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

描述（由申请人提供）：我们的12个联邦拨款（r01型）的长期目标是解决神经科学和心脏生理学中的重要和独特问题，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环境中的其他变化做出反应。目前的应用程序是为了响应我们的8个主要用户的重要需求，以获得共聚焦图像和电生理记录，通常同时，从活的神经，感觉和心脏细胞或组织切片。具体需求包括以下主要功能：活组织切片中Ca2+瞬态和荧光标记物的共聚焦成像；钙离子瞬态的比例Fura-2成像；在伴随膜片钳记录离子通道活性时，Ca2+成像的高时空分辨率；和紫外闪光光解笼化化合物。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。总的来说，我们已经发表了一百多篇使用一种或多种这些技术的论文，其中包括同时使用功能共聚焦成像和活细胞电生理记录的15篇论文。激光共聚焦显微镜固有的问题是非常强烈的激发光，导致大量的染料分解（光漂白）在活细胞成像。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。它采用旋转盘共聚焦技术，采用新一代超冷EM-CCD相机，生成具有高时空分辨率的图像。广谱光源产生的光漂白比激光少得多。此外，该系统还增加了一个用于光活化（笼状）化合物光解的UV闪光照明器，完全集成了用于同时进行电生理记录的膜片钳仪器，以及用于组织切片工作的直立支架。其结果是一个非常具有成本效益和灵活的解决方案，响应所有描述的主要用户的需求。在肯塔基大学或附近没有其他仪器可以提供这样的特征。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，并为系统提供维护。该系统将由扫描磁盘技术和电生理学专家在肯塔基大学的一个大型成熟的成像设施内安装。该系统将使主要用户能够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.