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。在过去的一年中，我们取得了以下主要发现：1)我们最近已经确定，线粒体在骨骼肌细胞中形成了一个网状结构，允许肌肉收缩的势能在细胞间快速传递。这对肌肉收缩的基本能量支持至关重要。我们今年发表了心脏细胞的线粒体网状结构，它与骨骼肌细胞明显不同。在心脏，这个网络在肌肉的长轴上连接得更紧密，而不是像骨骼肌那样在短轴上连接。我们认为这是由于与心肌相比，心脏细胞的直径减小了。我们目前正试图寻找与心脏细胞线粒体网形成相关的蛋白质。2)线粒体在所有肌肉细胞中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。我们最近发表的文章（肌肉线粒体网的电网保护参见参考书目）指出，一个快速故障安全系统已经到位，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。与线粒体从网络中快速移除相关的机制是实验室正在进行的工作。3)我们还发表了一个模型（线粒体网i波段段的电化学传输：见参考书目），关于导电如何在线粒体网中发生，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。4)利用我们快速监测活体动物亚细胞事件的能力，我们与约翰霍普金斯大学的Sinnis博士合作，通过模拟蚊子的喙来监测注射后疟疾寄生虫的贩运。模拟喙是一种特殊设计的荧光玻璃吸管，我们可以用双光子激发显微镜对其进行监测。使用带有绿色荧光蛋白的基因标记的寄生虫和小鼠（带有血管壁荧光蛋白），我们已经能够观察接种过程的最初几秒钟，并观察寄生虫在皮肤下的行为。我们希望这些研究将揭示寄生虫如何找到并穿透血管从而引发疟疾感染。这一认识可能为接种后立即预防疟疾感染提供新的策略。

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. The following major findings were made over the last year: 1) We have recently established that the mitochondria forms a reticulum across the skeletal muscle cell that permits the rapid transmission of potential energy for muscle contraction across the cell. This is critical for the fundamental energy support of muscle contraction. We published this year the mitochondrial reticulum structure of the heart cell which was distinctly different from the skeletal muscle cell. In the heart this network coupled more tightly across the long axis of the muscle rather than the short axis as in the skeletal muscle. We reasoned this to be due to the reduced diameter of the heart cell when compared to the heart muscle. We are currently attempting to look for the proteins associated with the formation of the mitochondria reticulum in heart cells. 2) This tight coupling of mitochondria across all of the muscle cells 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 recently published (Power Grid Protection of the Muscle Mitochondrial Reticulum see bibliography) 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. The mechanisms associated with this rapid removal of mitochondria from the network is an ongoing effort in the laboratory. 3) We have also published a modeled (The electrochemical transmission in I-Band segments of the mitochondrial reticulum: see bibliography) on 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. 4) Using our ability to monitor subcellular events rapidly in the living animal, we have initiated a collaboration with Dr. Sinnis at Johns Hopkins to monitor the trafficking of malaria parasites upon inject via a simulated mosquito proboscis. The simulated proboscis is a specially designed fluorescent glass pipet that we can monitor in the animal with a 2-Photon excitation microscope. Using genetically labeled parasites with green fluorescent protein and mice ( with a vessel wall fluorescent protein) we have been able to observe the first few seconds of the inoculation process and observe how the parasites behave under the skin. We hope these studies will reveal how the parasites find and penetrate blood vessels to initiate the malaria infection. This understanding may provide a new strategy in preventing the malaria infection immediately after the inoculation.