Tools for High Resolution Respirometry

我们的申请概述了4名生物学家对高分辨率呼吸测量(HRR)设备的请求，该设备对于检验有关应激源和局部麻醉剂对线粒体功能的影响的假说至关重要。该设备将能够实时准确、定量地测量线粒体和细胞中的呼吸、膜电位、钙、一氧化氮和活性氧。它将支持以下研究项目：(A)鱼类体内金属、缺氧和热应激的相互作用，(B)局部麻醉剂对鱼类线粒体生物能量学的影响，(C)有机污染物对水生系统的影响，以及(D)病毒感染对鱼类的影响。我们的研究有望产生关于细胞能量平衡在处理不同来源的应激源中的作用的重要信息。与市场上的同类系统不同，所要求的HRR系统是由最大限度地减少氧气扩散的材料建造的，并具有先进的电子设备，可以为氧气传感器提供高信号稳定性。高的信号稳定性最大限度地减少了所需的生物样本量，并为在生理上相关的低氧分压范围内的分辨率提供了基础。虽然该仪器主要是为测量呼吸作用而设计的，但它包括荧光、电位和安培模块，这些模块提供了测量膜电位、钙、pH和一氧化氮等阳离子的能力。事实上，可以根据需要购买其他附加组件，以增加测量内容。总体而言，该设备将使我们的研究人员能够生成一个全面的框架，使用线粒体功能指数来预测生物健康，这将对关于多种应激源和麻醉剂对能量稳态的影响的基础研究产生积极影响，并为水生污染和疾病管理的应用研究提供信息。此外，这一设备对于培训高素质人员掌握进步的研究方法至关重要，在当前竞争激烈的就业市场是必不可少的。

Our application outlines a request by a group of 4 biologists for high resolution respirometry (HRR) equipment that is essential to test hypotheses regarding effects of stressors and local anesthetics on mitochondrial function. This equipment will allow accurate, quantitative measurements in real-time of respiration, membrane potential, calcium, nitric oxide and reactive oxygen species in mitochondria and cells. It will support research programs focusing on (a) interactions of metals, hypoxia and thermal stress in fish, (b) effects of local anesthetics on mitochondrial bioenergetics in fish, (c) effects of organic pollutants on aquatic systems, and (d) effects of viral infections on fish. Our research is expected to generate important information regarding the role of cellular energy homeostasis in handing stressors of diverse origin. Unlike comparable systems in the market, the requested HRR system is constructed of materials that minimize oxygen diffusion and has advanced electronics to provide high signal stability of the oxygen sensors. The high signal stability minimizes the amount of required biological sample and provides the basis for resolution in the physiologically relevant low-oxygen tension range. Although this instrument is designed primarily to measure respiration, it includes fluorescence, potentiometric and amperometric modules that provide the capability to measure membrane potential, cations such as calcium, pH and nitric oxide. Indeed, other add-ons can be purchased as required to increase the repertoire of measurements. Overall, the equipment will enable our researchers to generate a comprehensive framework for using mitochondrial functional indices to predict organismal health that will positively impact fundamental studies on the effect of multiple stressors and anesthetics on energy homeostasis, as well as inform applied studies on management of aquatic pollution and diseases. Moreover, this equipment will be crucial for the training of highly qualified personnel in progressive research methodologies essential in the current competitive job market.