Intra-vital microscopy using non-linear optical techniques

这些研究的目的是发展成像技术来监测亚细胞结构和过程，在体内。我们一直在系统地开发一种适应生物组织和结构的体内光学显微镜系统，而不是强迫动物在传统的显微镜台上。今年，我们通过战略合作，在实验室中增加了三维电子显微镜和超光学分辨率成像技术的使用。在过去的一年中，我们取得了以下主要发现：1)我们完成了使用3D电子显微镜，组织化学和超分辨率显微镜表征小鼠骨骼肌和心肌细胞线粒体结构的重要研究。这些研究揭示了线粒体网的存在，我们提出，线粒体网通过线粒体膜电位而不是分子的缓慢扩散，在整个细胞中迅速分配能量。骨骼肌网和心脏网的相似之处在于，线粒体通过它们的膜电位在细胞离散区域的大量线粒体上偶联。骨骼肌偶联是通过线粒体接触位点和长管状结构在细胞短轴上运行许多微米发生的。在心脏中，主要的偶联机制是通过沿细胞长轴的线粒体接触位点。在这两种系统中，我们都能够证明线粒体膜电位在细胞的大部分区域有快速传导。这种电耦合，就像房子里的电线一样，允许ATP生产的初级势能在细胞内快速分布，并将能量从许多线粒体汇总到细胞内的特定区域。细胞内线粒体的紧密耦合也是一种风险。如果一个线粒体出现故障，就会使整个线粒体网络瘫痪，就像房子里的短路一样。我们发现了一个快速故障安全系统，它可以迅速从网络中移除受损的线粒体。我们目前的假设是，这种故障安全或断路器机制本质上是结构性的，代表了线粒体与网络的物理解耦。这种能量在肌肉细胞内的快速分布与早期依赖于缓慢的高能代谢物扩散的模型形成鲜明对比，并提供了另一种在不同临床条件下评估的参数。我们也模拟了电导是如何在线粒体网中发生的，并得出结论，主要离子，如钾、钠和氯离子必须通过适当的质子-离子交换系统携带这种电流。线粒体网在与心脏代谢不全相关的不同疾病状态（如心力衰竭或缺血）中的作用尚待研究。

The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. This year we added the use of 3 dimensional electron microscopy and super optical resolution imaging to the technologies in the lab through strategic collaborations. The following major findings were made over the last year: 1) We completed a significant study in characterizing the structure of mitochondria in skeletal muscle and cardiac cells of the mouse using 3D electron microscopy, histochemical and super resolution microscopy. These studies revealed the presence of a mitochondrial reticulum that we proposed distributes energy rapidly throughout the cell via the mitochondrial membrane potential rather than the slow diffusion of molecules. The reticulum of the skeletal muscle and heart were similar in that the mitochondria are coupled via their membrane potential over large number of mitochondria in discrete regions of the cell. The skeletal muscle coupling occurred via both mitochondrial contact sites as well as long tubular structures running many microns across the short axis of the cell. In heart, the primary coupling mechanism was through mitochondrial contact sites along the long axis of the cell. In both systems we were able to demonstrate that there is a rapid conduction of the mitochondrial membrane potential across large regions of the cell. This electrical coupling, much like the wiring in a house, permits the rapid distribution of this primary potential energy for ATP production across the cell and summation of the energy from many mitochondria to specific regions in the cell. This tight coupling of mitochondria across the cell is also a risk. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have found that a rapid fail safe system is in place that rapidly removed damaged mitochondria from the network. Our current hypothesis is that this fail safe, or circuit breaker, mechanism is structural in nature representing the physical uncoupling of the mitochondria from the network. This rapid distribution of energy within the muscle cell contrasts with earlier models relying on slow high energy metabolite diffusion and provides another parameter to evaluate in different clinical conditions. We have also modeled how the electrical conduction occurs across the mitochondrial reticulum and reached the conclusion that the dominate ions, such as potassium, sodium and chloride must carry this current with appropriate proton-ion exchange systems. The role of the mitochondrial reticulum in different disease states associated with cardiac metabolic inadequacies, such as heart failure or ischemia, is yet to be investigated.