COBRE: UNV MED SCH: CORE D: DYNAMIC IMAGING FACILITY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 核心D将支持需要荧光成像专业知识的项目1、2和4。这个核心已经购买了两个荧光高速成像系统，可以用来检查完整组织和分离细胞的活动。其中一种成像装置以立式显微镜(尼康TS100)为基础，配有水浸透镜，并具有用于比率测量(Fura 2-AM)成像的波长切换器(DG-5)。后者允许校准加压血管、完整组织中的细胞和分离细胞中的钙活性。第二种成像系统以倒置显微镜为基础，并结合了电生理学装置。该系统用于确定电压与钙荧光信号之间的关系。这两个系统都使用了高灵敏度的背光摄像头(Cascade 512)。这些成像系统将支持需要检查细胞内和细胞间钙波传播的项目，这些钙波分别调节完整但孤立的眼底和结肠的平滑肌层的膜电位和收缩活动。项目2将检查这些波是否是修改过的原球蛋白基因敲除小鼠，并与它们的野生型小鼠进行比较。还将确定CaMKII抑制剂是否影响这些波的传播和持续时间。项目4将使用该设备来检测肠道肥厚区域的平滑肌层中细胞内和细胞间钙波传播的变化。此外，项目4将使用共聚焦钙成像来确定活动通过肠道神经系统神经元的传播是否发生变化。这是可能的，因为[Ca2+]i是动作电位依赖性肠神经元电活动变化的可靠指标。项目4还将结合膜片钳研究使用这项技术，以确定从部分梗阻的肠道不同区域分离的平滑肌细胞的自发钙瞬变(火花和泡芙)下的离子电导是否有变化。项目1将利用比率测量/加压血管成像系统来确定与野生型小鼠相比，缺乏dystrophin的MDX小鼠的动脉中的钙信号和血管反应性是否发生了变化。此外，还将利用结合膜片钳技术的单细胞钙成像实验，详细了解整合素如何影响分离的血管平滑肌细胞中的钙信号和电流。我们将在mdx和野生型小鼠分离的心肌细胞中比较钙离子通透性(L类型和非选择性)通道。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Core D will support Projects 1, 2 and 4 requiring expertise in fluorescent imaging. This core has purchased two fluorescent high speed imaging systems that can be used for examining activity in both intact tissues and in isolated cells. One imaging set up is based around an upright microscope (Nikon TS100) with water immersion lenses and has a wavelength switcher (DG-5) for ratio-metric (Fura 2-AM) imaging. The latter allows for calibrating calcium activity in pressurized blood vessels, cells in intact tissues and in isolated cells. The second imaging system is based around an inverted microscope and incorporates an electrophysiology set up. This system is used for determining the relationship between voltage and fluorescent calcium signals. Both systems use a highly sensitive back illuminated camera (Cascade 512). These imaging systems will support projects that need to examine the spread of intracellular and intercellular calcium waves that regulate the membrane potential and contractile activity respectively of the smooth muscle layers of the intact but isolated fundus and colon. Project 2 will examine whether these waves are modified PLB knockout mice compared to their wild type littermates. It will also be determined whether CaMKII inhibitors affect the propagation and duration of these waves. Project 4 will use this apparatus to examine the changes in the spread of intracellular and intercellular calcium waves in the smooth muscle layers from hypertrophied regions of bowel. In addition, Project 4 will use confocal calcium imaging to determine if changes occur to the spread of activity through the neurons of the enteric nervous system. This is possible because [Ca 2+]I is a reliable indicator of action potential dependent changes in the activity of enteric neurons. Project 4 will also use this technology in combination with patch clamp studies to determine if there are changes in the ionic conductances underlying spontaneous Ca2+ transients (Sparks and Puffs) in isolated smooth muscle cells taken from different regions of bowel with a partial obstruction. Project 1 will utilize the ratio-metric/pressurized vessel imaging system to determine whether Ca2+ signaling and vascular reactivity are altered in arteries from mdx mice lacking dystrophin compared to their wild type litter mates. In addition, single cell Ca2+ imaging experiments combined with patch clamp techniques will be utilized to provide a detailed understanding of how integrins affect Ca2+ signaling and currents in isolated vascular smooth muscle cells. Ca2+-permeable (L-type and nonselective) channels will be compared in isolated myocytes from mdx and wildtype mice.