Fast super-resolution/confocal microscopy for GI cell biology

项目总结：AIRYSCAN成像技术 新设备的目标是将高生产率的调查人员与最先进的光学技术联系起来 促进科学进步和新突破的技术和应用。的核心用户群 著名的细胞生物学家是美国国立卫生研究院资助的梅奥临床气体细胞信号中心的成员。 肠道病毒学(C-Sig)。这笔赠款的具体目标有三个。首先，提供可靠、可访问、 向用户群体提供最先进的成像技术，这将促进GI细胞信号的研究- 卡迪斯。第二，教育和培训小组的个别成员使用复杂的细胞图像- ING方法。重点是提供技术指导和教育调查人员如何 这些方法可以扩大其独立研究项目的范围和广度。第三，发展和发展 应用创新的荧光技术，包括生物传感器和生命染料，以研究胃肠道组织和/或 细胞在健康和疾病发病机制中的作用。这项工作需要对高动态和/或PHO进行高级成像 同时还定义了分子机械组件的定位 高灵敏度和低光毒性。新开发的AiryScan技术是一项重大突破， 通过以下方式实现这一点：i)由于其创新的探测器阵列提供具有最高灵敏度的完美光学部分， 二)将信噪比和空间分辨率提高到同类最佳，从而能够检测相互作用并促进 从未有过的过程，iii)提高灵敏度，以促进光敏过程的实时成像和 长时间(过夜)的结构，同时将光损害降至最低，以及iv)加快图像采集 具有卓越的时间分辨率，可捕获高度动态的事件和瞬时交互。新的 技术将成为C-Sig光学显微镜核心的一部分，共焦显微镜的使用已经 在过去的10年里增长了两倍多。该核心还提供指导、技术咨询、数据解释、 并开发新的、创新的光学方法来研究胃肠道细胞和组织中的信号通路。 起诉。这些服务涵盖广泛的主题，包括：结合计算机的共聚焦显微镜- 基于三维图像重建；荧光共振能量转移(FRET)在测量中的应用 动态蛋白质-蛋白质相互作用；光漂白后荧光恢复(FRAP)，允许 蛋白质募集/周转的定量；光漂白(FLIP)中的荧光损失；Liv- 以荧光为基础的生物探针的表达和使用，有助于研究和定位SPE- CICIC信号蛋白和脂类；特定的光激活笼状蛋白的开发和应用- 允许在活细胞中精确地在时间和空间上激活所需信号分子的化合物， 还有其他的。除了支持由NIH资助的生产性用户群外，还为以下用户提供优先访问 有前途的初级教员从事与消化系统疾病相关的研究并寻求独立资助。

PROJECT SUMMARY: AIRYSCAN IMAGING TECHNOLOGY The objective of the new equipment is to connect highly productive investigators with state-of-the-art optical technology and applications that facilitate scientific progress and novel breakthroughs. The core user group of well-established cell biologists are members of the NIH-funded Mayo Clinic Center for Cell Signaling in Gas- troenterology (C-SiG). The Specific Aims of the grant are threefold. First, to provide a reliable, accessible, state-of-the-art imaging technology to a user group that will facilitate the study of GI cellular signaling cas- cades. Second, to educate and train individual members of the group in the use of sophisticated cellular imag- ing methods. Emphasis is placed on providing technical instruction and educating investigators on how such approaches can expand the scope and breadth of their independent research programs. Third, to develop and apply innovative fluorescence technologies, including biosensors and vital dyes, to study GI tissues and/or cells in health and disease pathogenesis. This work requires advanced imaging of highly dynamic and/or pho- tosensitive processes within cells while also defining the localization of molecular machinery components with high sensitivity and low phototoxicity. The newly developed Airyscan technology is a major breakthrough that enables this by: i) providing a perfect optical section with highest sensitivity due to its innovative detector array, ii) improving signal-to-noise and spatial resolution to best-in-class, allowing detection of interactions and pro- cesses never before seen, iii) increasing sensitivity to facilitate live imaging of photosensitive processes and structures for long periods (overnight) while minimizing photo damage, and iv) speeding up image acquisition rates for superior temporal resolution to capture highly dynamic events and transient interactions. The new technology will be a part of the C-SiG Optical Microscopy Core, for which confocal microscope utilization has more than tripled in the past 10 years. The Core also provides instruction, technical advice, data interpretation, and development of novel, innovative optical approaches to the study of signaling pathways in GI cells and tis- sues. These services cover a wide range of topics including: confocal microscopy coupled with computer- based 3-D image reconstruction; Fluorescence Resonance Energy Transfer (FRET) applications to measure dynamic protein-protein interactions; Fluorescence Recovery After Photobleaching (FRAP) that allows the quantitation of protein recruitment/turnover; Fluorescence Loss in Photobleaching (FLIP); microinjection of liv- ing cells; expression and use of fluorescence-based bioprobes that facilitates the study and localization of spe- cific signaling proteins and lipids; the development and application of specific photo-activatable caged- compounds that allow a precise temporal and spatial activation of desired signaling molecules in live cells, among others. In addition to supporting a productive NIH-funded user base, prioritized access is provided for promising junior faculty engaged in digestive disease-related research and seeking independent funding.