (mice, Seung Kim). These studies investigate critical functional and structural questions in a variety of model organisms and human tissues and cover areas of biomedical research with implications for diverse aspects of human health and disease, ranging from cancer and viral pathogenesis to understanding the cellular and molecular basis of epilepsy, sensory biology and organ development. All require 2-photon instrumentation for imaging live samples which is most effectively provided by the requested Prairie Technologies' Ultima 2- photon imaging system. PUBLIC HEALTH RELEVANCE: The studies currently being undertaken by the NIH user group researchers, investigate critical functional and structural questions in a variety of model organisms and human tissues and cover areas of biomedical research with implications for diverse aspects of human health and disease, ranging from cancer and viral pathogenesis to understanding the cellular and molecular basis of epilepsy, sensory biology and organ development. All require 2-photon instrumentation for imaging live samples over extended time periods which is most effectively provided by the requested Prairie Technologies Inc., Ultima 2-photon microscope equipped with the Coherent Inc. Chameleon Ultra II ultra-fast pulsed laser.