Olympus BX-DSU Live-Cell Disk Scanning Imaging / Electrophysiology System

描述(由申请人提供)：我们的12项联邦拨款(R01类型)的长期目标是解决神经科学和心脏生理学中重要而独特的问题，包括更好地了解神经系统和心脏中的活细胞如何对药物或其他环境变化做出反应。目前的应用是为了满足我们的8个主要用户的重大需求，即通常同时从活的神经、感觉和心脏细胞或组织切片获得共焦图像和电生理记录。具体需求包括以下主要功能：对活体组织切片中的钙瞬变和荧光标记物进行共聚焦成像；比率测量型Fura-2对钙瞬变的成像；伴随膜片钳记录离子通道活动期间的高时间和空间分辨率的钙成像；以及笼状化合物的UV闪光光解。所有主要用户在活细胞钙成像、电生理学和/或共聚焦显微镜方面都有丰富的经验。我们总共发表了100多篇使用其中一种或多种技术的论文，其中包括15篇同时使用活细胞功能共聚焦成像和电生理记录的论文。激光共聚焦显微镜的一个固有问题是非常强烈的激发光，导致活细胞成像过程中大量的染料分解(光漂白)。提出的奥林巴斯BX-DSU活细胞磁盘扫描成像系统通过优化信号检测克服了这一问题。它使用旋转圆盘共焦技术和新一代过冷EM-CCD相机来生成高时间和空间分辨率的图像。与激光相比，广谱光源产生的光漂白要少得多。此外，该系统增加了用于光活化(笼式)化合物光解的UV闪光灯，完全集成了用于伴随电生理记录的膜片钳仪器，以及用于组织切片工作的立式支架。结果是一个非常经济高效和灵活的解决方案，满足了主要用户的所有描述需求。肯塔基大学附近或附近没有其他仪器可以提供这种星座特征。大学将提供适当的空间，为有经验的技术人员提供工资支持，并对系统进行维护。该系统将由扫描盘技术和电生理学专家在肯塔基大学一个大型、完善的成像设备中建立。该系统将使主要用户继续在更好地理解和治疗慢性疼痛、肥胖、听觉障碍、嗅觉障碍、肺部疾病、脑损伤后果和心血管疾病方面取得新的发现。

DESCRIPTION (provided by applicant): The long-term objectives of our 12 federal grants (R01-type) are to address important and unique problems in neuroscience and cardiac physiology, including a better understanding how living cells in the nervous system and heart respond to drugs or other changes in the environment. The current application is in response to a significant need of our 8 Major Users to obtain confocal images and electrophysiological recordings, often simultaneously, from live neural, sensory and cardiac cells or tissue slices. Specific needs include the following major functional capabilities: Confocal imaging of Ca2+ transients and fluorescent markers in live tissue slices; ratiometric Fura-2 imaging of Ca2+ transients; high temporal and spatial resolution of Ca2+ imaging during concomitant patch clamp recordings of ion channel activity; and UV Flash photolysis of caged compounds. All of the Major Users have extensive experience with live cell calcium imaging, electrophysiology, and/or confocal microscopy. Collectively, we have published over a hundred papers using one or more of these techniques, including 15 papers that simultaneously used functional confocal imaging and electrophysiological recording in live cells. An inherent problem of laser confocal microscopes is the very intense excitation light, leading to substantial dye decomposition (photobleaching) during live-cell imaging. The proposed Olympus BX- DSU Live-Cell Disk Scanning Imaging System overcomes this problem with optimization of signal detection. It uses spinning disk confocal technology with a new generation, super-cooled EM-CCD camera to generate images with high temporal and spatial resolution. The broad spectrum light source produces substantially less photobleaching than do lasers. Furthermore, this system adds a UV flash illuminator for photolysis of photoactivatable (caged) compounds, complete integration of patch-clamp instrumentation for concomitant electrophysiological recordings, and an upright stand preferred for tissue slice work. The result is a very cost-effective and flexible solution that responds to all described needs of the Major Users. No other instrument at or near the University of Kentucky can provide this constellation of features. The University will provide appropriate space, salary support for experienced technical staff, and maintenance for the system. The system will be set up within a large well-established Imaging Facility at the University of Kentucky by experts in scanning disk technology and electrophysiology. This system will allow the Major Users to continue to achieve new discoveries towards the better understanding and treatment of chronic pain, obesity, auditory disorders, olfactory disorders, pulmonary disease, consequences of brain injury, and cardiovascular disease.