Fast super-resolution/confocal microscopy for GI cell biology

项目概要：AIRYSCAN成像技术 新设备的目标是将高生产力的调查人员与最先进的光学设备连接起来。 促进科学进步和新突破的技术和应用。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马约诊所气体细胞信号中心的成员， 胃肠病学（C-SiG）。补助金的具体目标有三个方面。第一，提供一个可靠的，可访问的， 国家的最先进的成像技术，以用户组，这将有助于研究胃肠道细胞信号传导cas- cades.第二，教育和培训小组的个别成员使用先进的细胞图像， ing方法。重点放在提供技术指导和教育调查人员如何这样做， 这些方法可以扩大其独立研究方案的范围和广度。第三，发展和 应用创新的荧光技术，包括生物传感器和活体染料，研究胃肠道组织和/或 细胞健康和疾病的发病机制。这项工作需要先进的成像的高度动态和/或pho- 细胞内的敏感过程，同时也定义了分子机器组件的定位， 灵敏度高、光毒性低。新开发的Airyscan技术是一项重大突破， 通过以下方式实现：i）由于其创新的检测器阵列而提供具有最高灵敏度的完美光学部分， ii）将信噪比和空间分辨率提高到同类最佳水平，允许检测相互作用和促进 iii）增加灵敏度以促进光敏过程的实时成像，以及 结构，同时使光损伤最小化，以及iv）加速图像采集 速度为上级时间分辨率捕捉高度动态的事件和瞬态相互作用。新 该技术将成为C-SiG光学显微镜核心的一部分，其中共聚焦显微镜的利用率已 在过去十年里增长了两倍多。核心还提供指导，技术咨询，数据解释， 和发展新的，创新的光学方法来研究信号通路在胃肠道细胞和组织， - 是的这些服务涵盖广泛的主题，包括：共聚焦显微镜与计算机- 基于三维图像重建;荧光共振能量转移（FRET）应用于测量 动态蛋白质-蛋白质相互作用;光漂白后的荧光恢复（FRAP）， 蛋白质募集/周转的定量;光漂白中的荧光损失（FLIP）;活体显微注射， ing细胞;表达和使用基于荧光的生物探针，促进spe的研究和定位， cific信号蛋白和脂质;特异性光激活笼状蛋白的开发和应用; 允许在活细胞中精确时间和空间激活所需信号分子的化合物， 还有其他的除了支持生产NIH资助的用户基础，优先访问提供给 有前途的年轻教师从事消化系统疾病相关的研究，并寻求独立的资金。

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.